Dengue Virus Disease From Origin to Outbreak 2020

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Dengue Virus DiseaseFrom Origin to OutbreakEdited byAdnan I. QureshiExecutive Director, Zeenat Qureshi Stroke Institute,St. Cloud, MN, United StatesProfessor, Department of Neurology at University of Missouri,Columbia, United StatesOmar SaeedClinical Research Fellow, Zeenat Qureshi Stroke Institute,St. Cloud, MN, United StatesResident Physician, University of Tennessee Health Science Center inMemphis, Tennessee, United StatesAcademic Press is an imprint of Elsevier125 London Wall, London EC2Y 5AS, United Kingdom525 B Street, Suite 1650, San Diego, CA 92101, United States50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United StatesThe Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United KingdomCopyright � 2020 Elsevier Inc. All rights reserved.No part of this publication may be reproduced or transmitted in any form or by any means,electronic or mechanical, including photocopying, recording, or any information storageand retrieval system, without permission in writing from the publisher. Details on how toseek permission, further information about the Publisher’s permissions policies and ourarrangements with organizations such as the Copyright Clearance Center and the CopyrightLicensing Agency, can be found at our website: www.elsevier.com/permissions.This book and the individual contributions contained in it are protected under copyright bythe Publisher (other than as may be noted herein).NoticesKnowledge and best practice in this field are constantly changing. As new research andexperience broaden our understanding, changes in research methods, professionalpractices, or medical treatment may become necessary.Practitioners and researchers must always rely on their own experience and knowledge inevaluating and using any information, methods, compounds, or experiments describedherein. In using such information or methods they should be mindful of their own safetyand the safety of others, including parties for whom they have a professional responsibility.To the fullest extent of the law, neither the Publisher nor the authors, contributors, oreditors, assume any liability for any injury and/or damage to persons or property as a matterof products liability, negligence or otherwise, or from any use or operation of any methods,products, instructions, or ideas contained in the material herein.Library of Congress Cataloging-in-Publication DataA catalog record for this book is available from the Library of CongressBritish Library Cataloguing-in-Publication DataA catalogue record for this book is available from the British LibraryISBN: 978-0-12-818270-3For information on all Academic Press publications visit our website athttps://www.elsevier.com/books-and-journalsPublisher: Andre Gerhard WolffAcquisition Editor: Kattie WashingtonEditorial Project Manager: Anna DubnowProduction Project Manager: Sreejith ViswanathanCover Designer: Mark RogersTypeset by TNQ Technologieshttp://www.elsevier.com/permissionshttps://www.elsevier.com/books-and-journalsContributorsIqra Naveed Akhtar, Clinical Research Fellow, Department of Neurology, Universityof Missouri, Columbia, MO, United States; Zeenat Qureshi Stroke Institute,St. Cloud, MN, United StatesMohammad Ali Arif, Internal Medicine Shaheed Zulfiqar Ali Bhutto MedicalUniversity/Pakistan Institute of Medical Sciences, Islamabad, Punjab, Pakistan;Internal Medicine, Ali Medical Centre, Islamabad, Punjab, PakistanAhmer Asif, Zeenat Qureshi Stroke Institute, St. Cloud, MN, United States; Depart-ment of Internal Medicine, University of Oklahoma, Oklahoma City, United StatesSachin M. Bhagavan, Resident Physician, Department of Neurology, University ofMissouri Health Care, Columbia, MO, United StatesMohammad Rauf A. Chaudhry, Resident Physician, Texas Tech University HealthSciences Center, El Paso, TX, United States; Zeenat Qureshi Stroke Institute,St. Cloud, MN, United StatesMohammad F. Ishfaq, Resident physician, University of Tennessee Health Sciencecenter, Memphis, Tennessee, United States; Zeenat Qureshi Stroke Institute,St. Cloud, MN, United StatesJahanzeb Liaqat, Lt. Colonel, Pak Emirates, Military Hospital, Rawalpindi, PakistanIryna Lobanova, Project Manager, Dengue virus disease project, Zeenat QureshiStroke Institute, St. Cloud, MN, United States; University of Missouri, Columbia,MO, United StatesNgan Nguyen, Department of Internal Medicine, University of Tennessee HealthScience Center, Memphis, Tennessee, United StatesAdnan I. Qureshi, Executive Director, Zeenat Qureshi Stroke Institutes, St. Cloud,MN, United States; Professor, Department of Neurology at University of Missouri,Columbia, United StatesIhtesham Qureshi, Resident Physician, Department of Neurology, Texas TechUniversity of Health Sciences Center, Paul L. Foster School of Medicine, El Paso,TX, United StatesMushtaq H. Qureshi, Texas Tech University Health Sciences Center El Paso,Neurology, Texas Tech University, El Paso, TX, United States; Zeenat QureshiStroke Institute, St. Cloud, MN, United StatesArbaab Qureshi, Clinical Research Fellow, Department of Neurology, Texas TechUniversity of Health Sciences, El Paso, TX, United StatesxiOmar Saeed, Resident Physician, Department of Neurology, University of TennesseeHealth Science Center, Memphis, Tennessee, United States; Zeenat Qureshi StrokeInstitute, St. Cloud, MN, United StatesMuhammad A. Saleem, Resident Physician, Family Medicine, Mercyhealth,Janesville, WI, United StatesSargun Singh Walia, Clinical Research Fellow, Zeenat Qureshi Stroke Institute,St. Cloud, MN, United States; Department of Neurology, University of Missouri,Columbia, MO, United Statesxii ContributorsChapter 1Dengue virus infectionAdnan I. QureshiExecutive Director, Zeenat Qureshi Stroke Institute, St. Cloud, MN, United States; Professor,Department of Neurology at University of Missouri, Columbia, United StatesIntroductionThe World Health Organization website states “Dengue is a mosquito-borneviral disease that has rapidly spread in all regions of World Health Organiza-tion in recent years”. In April 2016, World Health Organization issued a con-ditional recommendation on the use of the vaccine for areas, in which Denguevirus infection is highly endemic, which is defined by population seroprevalenceof 70% or higher. Dengue virus infection mainly causes a self-limiting flu-likeillness and may remain asymptomatic (Fig. 1.1). A reader may wonder why abook should be dedicated to this disease. There are several reasons: 1. Theinfection can develop into a potentially severe Dengue virus illness which canbe fatal and now a major cause of severe illness among children; 2. The diseaseis increasing exponentially in prevalence; 3. There is no specific treatment forthe disease. The global incident map website provides a detailed list of hundredsof Dengue viral illness outbreaks since 2010. The first and last page of thelisting is provided to give a better perspective of large number of incident casesin the world. The global map from new Dengue virus disease cases from May2019 also provides a regional perspective on the disease (Fig. 1.2).Health officials in the Philippines in July 2019 declared a national emer-gency after a record-breaking 106,630 cases of Dengue viral fever were re-ported since January 2019 [1]. This represented a 85% increase than thenumber of cases in the same period in 2018. Approximately 500 people havedied from the Dengue viral illness in 2019. Military hospitals and clinics wereput on alert for a possible surge in Dengue viral illness patients [2].World Health Organization/Department of Control of Neglected TropicalDiseases released a detailed document “The Global Strategy for dengue pre-vention and control, 2012e20”. (Fig. 1.3) The executive summary of thedocument states:“Dengue is a major public-health concern throughout tropical and sub-tropical regions of the world. It is themost rapidly spreading mosquito-Dengue Virus Disease. https://doi.org/10.1016/B978-0-12-818270-3.00001-1Copyright © 2020 Elsevier Inc. All rights reserved. 1https://doi.org/10.1016/B978-0-12-818270-3.00001-1FIGURE 1.1 List of country with Dengue hemorrhagic fever [9].FIGURE 1.2 Map of Dengue virus disease outbreak around the world [8].2 Dengue Virus Diseaseborne viral disease, with a 30-fold increase in global incidence over the past50 years. The World Health Organization (WHO) estimates that 50e100million Dengue infections occur each year and that almost half the world’spopulation lives in countries where dengue is endemic. In some countries, theburden of dengue is comparable to that of tuberculosis and other communi-cable diseases with high disease burdens; unexpected surges in cases and thechallenge to health systems of triaging thousands of cases without knowingwhich severe cases will require hospital care are additional challenges . ThisGlobal strategy for dengue prevention and control, 2012e20 aims to addressthis need.Dengue morbidity can be reduced by implementing improved outbreakprediction and detection through coordinated epidemiological and entomo-logical surveillance; promoting the principles of integrated vector managementand deploying locally-adapted vector control measures including effectiveurban and household water management. Effective communication can ach-ieve behavioural outcomes that augment prevention programmes.FIGURE 1.3 List of country with Dengue hemorrhagic fever [9].Dengue virus infection Chapter | 1 3Research will continue to play an important role in reversing the trend indengue, a neglected tropical disease, by improving methods and systems forsurveillance, prevention and control.Reversing the trend requires commitments and obligations from partners,organizations and countries, as well as leadership by WHO and increasedfunding. Dengue prevention and management can now exploit opportunitiespresented by promising advances in vector control technology interventions,diagnostics, prognostic systems for triage, evidence-based clinical in-terventions and candidate vaccine developments.”TDR, the Special Program for Research and Training in Tropical Diseases,which is a scientific collaboration supports efforts to combat diseases ofpoverty has a dedicated section on Dengue virus disease. The program wasdeveloped by World Health Organization, and is sponsored by the UnitedNations Children’s Fund, the United Nations Development Program, and theWorld Bank. On the programs by TDR is the web based tool “OperationalGuide: The Early Warning and Response System (EWARS) for DengueOutbreaks” which acts as a resource for: (i) analysis of historic denguedatasets; (ii) identify appropriate alarm indicators that can predict forthcomingoutbreaks; and (iii) use these results and analyses to build an early warningsystem to detect dengue outbreaks (Fig. 1.4). Another handbook by TDR“Technical handbook for dengue surveillance, dengue outbreak prediction/detection and outbreak response” provides a “model contingency plan” is toassist program managers and planners in developing a Dengue virus illnessoutbreak response plan through clearly defined and validated alarm signals andFIGURE 1.4 Website of special program for research and training in tropical disease [8].4 Dengue Virus Diseaseorganize an early response “emergency response” once an outbreak has started(Fig. 1.5).Another controversy is whether donated blood should be screened forpresence of Dengue virus [3]. Dengue virus like West Nile virus and Chi-kungunya virus may be transmitted through blood transfusions. One studytested 15,350 blood donation samples; Dengue virus RNA was detected in 29samples for a prevalence of 1 per 529 (0.19%). Dengue virus types 1, 2, and 3with viral titers of 105e109 copies/mL were detected by type specific reversetranscriptase polymerase chain reaction in 12 samples of which all were in-fectious in mosquito culture [4]. Another study in Portugal found that 43 of1948 blood donations tested positive for Dengue virus genome (further iden-tified as Dengue virus serotype-1) [5]. Pozzetto et al. reported that only fiveFIGURE 1.5 World Health organization publication of Dengue viral illness prevention andcontrol [8].Dengue virus infection Chapter | 1 5cases of transfusion-transmitted Dengue viral illness have been confirmed [6].One patient who received red blood cells containing 108 copies/mL Dengueviral serotype-2, developed Dengue hemorrhagic fever 3 days after trans-fusion. Both donor and recipient were shown to harbor viruses with the sameenvelope sequence. Pozzetto et al. [6] recommended the following steps toreduce transfusion related transmission of Dengue viral illness [1] the clinicalselection of donors [2]; the implementation of screening tests specific forDengue virus; and [3] the nonspecific reduction or inactivation of pathogens bythe use of physical or chemical treatments applied to blood products. Inendemic areas, excluding donors who may be at higher risk of infection is notpractical as exposure to mosquito bite is unpredictable. The presence of feverin donors of blood products is a usually a contraindication of blood donation.Gen-Probe Inc. (San Diego, CA, United States) developed a prototypetranscription-mediated amplification assay for large-scale screening purpose asin blood donors, with ability to detect approximately 15 copies/mL for eachserotype in blood sample. Transcription Mediated Amplification technologysimplifies nucleic acid testing by enabling simultaneous detection of multipleviruses in blood sample. Techniques are available to treat blood products inorder to inactivate some pathogens. Solvent-detergent treatment to disruptviral envelopes, exposure to light activated dyes containing phenothiazine likemethylene blue, leading to oxidation of guanine present in viral genomes, andnanofiltration to retain viral particles, and photoactivation of compounds byultraviolet rays. In 2009, the American Association of Blood Banks stratifiedin four levels (red, orange, yellow and green) the emergent or reemergentinfectious agents [7]. The stratification was performed to identify agents thatcould represent a potential threat to transfusion in North America for the nextyears. The criteria was based on [1] presence in blood at least for a few hoursor days [2]; asymptomatic donors to avoid clinical selection [3]; ability toinduce a severe disease; and [4] finally, resist to inactivation by immunity ofthe donor. Dengue virus was classified in the upper red level, together withBabesia sp and the human variant of CreutzfeldteJakob disease.Vaccination for Dengue viral infection has a fascinating story. The Foodand Drug Administration on May 1st, 2019 approved the first vaccine“Dengvaxia” from Sanofi Pasteur against Dengue viral illness which wasapproved by European Commission last year. Dengvaxia is licensed for use in19 countries, plus the eligible parts of the European Union. The vaccinationcan increase the risk of severe Dengue viral illness in some individuals. Aphenomenon that has resulted in suspension of vaccine and revoking of licensein Philippines, one of the countries with largest exposure to the vaccine. Sanofiemployees, and several current and former Philippines health officials arebeing investigated over deaths that could be linked to use of the vaccine. TheWorld Health Organization in September 2018 replaced the 2016 positionpaper in particular due to new evidence of a greater precipitating severeDengue viral illness in trial participants who were seronegative compared with6 Dengue Virus Diseasethose who are seropositive before vaccination. The World Health Organizationin September 2018 also recommended “only persons with evidence of a pastDengue infection would be vaccinated (based on an antibody test, or on adocumented laboratory confirmed Dengueinfection in the past). Only if pre-vaccination screening is not feasible, vaccination without individualscreening could be considered in carefully selected areas with recent docu-mentation of seroprevalence rates of at least 80% by the age of 9 years.” Theposition paper also identified the development of highly specific and sensitiverapid tests for determination of Dengue virus serological status as a researchpriority. Furthermore, research is also needed to evaluate vaccine scheduleswith fewer doses, and to assess the need for booster doses. The development ofsafe, effective, and affordable Dengue vaccines for use irrespective of seros-tatus remains a high priority according to World Health Organization. TheCenters for Disease Control and Prevention also is tracking cases in UnitedStates as 112 cases have been reported.References[1] Fox news. Philippines declares national emergency after more than 100,000 people contractDengue fever Fox News Flash top headlines for July 17. Fox News; 2019. Available from:foxnews.com/world/philippines-national-emergency-dengue-fever.[2] Dayaram S. Philippines declares national alert after 456 die from dengue fever Online: CNNhealth. 2019. Available from: https://www.cnn.com/2019/07/16/health/philippines-dengue-national-alert-hnk-intl/index.html.[3] Lanteri MC, Busch MP. Dengue in the context of “safe blood” and global epidemiology: toscreen or not to screen? Transfusion 2012;52(8):1634e9.[4] Stramer SL, Linnen JM, Carrick JM, Foster GA, Krysztof DE, Zou S, et al. Dengue viremiain blood donors identified by RNA and detection of dengue transfusion transmission duringthe 2007 dengue outbreak in Puerto Rico. Transfusion 2012;52(8):1657e66.[5] Control ECfDPa. Dengue outbreak in Madeira, Portugal. March 2013. Online.[6] Pozzetto B, Memmi M, Garraud O. Is transfusion-transmitted dengue fever a potential publichealth threat? World J Virol 2015;4(2):113e23.[7] Stramer SL, Hollinger FB, Katz LM, Kleinman S, Metzel PS, Gregory KR, et al. Emerginginfectious disease agents and their potential threat to transfusion safety. Transfusion2009;49(Suppl. 2):1se29s.[8] https://www.who.int[9] https://outbreaks.globalincidentmap.com/Dengue virus infection Chapter | 1 7http://foxnews.com/world/philippines-national-emergency-dengue-feverhttps://www.cnn.com/2019/07/16/health/philippines-dengue-national-alert-hnk-intl/index.htmlhttps://www.cnn.com/2019/07/16/health/philippines-dengue-national-alert-hnk-intl/index.htmlhttps://www.who.inthttps://outbreaks.globalincidentmap.com/Chapter 2Dengue virus disease; theoriginsOmar Saeed1,2, Ahmer Asif2,31Resident Physician, Department of Neurology, University of Tennessee Health Science Center,Memphis, Tennessee, United States; 2Zeenat Qureshi Stroke Institute, St. Cloud, MN, UnitedStates; 3Department of Internal Medicine, University of Oklahoma, Oklahoma City, United StatesIncredibly, just one mosquito species, Aedes aegypti is responsible for the spreadof four known different deadly viral diseases to human beings, yet this mosquitohas been allowed to infest densely-populated urban centers.T.K. Naliaka.Another member of the Flaviviridae family with Chikungunya, Yellow fever,and Zika virus is the Dengue virus. This particular virus has seen exponentialgrowth in terms of a global spread over the past several decades. The origins of thewordDengue remain unclear; however, the use of the wordDengue to describe thedisease for the very first time was in Spain in 1801. A most likely theory is that it isderived from the Swahili phrase “Ka-dinga pepo”, meaning “cramp-like seizurecaused by an evil spirit” [1,2]. The Swahili word “dinga” may possibly have itsorigin in the Spanish word “Dengue” meaning fastidious or careful, which woulddescribe the gait of a person suffering from the bone pain of Dengue fever. Slaves inthe West Indies who contracted Dengue viral illness were said to have theposture and gait of a dandy, and the disease was known as “Dandy Fever.” Duringthe 1828 epidemic in Cuba, the illness was first called dunga, but later it waschanged to Dengue, the name by which we have been addressing this disease eversince.The first suspected outbreaks of Dengue-like disease were reported in 1635in Martinique and Guadeloupe and 1699 in Panama [3e5]. However, reportsof illnesses compatible with Dengue fever occurred even earlier. The earliestrecord found to date was in a Chinese “encyclopedia of disease symptoms andremedies,” first published during the Chin Dynasty (AD 265e420) andformally edited in AD 610 (Tang Dynasty) and again in 992 during theNorthern Sung Dynasty [6]. The Chinese used the name water poison for thedisease as it was thought to have a connection with flying insects related towater [30]. Hence, before the 18th century, the Dengue-like disease had a widegeographic distribution where major epidemics occurred widely.Dengue Virus Disease. https://doi.org/10.1016/B978-0-12-818270-3.00002-3Copyright © 2020 Elsevier Inc. All rights reserved. 9https://doi.org/10.1016/B978-0-12-818270-3.00002-3The disease emerged from Africa during the slave trade in the 15th through19th centuries and spread into the Americas through commercial exchanges inthe 17th, 18th, and 19th centuries. The Aedes aegypti became highly adaptedto the humans and metropolitan environments and sailing ships were the majorreason for the spread of this mosquito species throughout the tropics of theworld. The infestation first started from the port cities and later moved into theinland. Expansion of the urbanization was solely responsible for it. Over time,A. aegypti became intimately associated with the humans, i.e., feeding on themand sharing their living places, making it an efficient epidemic vector ofDengue and Yellow fever viruses [3]. Due to this setting, the major epidemicsof Dengue occurred during the 18th till the early 20th centuries (Fig. 2.1).Early on, after the documentation of mosquitoes as vectors for Yellowfever, many workers in this field suspected mosquitoes to be the vectors forDengue fever as well. However, in the previrology era due to the slow progressof work and use of human volunteers, its documentation took long. At thebeginning of 19th century, the documentation of transmission of Dengue viralillness by mosquitoes was done by Graham in 1903, Bancroft in 1906, andthen by Cleland in 1918.Since after the documentation that mosquitoes transmit the Dengue virus,they were not isolated until the 1940s. During World War II [7e10], in the year1943, the Dengue virus was isolated for the first time. At that time, Denguefever was chiefly responsible for illness among Japanese and their alliedsoldiers in the Asian and Pacific regions. There are four different strains ofDengue virus (DENV1-4) and they are antigenically and phylogeneticallydifferent from one another. They were reported for the first time in differentregions, as follows:- DENV1: This strain of Dengue virus was first reported in Japan and FrenchPolynesia in 1943 followed by Hawaii in 1945 [8]. Over time, the reportingof DENV1 in the Asian territories kept on increasing and later in Africa. Inthe Americas, it was first reported in 1977.- DENV2: This strain was first identified in 1944 in Papua New Guinea. andIndonesia. Later it was reported in the Philippines, Malaysia and thenThailand. In the Americas, it was first reported in 1953 [11,12].- DENV3: This strain was reported for the first time in the Philippines andThailand in 1953. Since then it is been very commonly reported in theAsian territory. The first reports in the Americas were in the year 1963 [13].- DENV4: This strain was also reported for the first time in the Philippinesand Thailand in 1953. Since then it has been reported very commonly in theAsian regions especially Indonesia and Sri Lanka. In the Americas, it wasfirst reported in 1981.The incidence of a severe form of Dengue fever knownas Dengue hem-orrhagic fever is not limited to the 20th century. These patients with symptomsof Dengue hemorrhagic fever have been reported since 1780, i.e., in the10 Dengue Virus DiseasePhiladelphia epidemic [14]. Since then, several cases of Dengue hemorrhagicfever have been reported in the form of subsequent epidemics including theones in Australia (1897), Beirut (1910), Taiwan (1916), and Greece (1928)FIGURE 2.1 Dengue viral illness occurrence in Africa and the Middle East.Dengue virus disease; the origins Chapter | 2 11[15,16]. Unlike the Dengue fever, these epidemics occurred relatively rarelyand had long intervals between them. It made them less important as a longterm and continuous public health problem.Shifting patterns of dengue feverUntil the 1940s, the Dengue viral illness outbreaks were relatively infrequentbut World War II, especially in Southeast Asia, lead to a changed pattern of thedisease due to the ecological disturbance. During the years after the war,significant urbanization and economic progress offered ideal conditions for theoverwhelming spread of mosquito-borne diseases that in turn led to the start ofa global pandemic of Dengue viral illness. Due to the increased epidemicspread and flow of people across the countries, hyperendemicity i.e., cocir-culation of multiple Dengue virus strains, developed in the South Asia regionleading to the emergence of epidemic Dengue hemorrhagic fever [17]. Firstknown Dengue hemorrhagic fever epidemic occurred in 1953 in Manila and itbecame more intense by its spread over the next 20e30 years in SoutheastAsia. In Asia, the Dengue epidemics geographically stretched to India,Maldives, Pakistan, and Southeast Asian countries east to China [3,17].Numerous island countries of the Central and South Pacific regions (NewCaledonia, Tahiti, Cook Islands, Palau, Niue, Yap, and Vanuatu) have alsoexperienced a number of minor and major Dengue hemorrhagic fever out-breaks [18] (Fig. 2.2).In the Americas, the changes in the epidemiology were the most histrionic.During the 1960s and 1970s, the epidemics of Dengue viral illness were rare inthe American territories due to the eradication of A. aegypti from the Southernand Central parts of America [19e21]. In the early 1970s, the eradicationprogram was stopped which led to the reinvasion of the infection in thecountries from where it was already eradicated [20,21]. Until the 1990s,the A. aegypti reinfested that geographic distribution again. During this time,the American region was facing the major Dengue viral illness epidemics thathad been free of the disease for the past 100 years [19,20,22]. Similar to theSoutheast Asian region, the development of hyperendemicity due to thepeaked epidemic activity in the American region also led to the emergence ofepidemic Dengue hemorrhagic fever. Since the year 1981e2015, Denguehemorrhagic fever was confirmed on laboratory reports in 24 Americancountries (Fig. 2.3) [22,23].In Africa, the sporadic cases of Dengue hemorrhagic fever occurred morecommonly than having major epidemics. This is due to the remarkable in-crease in the Dengue fever epidemic in the past 25 years in this region leadingto severe disease. Until the 1980s, very little was known about the spread ofDengue fever disease viruses in Africa. Since then, major Dengue fever epi-demics have occurred in both the Western and Eastern parts of Africa [18,24],which involved all four viral strains. In the 1990s, these outbreaks were more12 Dengue Virus Diseasecommon in the Middle East and East Africa, having the major ones in Djiboutiin 1991 and in Jeddah, Kingdom of Saudi Arabia in 1994 [18].FIGURE 2.2 Dengue viral illness occurrence in Asia and Oceania.FIGURE 2.3 Dengue viral illness occurrence in the Americas and Caribbean.Dengue virus disease; the origins Chapter | 2 13In 1997, the A. aegypti mosquitoes and Dengue viruses had globaldissemination in tropics and more than 2.5 billion humans dwell in regionswhere the Dengue fever is endemic [23,25,26]. Presently, this virus isresponsible for causing more morbidity and mortality than any other arbovirusillness in humans. Due to these epidemics, every year approximately 100million cases of Dengue fever and many hundred thousands of Denguehemorrhagic fever occur [23,25,27].Factors responsible for increased incidenceDengue fever and Dengue hemorrhagic fever outbreaks have been globalpublic health problems over the past 17 years. Although a number of factorsare responsible for the significant resurgence and emergence of these out-breaks but still the precise determination of these factors is complex and notwell understood. Nevertheless, over the past 50 years [18,23,24] this resur-gence seems to be narrowly linked with the demographic and societal changes.Following are the four major factors responsible for the increase in incidence:1) One of the major factors has been the extraordinary growth of the globalpopulation. This population growth has been the main driving force for theuncontrolled and unplanned urbanization, particularly in tropical countries.In this regard, the substandard housing, overcrowding of cities, and declinein sewer, water, and waste management systems are aiding to provide anideal environment for the increased transmission of vector-borne diseases.2) A second major factor is the deficiency of an effective mosquito controlprogram in Dengue-endemic areas [19,20,23,24]. Since the past 25 years,spraying the spaces with insecticides to kill the mosquitoes is being usedbut it has proven to be ineffective over time [20,28,29]. In addition to that,because of the augmented amount of mosquito larval habitations, thepopulation density and terrestrial distribution of A. aegypti has alsoincreased, especially in the tropics.3) Another most important factor contributing to the increase in the emer-gence of Dengue fever and Dengue hemorrhagic fever outbreaks isincreased travel by airplanes. Air travel provides an ideal way for thetransportation of viruses like dengue and other urban pathogens amongdifferent population centers of the world [23,24].4) Fourth important factor responsible for the resurgence of Dengue outbreakshas been the deterioration of public health infrastructures in the underde-veloped countries over the last 30 years. Scarcity of resources has led to anoverwhelming shortage of qualified physicians who can propose and14 Dengue Virus Diseasedevelop effective prevention and control programs for the mosquito andother vector-borne diseases.In summary, the societal and demographic changes, lack of effectivemosquito control programs, scarce resources for the vector-borne diseaseprevention and control, and alterations in the public health program have allled to the increased Dengue epidemic activity, the hyperendemicity develop-ment, and the incidence of Dengue hemorrhagic fever epidemic. As of 2019,the global burden of Dengue viral illness is truly immense affecting approx-imately 2.5 billion or 40% of the world’s population being endemic in over100 countries including Asia, Pacific, the Americas, Africa, and Caribbean.According to World Health Organization, there are anywhere from 50 to 100million cases every year with staggering 22,000 deaths due to the Dengue viralillness.References[1] Christie J. On epidemics of dengue fever: their diffusion and etiology. Glasgow Med J1881;16(3):161.[2] Christie J. Remarks on “Kidinga Pepo”: a peculiar form of exanthematous disease. BritishMed J 1872;1(596):577.[3] Gubler DJ. Dengue and dengue hemorrhagic: its history and resurgence as a global publichealth problem. In: Dengue and dengue hemorrhagic fever. London: CAB International;1997. p. 1e22.[4] McSherry JA. Some medical aspects of the Darien scheme: was it dengue? Scott Med J1982;27(2):183e4.[5] Halstead S. Dengue: overview and history. In: Tropical medicine: Science and practice.London: ImperialCollege Press; 2008.[6] Nobuchi H. The symptoms of a dengue-like illness recorded in a Chinese medical ency-clopedia. 1979. p. 422e5.[7] Kimura R, Hotta S. Studies on dengue: anti-dengue active immunization experiments inmice. Jpn J Bacteriol 1944;1:96e9.[8] Hotta S. Experimental studies on dengue: I. Isolation, identification and modification of thevirus. J Infect Dis 1952;90(1):1e9.[9] Sabin AB. Research on dengue during world war II. Am J Trop Med Hyg 1952;1(1):30e50.[10] Sabin AB, Schlesinger RW. Production of immunity to dengue with virus modified bypropagation in mice. Science 1945;101(2634):640e2.[11] Rico-Hesse R, Harrison LM, Salas RA, Tovar D, Nisalak A, Ramos C, et al. Origins ofdengue Type 2 viruses associated with increased pathogenicity in the Americas. Virology1997;230(2):244e51.[12] Cologna R, Armstrong PM, Rico-Hesse R. Selection for virulent dengue viruses occurs inhumans and mosquitoes. J Virol 2005;79(2):853.[13] Messer WB, Gubler DJ, Harris E, Sivananthan K, De Silva AM. Emergence and globalspread of a dengue serotype 3, subtype III virus. Emerg Infect Dis 2003;9(7):800.[14] Rush B. An account of the bilious remitting fever: as it appeared in philadelphia, in thesummer and autumn of the year 1780. Am J Med 1951;11(5):546e50.Dengue virus disease; the origins Chapter | 2 15[15] Copanaris P. L’Epidémie de dengue en Grèce au cours de l’été 1928. In: Par P. Copanaris:Office international d’hygiène publique; 1928.[16] Akashi K. A dengue epidemic in the Tainan District of Taiwan in 1931. Taiwan No Ikai1932;31:767.[17] Halstead SB. Dengue haemorrhagic feverda public health problem and a field for research.Bull World Health Organ 1980;58(1):1.[18] Gubler DJ. Dengue and dengue hemorrhagic fever: its history and resurgence as a globalpublic health problem. In: Dengue and dengue hemorrhagic fever; 1997.[19] Gubler D. Epidemiology of arthropod-borne viral diseases. Boca Raton, USA: CRC PressInc; 1988.[20] Gubler DJ. Aedes aegypti and Aedes aegypti-borne disease control in the 1990s: top downor bottom up. Am J Trop Med Hyg 1989;40(6):571e8.[21] Pinheiro FP. El dengue en las Américas: 1980e1987. 1989.[22] Pinheiro FP, Corber SJ. Global situation of dengue and dengue haemorrhagic fever, and itsemergence in the Americas. World Health Stat Q 1997;50:161e9.[23] Gubler D. The global pandemic of dengue/dengue haemorrhagic fever: current status andprospects for the future. Ann Acad Med Singapore 1998;27(2):227e34.[24] Gubler D, Trent D. Emergence of epidemic dengue/dengue hemorrhagic fever as a publichealth problem in the Americas. Infect Agents Dis 1993;2(6):383e93.[25] Gubler DJ, Clark GG. Dengue/dengue hemorrhagic fever: the emergence of a global healthproblem. Emerg Infect Dis 1995;1(2):55.[26] Halstead SB. Pathogenesis of dengue: challenges to molecular biology. Science1988;239(4839):476e81.[27] Monath TP. Dengue: the risk to developed and developing countries. Proc Natl Acad Sci1994;91(7):2395e400.[28] Newton EA, Reiter P. A model of the transmission of dengue fever with an evaluation of theimpact of ultra-low volume (ULV) insecticide applications on dengue epidemics. Am J TropMed Hyg 1992;47(6):709e20.[29] Reiter P, Gubler DJ. Surveillance and control of urban dengue vectors. New York: Dengueand dengue hemorrhagic fever CAB International; 1997. p. 425e62.[30] Bock G, Goode J, editors. New treatment strategies for dengue and other flaviviral Diseases:Novartis Foundation Symposium 277, vol. 277. First published: 25 August 2006. HistoryPage 3e4.Further reading[31] Gubler DJ. Dengue and dengue hemorrhagic fever. Semin Pediatr Infect Dis 1997;8(1):3e9.16 Dengue Virus DiseaseChapter 3Dengue virus infectionoutbreak: comparison withother viral infection outbreakMohammad Rauf A. ChaudhryResident Physician, Texas Tech University Health Sciences Center, El Paso, TX, United States;Zeenat Qureshi Stroke Institute, St. Cloud, MN, United StatesDengue virus infection is one of the most common arthropod-borne viraldisease causing about 50e100 million infections per year [1]. About 40%world population live in areas with high risk of Dengue virus transmission.There are about 100 countries in Asia, the Pacific, the Americas, Africa, andthe Caribbean where Dengue virus infection is endemic [2]. Dengue virusinfection is caused by any of the four narrowly related serotypes: Dengueviruses 1e4. Infection with one serotype does not provide immunity againstother serotypes. In fact it increases the risk for Dengue hemorrhagic fever andDengue shock syndrome [3]. The four serotypes originated in monkeys andindependently made a cross over to humans in Africa or South East Asia about100e800 years ago [4].Dengue virus infection can be asymptomatic or a self-limited, varying inseverity, classical form is characterized by high fever, headache, stomach ache,rash, myalgia, and arthralgia. Dengue hemorrhagic fever and Dengue shocksyndrome are severe forms of Dengue virus infection, accompanied bythrombocytopenia, vascular leakage, and hypotension [1]. Mechanisms un-derlying the severe form of disease are still not well understood despite theintensive research. The lack of understanding is partly due to lack of appro-priate animal models of infection and disease. Due to lack of vaccine andantiviral drugs, only control measure is limiting the Aedes mosquito vectorsspread [1].Epidemic versus pandemicEpidemic and pandemic are primarily different in terms of spread of conta-gious, infectious, or viral illness. Epidemic is limited to one specific region andDengue Virus Disease. https://doi.org/10.1016/B978-0-12-818270-3.00003-5Copyright © 2020 Elsevier Inc. All rights reserved. 17https://doi.org/10.1016/B978-0-12-818270-3.00003-5is an event in which a disease is actively spreading while pandemic describe adisease affecting the whole country or the entire world. In simple words, whenepidemics falls short in describing the scale of a problem, it is better to usepandemic [5].Common features of epidemicsAccording to our previous work on Ebola virus infection epidemic byQureshi et al. [6], there are several factors that can start an epidemic listedbelow:1 Disasters (e.g., wars, famine, floods, and earthquakes)2 Temporary population settlements3 Preexisting diseases in the population4 Ecological changes like floods and cyclones5 Resistance potential of the host (i.e., nutritional and immunization status ofthe host)6 Damage to public utility and interruption of public health servicesQureshi et al. mentioned that there are three patterns of disease continuity:1 Saw tooth pattern2 Tooth necklace pattern3 Tooth eruption patternSaw tooth patternIt represents an intermittent outbreak of a disease that recedes in intensity, butthe disease is not eradicated from the population. The smallpox epidemics inAfrica during 1920e1950s would be an example of such a patternFigs. 3.1e3.3.Tooth necklace patternIt constitutes where the disease is eradicated from the population, but pathogenspecies is kept alive under controlled circumstances for preparation of vac-cines and biological studies. While the escape of pathogen from confinementsof laboratories has been the subject of numerous conspiracy theories, vacci-nation with live attenuated viruses is more likely to be the string to maintainthe continuity.Tooth eruption patternIt constitutes where, like the tooth hidden within the gums and emerging in-dependent of other teeth, the pathogen emerges and is exterminated without18 Dengue Virus Diseaseany relation to previous occurrences. The Dengue virus infection epidemicfollows the “tooth eruption” pattern.Why epidemics die their deaths?It is generally believed that measures like such as vaccination of at-risk in-dividuals, quarantine of diseased persons, and acute and timely treatment helpto control all the epidemics. However, facts do notsupport this conclusion. InFIGURE 3.1 Map showing the estimated global distribution ofDengue, Zika, andChikungunya [9].ScleraChoroldCiliary body andciliary muscleRetina(Retinitis, chorioretinitis, retinal edemaneuroretinitis)KFDVKFDVZIKVDENVMacula(Maculopathy)Vitreous bodyOptic nerve(Vitritis)(Optic neuritis)DENVZIKVDENVWNVYFVZIKVDENV(Uveitis)(Keratitis)WNVZIKVKFDVKFDV(Lens Opacity)(Conjunctivitis)KFDVDENVRetinal blood vessels(Retinal hemorrhage)JEVZIKVKFDVZIKVKFDVDENVIrisPupilUveaAnterior chamberCorneaLensPosterior chamberConjunctivaFIGURE 3.2 Eye anatomy and ocular complications caused by flaviviruses. Various componentsof the human eye are labeled in black. The flaviviruses responsible for causing ocular manifes-tations are shown in green whereas specific ocular tissue pathology is highlighted in red [11].Dengue virus infection outbreak Chapter | 3 19fact, the largest epidemics, such as the Peloponnesian War Pestilence, Anto-nine Plague, Plague of Justinian, Black Death of the 14th century, and Spanishflu, came to an end without widespread use of any of these strategiesmentioned above.Qureshi et al. [6], came up with three theories for spontaneous remission ofepidemics which are mentioned below:1 There are two types of people within the exposed population: some morevulnerable and some more resistant. The people who may be resistant to thedisease may be so because of previous exposure to viruses with similarstructures, resulting in the development of immune responses that areadequate for multiple pathogens. They might also be resistant due to su-perior health, including age, nutritional status, and occupational advantages.The virus might eventually be faced with a population that is completelyresistant to the infection.2 Changing environment within habitats that are not conducive to the survivalor propagation of viruses or other pathogens. Weather changes, includingtemperature and humidity fluctuations, may significantly influence thesurvival or propagation of a virus outside the body. Elimination of reservoirsthat carry pathogens including animals, insects, food, or water, by chance ordesign, may disrupt the cycle of propagation. Such elimination of infectionis less likely to occur within an epidemic because of diverse factors andgeographical areas involved.3 The most likely explanation is the “Sand Filter Theory,” a term coined byDr. Qureshi. This theory reflects the similarity between retention of par-ticulate matter during filtration based on density of sand particles, which canbe compared to pathogens within a population based on population density.FIGURE 3.3 Comparison of the major ocular findings among the different flaviviruses infections[11].20 Dengue Virus DiseaseMost epidemics are composed of diseases that require close contact betweendiseased and healthy individuals for continued propagation of pathogens.Unlike natural disasters, such as hurricanes, floods, volcanoes, and changesin climate that exist independent of population density, epidemics dependupon population density, a feature shared with reproduction rates, migra-tions, and predation. After population density reduces below a critical limit,such contact may not be available enough for continued propagation ofpathogens.Review of the factors modulating Dengue virus infectiontransmissionUnderstanding of factors, which play important role in Dengue virus infectiontransmission, is very important for planning more effective strategies forprevention and control of this disease [7].Relations among rainfall, vector density, and Dengue virusinfection incidenceA positive association between rainfall or larval density and Dengue viralillness incidence has been reported by few studies but it only hold true forcountries just above and below the equator. Generalization of this associationto drier regions has been flawed. For example, Dengue virus infection epi-demics have been recorded in regions where rainfall or larval indices wereunusually low or where due to availability of piped water, water jars/wellswere rare. In northeastern Thailand in 1987, Dengue hemorrhagic feverepidemic happened in dry and hot season and was over before the rainyseason. It is important to note that Dengue hemorrhagic fever incidence washighest in that region of the country [7].TemperatureBlanc and Caminopetros reported that Aedes aegypti mosquitoes canonly transmit the Dengue virus at temperature above 20�C. For example,the Dengue virus infection epidemics ceased when the temperaturedropped to 14e15�C in winter. Global warming may affect this seasonalvariation and vector distribution. As Aedes aegypti mosquitoes infectmostly domesticated species, as a result outdoor temperature maynot always affect their distribution. That is why it was not surprisingto have Dengue virus infection outbreak in Mexico at an altitude of1700 m [7].Dengue virus infection outbreak Chapter | 3 21Minimum threshold of vector density for Dengue virusinfection transmissionThough minimum vector density below which the Dengue virus infectiontransmission ceases is possible but, minimum threshold has not been definedyet. The validity of using the minimum vector density to access the success ofprograms directed toward Dengue virus infection has been questioned [7].Vector movementFlight rangeThe flight range for Aedes aegypti mosquito has been variable from as long as2.5 km over 24 h, to as short as 25 m in desert and urban environments,respectively The possible reasons for this variation could be finding suitablesources of food such as nectar, human hosts, an oviposition site, or restingplace along the flight path. In one study conducted in an African village, themajority of marked mosquitos remained in the house when they were recap-tured. In addition, the house entering behavior of mosquito is geneticallycontrolled [7].Transport of vectorsThree important modes of vector transmission are: water, land, and air. Before,the advent of air travel, ships were considered to be the principal source fortransmission of Dengue virus infection from Africa to Asia and Americas.Latter on development of efficient highways and other means of groundtransportation lead to infestation by the Aedes aegypti mosquito in townslocated along the roads and railways. Similarly, an extensive list of recordsshowing aircraft bringing Aedes aegypti mosquitoes to Dengue-free countriesis available [7].Mosquito-related factorsDensity (number) of Dengue viruseinfected adult female mosquitos perresidence is an important factor in transmission but is not the only factor. Forexample, in an isolated Dengue virus infection outbreak in a school inMalaysia, only three A. aegypti female mosquitoes were collected from ahostel when there 20 students were infected. Other factors include proportionof engorged females mosquitoes per residence, number of virus infected fe-males, and multiple feedings (bites). Multiple feeding per mosquito isconsidered to be an important factor for exponential spread of Dengue virusinfection epidemic, but it is also not the only factor. It depends if the firsthuman bitten had neutralizing antibody which could result in neutralizing ofvirus from a viremic person to a noninfected person, provided the second bite22 Dengue Virus Diseaseis within 6 h after first bite. Other factors include basic reproductive rate,extrinsic incubation period (the period between an insect’s feeding on aviremic person and it becoming infective), and amount of virus in infectivevector and amount injected by bite [7].Human factorsMore than one person per household increases the chance of transmission ofDengue virus infection epidemic. Transmission occurs if same mosquito in-fects other susceptible member of the household or an uninfected femalemosquito feeds on first victim who is still in viremicstage of illness. Thusmultiple infections in a single household accelerate the spread of infection inthe community. Vector-infested places: schools, commercial establishments,churches or temples, offices, military bases, factories, hospitals, prisons, andtheaters facilitate the Dengue virus infection spread [7].Herd immunityWith respect to role of protective antibody, it is difficult to determine the exactlevels of immunity required against a specific serotype for all age groups insecondary infection. In one study in Singapore, despite the low levels ofmosquito density, low levels of herd immunity was likely responsible forcontinuous spread of Dengue hemorrhagic fever in children under age 10years. Thus it was reported that intense vector control program in Singaporebrought the opposite effects with increase in outbreaks of Dengue fever as aresult of declining herd immunity. For some viral diseases such as measles andrubella, levels of immunization required for disease prevention range from 84to 96%. Thus knowing levels of herd immunity required for Dengue virusinfection prevention is vitally important [7].Breast milk as a possible route of Dengue virus infectionvertical transmissionBarthel et al. [8]. reported breast feeding as possible route for Dengue virusinfection transmission. They reported a case report where patient presentedto hospital in preterm labor and gave birth to a premature but healthy baby.On day 2 after birth, infant was fed on expressed breast milk after whichboth mother and baby experienced nonsevere acute Dengue infection withfever and severe thrombocytopenia but no signs of hemorrhage or plasmaleakage. Dengue virus was tested in breast milk and as result breast feedingwas stopped on day 4. The viremic period was prolonged (�10 days) innewborn which could be related to child’s prematurity. Breast feedingtransmission has been reported in other flaviviruses such as West Nile virusand Yellow fever virus. Therefore, recommendations have been made to stopDengue virus infection outbreak Chapter | 3 23breast feeding during the acute viremic phase after using a live attenuatedvirus vaccine [8].Dengue, Zika, and Chikungunya viruses: emergingarboviruses in the new worldDengue, Chikungunya, and Zika viruses are all three arboviruses, which inrecent years have expanded across the globe with large outbreaks in WesternHemisphere territories in close proximity to the United States. The increase inglobalization led to spread of these infections to populations with no nativeimmunity. These viruses spread to human through bite of Aedes speciesmosquito (Ae. aegypti and Ae. Albopictus). These mosquitoes are aggressivebitters during the day time but can also bite at night too. These mosquitos layeggs in standing water in things like buckets, bowls, animal dishes, flowerpots, and vases [9].Zika virusZika virus is named after an Ugandan forest in which it was first discoveredand is closely related to Dengue virus. For decades, it was of a little concernfor clinicians, until recently when a correlation between Zika virus infectionand fetal microcephaly was discovered in 2016 and World Health Organizationofficially declared it a “Public Health Emergency of International Concern.”First large outbreak of Zika virus infection occurred in 2007 in Yap, a smallisland in Micronesia. About 73% population of the island got infected duringthis outbreak. Subsequent outbreaks occurred across the Pacific Islands until2015 when Brazil reported the first case of Zika virus infection in the America.Other possible mechanisms other than mosquito biting are sexual transmissionand blood borne transmission likely during the viremia stage. Zika virus hasalso been isolated from urine, saliva, and breast milk of infected individualsbut no transmission has been documented from these sources yet. Incubationperiod for Zika virus about 2 weeks. During viremia, a mild disease consistingof fever, nonpurulent conjunctivitis, a maculopapular rash, arthritis/arthralgias,headache, and vomiting occurs. Zika virus infection has not been shown tocause the severe capillary leak syndrome or hemorrhagic fever. Almost 80%infections are asymptomatic. Since symptoms are clinically indistinguishablefrom Dengue virus infection initially, aspirin, nonsteroidal antiinflammatorydrugs, and steroids should be avoided as they may increase the severe hem-orrhage in cases of Dengue infection. Due to potential risk of fetal birth defectsin pregnant women due to Zika virus infection, it is strongly recommended byCenters for Disease Control and Prevention to avoid travel to endemic areas. Ifthe travel cannot be avoided, strict mosquito protection measures should betaken. Pregnant women with Zika virus infection should undergo serial ul-trasounds every 3e4 weeks throughout the pregnancy. In addition, there is also24 Dengue Virus Diseasea concern for possible correlation between Zika virus infection andGuillaineBarre syndrome. Some occurrences have been reported by severalcountries in the western Pacific and Americas [9].Chikungunya virusChikungunya virus was first isolated 1953 from a febrile patient in Tanzania.In 2004, large outbreaks occurred in throughout Africa and Asia. Chikungu-nya, an alphavirus of the Togaviridae family is a mosquito-spread virus andcause the symptoms similar to Dengue and Zika virus infections. Viremia andother symptoms occur after an incubation period of 1e12 days (typically3e7 days). Arthralgia almost occurs in all the cases with common jointsinvolved are ankles, wrists, and fingers. Arthralgia is worse in the morning,improves with mild exercise but worsens with strenuous exercise. Similar toDengue and Zika viral infections, management of Chikungunya viral in-fections is supportive. Acetaminophen is preferred for pain and fever controlover nonsteroidal antiinflammatory drug due to risk of possible misdiagnosisof Dengue virus infection [9].Dengue and other emerging flavivirusesMore than 70 flaviviruses have been identified [10], almost half of them causedisease in humans and only few are of major importance. There are three clinicalsyndromes caused by the flaviviruses: fever-arthralgia-rash, viral hemorrhagicfever with or without hepatitis, or central nervous system diseases. There are noantiviral drugs against the flaviviruses but vaccines do exist against a few of them.Mosquito-borne viruses tend to occur in warm while the tick-borne viruses incooler climates. Examples are mosquito-borne Japanese encephalitis virus oc-curs in southern and eastern Asia but tick-borne encephalitis virus occurs inEurope and Commonwealth of Independent States (the former Soviet Union). Inaddition, mosquito-borne viruses have shorter life cycles due to the fact thatmosquito have shorter life cycles than ticks. As a result, mosquito-borne virusesare evolving rapidly to fill in the ecological niches in new geographical areas.Flaviviruses other than Dengue and yellow fever viruses are enzootic meaningthey use birds or small mammals as the natural hosts as these animals have highreproductive rates which provide them a ready supply of immunologically naı̈vehosts. Humans get infected from these enzootic viruses when they come in closeproximity with the mosquitoes’ natural cycle. As humans do not produce highviremia, therefore act as a dead end hosts for them. Most of the flavivirus in-fections produce a mild disease with the exception of few diseases [10].Yellow fever virusEpidemics compatible with yellow fever virus have been described in WestIndies, Central and South America and, West Africa since 15th century. In theDengue virus infection outbreak Chapter | 3 25early 1900s, a team demonstrated that yellow fever was caused by a filterableagent (a virus), transmitted by mosquitoes. Yellow fever occurs in jungle andurban cycles in West and South America, transmitted to forestry and agri-cultural workers when bittenby mosquitoes. Due to high and prolonged vi-remias in humans, infected individuals will carry the disease to populatedareas where Aedes mosquitoes transmit the virus to cause “urban YellowFever.” Yellow fever is characterized with high grade fever, headache, backand muscle ache nausea, and vomiting with more disease as liver failure oc-curs, causing the mild jaundice which gives disease its name. Yellow fevervaccine was one of the first live attenuated vaccines with conferring immunityup to 10 years or more. Other effective measures included removal of breedingsites, treatment of stored water, and ultralow volumes spraying [10].Japanese encephalitisJapanese encephalitis is the most important viral encephalitis worldwide with50,000 cases and 10,000e15,000 deaths. The disease was recognized sinceepidemics of encephalitis in Japan in 1870s onwards and virus was isolatedfrom a fatal case in 1930s. Virus gets transmitted between birds and animals byCulex mosquitoes, especially Culex tritaeniorhynchus and humans get infecteddue to close proximity with this enzootic cycle. Serological studies haveshown that almost all individuals living in rural Asia get infected with Japa-nese encephalitis during the childhood but only 1 in 300 become symptomatic.Japanese encephalitis virus infections cause variable clinical presentationranging from headache, cough, and coryza but in some cases these febrileprodromes are followed by coma and convulsions. About 20%e30% patientsdie and more than half of the survivors are left with severe neuropsychiatricsequelae. Cerebrospinal fluid analysis will likely show lymphocytic pleocy-tosis but can be normal and computer tomography and magnetic resonanceimaging shows damage in thalamus, basal ganglia. Japanese encephalitisvaccine is available for 30 years now and strongly recommended for residentsof endemic areas and travelers planning to visit the endemic areas for morethan 3 weeks. Other important preventive measures include insect repellents;bed nets to avoid mosquitoes biting; and keeping pigs, chickens, and otherpotential animals away from human dwellings [10].West Nile encephalitisWest Nile virus, first isolated in 1930s, was considered benign until recentlyand is mostly found in much of Africa, much of Asia, Southern Europe, andrecently in North America. Like Japanese encephalitis, West Nile encephalitistransmitted by Culex mosquitoes and humans are dead hosts. Virus will causefever-arthralgia-rash syndrome mostly with conjunctivitis and lymphadenop-athy. Clinically, West Nile encephalitis is similar to Japanese encephalitis in26 Dengue Virus Diseaseterms of causing coma, convulsions, and mixture of causing upper and lowermotor neuron signs: flaccid paralysis of limb and respiratory muscles.Meningoencephalitis is considered a rare complication of West Nile virusinfection. A large outbreak occurred in Romania, in 1960s in which about 600people had the neurological disease but no one with rash or lymphadenopathy.Poor plumbing and sewerage under the apartments was considered to cause theoutbreak as it lead to explosive increase in population of Culex pipiens. All ageof people were affected but the attack rate and mortality was higher in olderadults [10].Other mosquito-borne flavivirusesOther important mosquito-borne flaviviruses include Murray Valley encepha-litis virus, Kunjin virus, Saint Louis encephalitis virus, and Rocio virus. MurrayValley encephalitis virus was the cause of polio like encephalomyelitis inAustralia in 20th century. The virus gets transmitted by Culex annulirostris,water birds, cattle, and some marsupials. Kunjin virus is widely distributedgeographically but cause a milder disease. St Louis encephalitis virus was animportant neurotropic flavivirus in United States with first epidemic reported in1930s. Though most of the areas of United States, Canada, and Mexico areaffected by this disease at some point in time, sporadic cases still occur. Rociovirus was first isolated from the brain of a fatal case from an outbreak in SaoPaulo State of Brazil in 1975 and is transmitted by Psorophora mosquitoes [10].Tick-borne flavivirusesTick-borne flaviviruses are less important in humans compared to animals asticks preferentially feed on animals compared to humans. Viruses are transmittedby Ixodidae (hard ticks): Dermacentor and Hemophysalis species. Otherimportant routes of transmission are ingestion of infected milk, and directtransmission from infected animal carcasses. If no host is available, ticks can actas virus reservoirs formonths and years. Tick-borne encephalitismostly circulateamong the small wild animals like rodents and transmitted to human by ingestionof goat milk in addition to tick bites. Up to 70% of the patients remember the tickbitewith tick-borne encephalitis presenting 1e2weeks after the high grade fever,headache, malaise, and myalgia. In latter cases, it can progress to flaccid pa-ralysis of upper limb and shoulder girdle. Respiratory muscles and bulbar (brainstem) involvement can lead to respiratory failure and death [10].Ocular manifestations of emerging flaviviruses and theblood-retinal barrierFlaviviruses, despite causing many systemic complications, has been docu-mented to cause multiple ocular abnormalities such as conjunctivitis, retinalDengue virus infection outbreak Chapter | 3 27hemorrhages, chorioretinal atrophy, posterior uveitis, optic neuritis, andmaculopathies [11].Eyes are protected from the systemic infections by presence of blooderetinal barriers. Flaviviruses modulate the retinal innate response and pene-trates the blooderetinal barriers to cause the ocular pathologies [11].Seroepidemiology of Dengue, Zika, and Yellow Feverviruses among children in the democratic republic of theCongoThe public health importance of arthropod-borne viruses is growing tremen-dously as they cause millions of infections in humans annually with physicalmanifestations ranging from birth defects, hemorrhage, shock, encephalitis,and even death. Dengue fever is causing about 400 hundred millions infectionsannually. In 2015, Zika virus infection in Latin America became an interna-tional public health emergency due to its adverse effects on developing fetuswhen expecting mothers were infected. The epidemiology of Dengue virusinfection and Zika virus infection in Asia and America better describedcompared with Africa. Finding Dengue and Zika virus infections in travelersreturning from Africa suggests that prevalence of these viral infections inAfrican population is largely underestimated. A recent outbreak of yellowfever virus in 2016 in Angola and surrounding countries despite the existenceof an effective vaccine since the 1930s represents a constant threat for yellowfever virus epidemic [12]. At least 42 deaths were reported in DemocraticRepublic of the Congo when the Angolan outbreak crossed country orders.Risk of Yellow fever virus outbreaks in the Democratic Republic of the Congoand lack of data on other flavivirus infections such as Dengue virus and Zikavirus prompted Willcox et al. [12] to conduct a seroepidemiological survey forthese three flaviviruses. They found that children despite the documented proofof receiving the Yellow fever virus vaccine, failed to show the evidence ofseroconversion. Evidence of low rate of seroconversion among children hasbeen previously reported but it consistently reported to be more than 80%. Theother possible reason could be that majority of children had administration ofMeasles vaccine with Yellow fever virus vaccine. It has been demonstratedthat coadministration of measles vaccine with yellow fever vaccine decreasesits immunogenicity [12]. Other reasons include malnutrition in children andinsufficiency of sensitivity of enzyme-linked immunosorbent assay andneutralization assays to detect the low levels of antibodies that are sufficientfor protection. Despite the possibility of abovementioned reasons, the factremains that Democratic Republic of the Congo continues to experience majoryellow fever outbreaks which questions the effectiveness of efforts to integrateyellow fever vaccine into routine childhood vaccination programs. In addition,the study found that Dengue, Zika, and yellow fever viruses are circulating inthe Democratic Republic of the Congo. Recommendations were made to28 Dengue Virus Diseaseconduct more studies to explore the detailed reasons for low rates of sero-conversion observed in vaccinated Congolese children and considering flavi-virus infection as an important etiology of acute febrile illness especially inpatients who test negative for malaria [12].Viremia and clinical presentation in Nicaraguan patientsinfected with Zika virus, Chikungunya virus, and DenguevirusAll the three viruses cocirculate in Nicaragua. Study was conducted tocompare the clinical presentation and quantify the levels of viremia. Out of263 patients tested positive: 192 tested positive for a single virus infection, 71for two, and 2 for all the three viral infections (coinfections). Viremia levelswere lower in Zika virus infections compared with Chikungunya virus orDengue virus. Zika virus infected patients were likely to develop rash but lesslikely to be febrile or hospitalized compared to chikungunya virus or Dengueviruseinfected patients. Due to lot of similarity in clinical presentation, itbecomes difficult to make accurate clinical diagnosis where the patient may beinfected with any of three viral infections. This supports the use of testingprotocol for sensitive, accurate, multiplex diagnostics for clinical care, diseaseresearch, and epidemiological surveillance of Zika, Chikungunya virus, andDengue virusesuspected cases [13].Concurrent outbreaks of Dengue, Chikungunya and Zikavirus infectionsdan unprecedented epidemic wave ofmosquito-borne viruses in the pacific 2012e14About 28 new documented outbreaks and circulation of Dengue, Chikungu-nya, and Zika virus infections have been reported between January 2012 and17 September 2014 and about 120, 000 people were affected in the Pacificregion. These outbreaks put extra burden on preexisting healthcare system inPacific Islands. The risk for further spread in the Pacific Region is high due toseveral reasons. First could be due to low immunity as Dengue virus serotype 3had been absent in this region since 1995. Secondly, in addition to Aedesaegypti and Aedes albopictus mainly in this region, local mosquitoes such asAedes polynesiensis or Aedes hensilli can also transmit these viruses andthirdly, the large population mobilization and airline travel facilitate thespread [14].Dengue and Chikungunya viruses infections: long-distance spread and outbreaks in naı̈ve areasOutbreaks of Dengue and Chikungunya virus infections are taking place inpreviously disease-free areas. The important factor for long-distance spread ofDengue virus infection outbreak Chapter | 3 29infectious disease is increased human mobility. Outbreaks were caused byinfected persons coming from endemic and epidemic areas, acting as a Trojanhorse for these germs. After the virus was incorporated in the new area, otherfactors like climate change, virus evolution, lack of vector control, socio-demographic changes, and environmental changes i.e., rapid uncontrolledurbanization, play an important role for geographic spread of mosquito-borneinfections [15].Identifying and diagnosing the patient with uncleardiagnosisDue to long turnaround time, serum testing is not effective for emergenttesting, therefore diagnosis of Dengue, Zika and Chikungunya virus infectionshould be made on clinical grounds. Even in strongly suspected cases of Zikaand Chikungunya virus infections, Dengue virus infection still needs to beruled out due to its life threatening complications. Acetaminophen should bepreferred over nonsteroidal antiinflammatory drug given the hemorrhagiccomplications in Dengue virus infection cases. All three entities: Zika, Chi-kungunya, and Dengue virus infections are reportable diseases. If the patientalso traveled to endemic for malaria, it is strongly recommended to rule outmalaria. On the basis of travel, other illnesses like yellow fever, typhoid,leptospirosis, and helminth needs to be ruled out too [9].Following testing algorithm is strongly recommended [9].Personal protection in endemic areasBoth as an issue to personal safety as well as public health measure, personalprotection in an endemic area cannot be emphasized. The use of EnvironmentalProtectionAgency-registered insect repellant such asN,N-diethyl-meta-toluamideEmergency department testing Sending out testingMalaria Smear/Point of care test Dengue virus infectionComplete blood count (CBC) Dengue NS1 polymerase chainreaction (PCR)Chem 7 Dengue Immunoglobulin MLiver Function Tests (LFTs) Chikungunya virus infectionProthrombin time/Partial thromboplastintime (PTT)Zika virus infectionUrine Analysis (UA)/human chorionicgonadotropin (Hcg)Chest radiograph30 Dengue Virus Disease(DEET) or picaridin are strongly recommended by Center for disease Controland Prevention. These agents are proven to be safe and effective for use in in-fants over two months, pregnant and breast feeding women [9]. A list ofapproved agents can be found at https://www.epa.gov/insect-repellents. Addi-tional use of screens and mosquito netting are also important.Discovery of fifth serotype of Dengue virus (DENV-5): anew public health dilemma in Dengue virus infectioncontrolRecently in October 2013, the fifth variant DENV-5 has been isolated whichfollow the sylvatic cycle contrary to other four serotypes, which follow thehuman cycle. Likely cause of emergence of these serotypes is due to geneticrecombination, natural selection, and genetic bottlenecks. DENV-5 is currentlypresent in India. Discovery of DENV-5 and more such sylvatic strains mayimpede the Dengue virus vaccine. Sustainable Dengue control then depends onintegrated vector management [16].Mosquito-borne diseases and cancer: what do we reallyknow?There are only few studies published about relationship between some types ofcancer and mosquito-borne diseases. The association between malaria andcancer may be explained by immune system suppression inducing by plas-modium. A second explanation by Lehrer [17,18] hypothesizes that Anophelesmosquitoes, a vector for malaria, could represent a source for brain tumorviruses. The evidence of association between Anopheles bites and brain tu-mors was reported from the link malaria outbreaks in USA and brain tumorincidence. Further research about this relationship is urgently needed as if themosquito-transmitted brain tumor viruses are identified, development of braintumor vaccine might be possible.Espina et al. [19] observed increased production of tumor necrosis factoralpha in Dengue virus (serotype DEN-2)-infected human monocyte cultures.Monocytes play an important role in defense mechanisms against viruses byviral phagocytosis of infected apoptotic cells and release of proinflammatorycytokines. Later on, Chen et al. [20] described high viral DEN-2 titer,macrophage infiltration, and tumor necrosis factor alpha production in thelocal tissues as important events leading to hemorrhages [21].Dengue virus infection in the United StatesAlmost all Dengue virus infection reported in United States were acquiredelsewhere by travelers and these imported cases rarely result in secondarytransmission due to infrequent contact between Aedes and people [4].Dengue virus infection outbreak Chapter | 3 31https://www.epa.gov/insect-repellentsDengue hemorrhagic fever d U.S.-Mexico border, 2005The last reported Dengue virus infection outbreak was in south Texas in 2005.A case of Dengue hemorrhagic fever was reported in a resident of Brownsville,Texas in July 2005. On June 24, 2005, patient had acute onset of fever,chills,headache, nausea, vomiting, abdominal pain, arthralgia, and myalgia. In heryouth, patient lived in across the border in the city of Matamoros in Tam-aulipas, Mexico. Due to her illness, patient moved across the border intoMatamoros. On June 28, she was hospitalized in Matamoros with the likelydiagnosis of Dengue virus infection and urinary tract infection. Patient hadthrombocytopenia (62,000 platelets/mm3) but no hemorrhagic manifestations,treated with antibiotics and discharged. On July 1, patient came back andsought treatment for continued fever, chills, vomiting, and abdominal pain andwas hospitalized in Brownsville, Texas. Blood pressure was 94/70 mm Hg,and laboratory testing indicated proteinuria, hematuria, with a further decreasein platelet count (43,000/mm3). The patient was treated with antibiotics for aurinary tract infection and fluid resuscitation. Platelets dropped to 39,000/mm3and albumin to 2.9 g/100 mL. A fecal occult blood test was positive, andpleural effusion was noted. Platelet count improved to 118,000/mm3. Patientwas discharged on July 4 with diagnosis of possible murine typhus or viralinfection and doxycycline prescription. Dengue virus infection was not diag-nosed despite the patient’s clinical characteristics (i.e., acute fever, plateletcount <100,000/mm3, evidence of bleeding [hematuria and fecal occultblood] and plasma leakage) consistent with World Health Organization(WHO) criteria for Dengue hemorrhagic fever. Subsequently, serum sampletaken on July 3 was positive for Dengue immunoglobulin M (IgM) by enzyme-linked immunosorbent assay (ELISA) and had an elevated titer of immuno-globulin G (IgG) antibodies to Dengue virus (1:655,350) and interpreted asindicative of a secondary Dengue virus infection. In August 2005, 1251 casesof Dengue fever, including 223 cases (17.8%) of Dengue hemorrhagic feverwere reported in the neighboring state of Tamaulipas, Mexico [22].A small outbreak of Dengue virus infection occurred in Hawaii in 2001.Dengue virus illness is endemic in Puerto Rico, the U.S. Virgin Islands, Sa-moa, and Guam. Large island-wide epidemics have been reported in PuertoRico since late 1960s with most recent one in 2007 when more than 1000 caseswere reported. The principal Dengue virus vector is Ae. aegypti in Puerto Ricoand most of the Caribbean Basin. In Puerto Rico, Dengue virus infectiontransmission follows a seasonal pattern: low transmission from March till Juneand high from August till November [4].Texas lifestyle limits transmission of Dengue virusDengue virus infection like illness was first noted in the New World as majoroutbreak Philadelphia in 1780 R. In 1922, about 500,000 cases were noted in32 Dengue Virus DiseaseTexas. From 1980 to 1999, only 64 locally acquired cases were reported inTexas whereas 62,514 suspected cases in three adjoining Mexican statesdCoahuila, Nuevo León, and Tamaulipas [23].Laredo, Texas, United States (population 200,000), and Nuevo Laredo,Tamaulipas, Mexico (population 289,000) are fundamentally a single citylocally known as “los dos Laredos” divided by a small river, the Rio Grande.In the summer of 1999, a seroepidemiologic survey was conducted to studythe factors affecting the Dengue virus infection transmission on either side ofthe border. IgM and IgG sero-positivity to Dengue virus was lower in Laredothan in Nuevo Laredo. Conversely, Texas side of the border has highernumber of mosquito-infested containers. About 82% homes were air-conditioned in Laredo compared to 24% in Nuevo Laredo. Laredo hadgreater proportion of houses with intact screens, greater average distancebetween houses and a few persons residing in each house. The difference intransmission rate between the two cities less likely related to climate vari-ation given their proximity. Ae. aegypti infestation rates in Laredo weremarkedly higher in despite the mosquito control programs. The likely rea-sons for lower transmission in Laredo are that most of the shops, houses, andrestaurants were air-conditioned and close windows leading to less oppor-tunity for mosquito/human contact. In Nuevo Laredo, most of the shops,bars, and restaurants are left open to street. Most of the buildings in UnitedStates are air-conditioned, fully glazed, and permanently closed. Even if theinfected mosquitos gain entry to such buildings, they are less likely tosurvive in artificially dried atmosphere. The dollar cost of electricity issimilar in Laredo and Nuevo Laredo but income per capita is much higher inTexas compared to Tamaulipas [23].It has been stated earlier that as a result of global warming, Dengue virusinfection, malaria and other mosquito-borne diseases will be more prevalent inUnited States [24e27]. These predictions are made from vectorial capacitymodel which population density, biting frequency, and daily survival proba-bility of the vector, and extrinsic incubation period of the pathogen [28,29] butlacks in counting the effect of factors like air-conditioning, use of evaporativecoolers, and behaviors of mosquitos and humans [30].Dengue surveillance in United StatesBeginning 2009, all the nationally diagnosed Dengue virus infection cases willbe reported to Centers for Disease Control and Prevention. Since 1915, sta-tistical data is being compiled regarding cases in Puerto Ric and since 1969,Center for Disease Control and Prevention’s Dengue Branch, located at SanJuan and operates the island-wide passive Dengue viral illness surveillancesystem (PDSS) in collaboration with Puerto Rico Department of Health.Passive Dengue viral illness surveillance system played an important role inidentifying the first case of Dengue virus infection in the Americas, the firstDengue virus infection outbreak Chapter | 3 33cluster of cases of Dengue virus infection, first laboratory-confirmation ofDengue virus infection and Dengue related death in Puerto Rico [4].References[1] Bäck AT, Lundkvist ÅKe. Dengue virusesean overview. Infect Ecol Epidemiol2013;3(1):19839.[2] Organization, W.H.. Dengue. 2019.[3] Yung C-F, et al. Dengue serotype-specific differences in clinical manifestation, laboratoryparameters and risk of severe disease in adults, Singapore. Am J Trop Med2015;92(5):999e1005.[4] Control, C.f.D.. Dengue. 2019.[5] Torrey T. Difference between an epidemic and a pandemic. 2019.[6] Qureshi AI. Ebola virus disease. 2019.[7] Kuno G. Review of the factors modulating dengue transmission. Epidemiol Rev1995;17(2):321e35.[8] Barthel A, et al. Breast milk as a possible route of vertical transmission of dengue virus?Clin Infect Dis 2013;57(3):415e7.[9] Patterson J, Sammon M, Garg M. Dengue, zika and chikungunya: emerging arboviruses inthe new world. West J Emerg Med 2016;17(6):671.[10] Solomon T, Mallewa M. Dengue and other emerging flaviviruses. J Infect2001;42(2):104e15.[11] Singh S, Kumar AJV. Ocular manifestations of emerging flaviviruses and the blood-retinalbarrier. Viruses 2018;10(10):530.[12] Willcox AC, et al. Seroepidemiology of dengue, zika, and yellow fever viruses amongchildren in the democratic republic of the Congo. Am J Trop Med Hyg 2018;99(3):756e63.[13] Waggoner JJ, et al. Viremia and clinical presentation in nicaraguan patients infected withZika virus, chikungunya virus, and dengue virus. Clin Infect Dis 2016;63(12):1584e90.[14] Roth A, et al. Concurrent outbreaks of dengue, chikungunya and zika virus infectionseanunprecedented epidemic wave of mosquito-borne viruses in the Pacific 2012e2014. EuroSurveill 2014;19(41):20929.[15] Rezza GJP. Dengue and chikungunya: long-distance spread and outbreaks in naı̈ve areas.Pathog Glob Health 2014;108(8):349e55.[16] Mustafa MS, et al. Discovery of fifth serotype of dengue virus (DENV-5): a new publichealth dilemma in dengue control. Med J Armed Forces India 2015;71(1):67e70.[17] Lehrer S. Anopheles mosquito transmission of brain tumor. Med Hypotheses2010;74(1):167e8.[18]Lehrer S. Association between malaria incidence and all cancer mortality in fifty U.S. Statesand the District of Columbia. Anticancer Res 2010;30(4):1371e3.[19] Espina LM, et al. Increased apoptosis and expression of tumor necrosis factor-alpha causedby infection of cultured human monocytes with dengue virus. Am J Trop Med Hyg2003;68(1):48e53.[20] Chen HC, et al. Both virus and tumor necrosis factor alpha are critical for endotheliumdamage in a mouse model of dengue virus-induced hemorrhage. J Virol2007;81(11):5518e26.[21] Benelli G, et al. Mosquito vectors and the spread of cancer: an overlooked connection?Parasitol Res 2016;115(6):2131e7.34 Dengue Virus Disease[22] Control, C.f.D. and Prevention. Dengue hemorrhagic fever–US-Mexico border, 2005.MMWR Morb Mortal Wkly Rep 2007;56(31):785.[23] Reiter P, et al. Texas lifestyle limits transmission of dengue virus. Emerg Infect Dis2003;9(1):86.[24] Watson R, et al. Impacts, adaptations & mitigation of climate change: scientific-technicalanalyses, vol. 25 (2/3); 1998. p. 133.[25] Jetten TH, Focks DA. Potential changes in the distribution of dengue transmission underclimate warming. Am J Trop Med Hyg 1997;57(3):285e97.[26] Patz JA, et al. Dengue fever epidemic potential as projected by general circulation models ofglobal climate change. Environ Health Perspect 1998;106(3):147e53.[27] Watson RT, et al. The regional impacts of climate change. 1998.[28] Macdonald GJTE, Malaria Co. The epidemiology and control of malaria. 1957.[29] Bailey NT. The mathematical theory of infectious diseases and its applications. 5a CrendonStreet, High Wycombe: Charles Griffin & Company Ltd; 1975. Bucks HP13 6LE.[30] Molineaux L. The pros and cons of modelling malaria transmission. Trans R Soc Trop MedHyg 1985;79(6):743e7.Dengue virus infection outbreak Chapter | 3 35Chapter 4Global health-care perspectiveof Dengue viral diseaseSachin M. BhagavanResident Physician, Department of Neurology, University of Missouri Health Care, Columbia, MO,United StatesIntroductionDengue viral illness is one the most important arboviral diseases of thehumans, occurring in tropical countries in more than 60 countries. An esti-mated 2.5 billion people worldwide are at risk of infection; approximately 975million of them live in urban areas in tropical and subtropical countries inSoutheast Asia, Africa, East Mediterranean, the Pacific, and the Americas.More than 50 million infections occur each year, including 500,000 hospi-talizations for Dengue hemorrhagic fever, mainly among children, with thecase fatality rate 10%e15% in endemic regions [1,2]. This chapter aims tohighlight the impact of the Dengue viral illness worldwide and the factors thatcontribute to its global spread. This chapter also sheds some light on themeasures that were taken in the past, reasons for their shortcoming, and at laston the research advances in the field of development of vaccines targetingDengue viral illness.Global epidemiology and impactDengue viral illness has been a big concern globally. The disease burden wasfirst estimated based on the constant infection rate annually on a crude pop-ulation at risk like 10% in one billion persons, another estimate of 4% in 2billion persons [3,4]. The estimated global infection rate in 1985 was esti-mated to be 80e100 million cases per year. More data being collected andsophisticated tools and methods are being used to identify the incidence of thedisease in recent years. Knowing the geographical distribution and amount ofdisease burden is a key to understand global mortality and morbidity to effi-ciently allocate the resources available for Dengue viral disease managementand in evaluating the impact of measures taken toward a goal of eliminatingthe disease.Dengue Virus Disease. https://doi.org/10.1016/B978-0-12-818270-3.00004-7Copyright © 2020 Elsevier Inc. All rights reserved. 37https://doi.org/10.1016/B978-0-12-818270-3.00004-7In 2009, the World Health Organization established new guidelines fordiagnosis and management, which changed the case definitions [1]. Thoughthey are more comprehensive, these definitions had a few shortcomings, likealtered hemostasis was not incorporated [2]. The actual numbers of viralillness cases are underreported because it takes into account only laboratorypositive cases, whereas a fraction of Dengueviral illness cases present with justviral prodrome-like illness. For example, estimates made in 2013 mentionedthat there are 390 million Dengue viral illness infections occurring every year(95% confidence interval 284e528 million), of which 24.6% manifest clini-cally with any severity of disease including Denguehemorrhagic fever/ Dengueviral illness shock syndrome which is 3 times more than estimated by theWorld Health Organization [5,6].To estimate the global incidence, various approaches combining historicaloccurrences and expert prediction have been used to identify areas that are atmaximum risk [7,8]. In the present time, various mathematical models havebeen used to estimate the global burden of the disease. These methodologiesare expected to help in estimating the economic impact of the disease,assessing morbidity and mortality, estimating amount of vaccines required,data for research and vaccine development.Fig. 4.1 compares the global incidence of Dengue viral illness in 2012 and2017 which has been developed from a model from the Global Health Data2017. This model used incidence from officially reported cases and adjustedraw estimates for underreporting based on published estimates of expansionfactors. To correct the underreporting, investigators used a three-phaseapproach. First, expected spatial distribution of the disease was definedbased on a principal component analysis of population-weighted probability ofDengue viral illness transmission. Secondly, the association between this ex-pected distribution and reported incidence was modeled using mixed-effectsbinomial modes; the model was calibrated by benchmarking those de-viations against published empirical expansion factors. Lastly, the modeldistributed total cases to cases based on ageesex groups by using the ageesexdistribution of Dengue viral illness cases captured by Hospital InformationSystem of the Brazilian Unified National Health System [9].Currently the incidence of the disease globally has been on the rise. Ac-cording to Global Health Data 2017 as shown in Fig. 4.2, the incidence per100,000 populations was less than 500 in 1990, which has risen in the last25 years to 1328 in 2017. This corresponds to more than 104 million new casesseen worldwide in just the year 2017 [10].As we can see from Fig. 4.3 and Table 4.1, the incidence has been on therise globally in the last 25 years in all the major regions of the world. Dengueviral illness has had significant impact in South Asia, Latin America, Africa,and the Middle East. South and Southeast Asian countries reported the highestincidence rate in the years with as high as 3670 per 100,000 populations ascompared with 1009 per 100,000 populations in the Americas. There is a38 Dengue Virus Diseasesignificant increase of reporting in the number of cases from India and theeastern Mediterranean region due to numerous outbreaks, with recent reportsof cases in Sudan, Yemen, Madagascar, Pakistan, and Saudi Arabia. AlthoughAfrica reported an incidence of 688 per 100,000 in 2017, the lack of appro-priate diagnostic test and adequate clinical suspicion still underestimated thetrue burden of the disease [1]. There has been prospective cohort studies inThailand and Nicaragua that show an incidence of Dengue virus infection of6%e29% per year [11,12]. There has been a resurgence of Dengue viral illnessFIGURE 4.1 Comparison of global incidence of Dengue viral disease in 2012 (top figure) and2017 (bottom figure).Global health-care perspective of Dengue viral disease Chapter | 4 39activitydocumented in Buenos Aires, Hong Kong, the Galápagos Islands,Easter Island, and Hawaii. Dengue viral illness has also been reported inFlorida, southeastern France, and Madeira Island [1,13]. Increasingly, coin-fections of Dengue virus infection have been occurring with malaria, humanimmunodeficiency virus/acquired immunodeficiency syndrome, leptospirosis,and Chikungunya., Dengue viral transmission by blood transfusion is alsoreported.[14].FIGURE 4.2 Incidence per 100,000 population globally.FIGURE 4.3 The incidence of Dengue viral illness among different regions in the last 2 decades.40 Dengue Virus DiseaseAlong with the increase in incidence, mortality has also been increasing inall regions of the world. According to Global Health Data 2017, Figs. 4.4 and4.5 show global mortality and region-specific mortality caused by Dengueviral illness. This model has used Cause of Death database and the Cause ofDeath Ensemble Modeling tool [15,16]. Covariates on basis of expected as-sociations with Dengue viral illness mortality and biological plausibility wereincluded like environmental covariates (rainfall, latitude, people in urbanareas, etc) and country level of development (income per capita, mean edu-cation level, access to health system). Population weighted mean probability ofDengue viral illness transmission was included. All cause mortality wasTABLE 4.1 The incidence of Dengue viral illness in different World HealthOrganization regions in 2017.Region Incidence per 100,000 in 2017African region 688.74Region of the Americas 1009.87Southeast Asia region 3670.69Eastern Mediterranean region 596.21Western Pacific region 483.65FIGURE 4.4 Trend in deaths due to Dengue viral illness globally.Global health-care perspective of Dengue viral disease Chapter | 4 41estimated and then within each sex-age-country-year group, the total of all-cause-specific death was constrained to equal the number of all-cause deaththrough a process CodCorrect [15].Mortality has been rising exponentially in the last 10 years with mortalityrate as high as 0.53 per 100,000 population in 2017 as compared to 0.34 per100,000 in 2005. As correlated with incidence, mortality was highest in thosecountries in South East Asia with as high as 1.76 per 100,000 populations ascompared to other regions where mortality was in the range of 0.05e0.017 (asshown in Fig. 4.4). Among the Southeast Asian countries, Indonesia, Malaysia,Philippines, Bali, and India were the most affected with mortality rate as highas 9 per 100,000 in certain regions.When we look into the morbidity of the disease, measured in terms ofDaily Adjusted Life Years, it has also been increasing in the last 10e15 years.According to Global Health Data 2017, Daily Adjusted Life Years in 2017 was38.25 per 100,000 population as compared to 29.02 per 100,000 in the year2005 as seen in Fig. 4.6. This model calculated Daily Adjusted Life Years asthe sum of Years to Live and Years Lived with Disability. Years to Live wasestimated as the difference between age at death and life expectancy for thosesurviving to that age at death [15]. Years Lived with Disability was estimatedby assigning a health state and corresponding disability weight to each case,which was based on pooled results from Global Burden of Disease 2010Disability Weights Measurement Study [17] and more recent EuropeanDisability Weights Study [18]. Each case was assigned two acute health states:moderate and severe episodes [19]. Proportion for the split between moderate2013 2014 2015 2016 2017African Region 0.005103633 0.005127765 0.005136629 0.005087465 0.005165073European Region 0.048761881 0.050273023 0.050971208 0.052334086 0.062462349Region of the Americas 0.124617261 0.124242324 0.128473305 0.127975749 0.127488048South-East Asia Region 1.559522856 1.637419794 1.671872119 1.720608131 1.768996711Western Pacific Region 0.125692711 0.124373739 0.127398976 0.126439226 0.17243881400.20.40.60.811.21.41.61.82Death per 100,000 populationFIGURE 4.5 Region-specific Dengue viral illness mortality rate in the last 5 years.42 Dengue Virus Diseaseand severe was derived from a meta-analysis of the subset of data that had bothtotal number of cases and number of severe cases. Lastly, for postDengue viralillness chronic fatigue was assigned the disability weight for infectious dis-easeand post acute consequences.Among the different regions of the world, Southeast Asia had the highesteffect with 114.25 per 100,000 population. Daily Adjusted Life Years indifferent regions of the world has been shown in Table 4.2.The disease burden mentioned above takes into account only the acuteillness of 7 days or less asstandard. There are increasing reports of long-termFIGURE 4.6 Trend in Disability Adjusted Life Years globally.TABLE 4.2 Daily Adjusted Life Years in various regions in 2017.RegionsDaily adjusted life years in 2017 per100,000 populationEuropean region 0.092188Southeast Asia region 114.8533165Eastern Mediterranean region 9.216687285Western Pacific region 16.95258218African region 7.122431707Region of the Americas 15.57597189Global health-care perspective of Dengue viral disease Chapter | 4 43postviral effects, such as depression and chronic fatigue syndrome, which addup further to the impact on productivity [20]. Also, disease burden does notaccount for lost work, absence from school, or lost tourism. The overallestimated disease burden is comparable to major diseases like tuberculosis ormalaria, although those diseases receive major funding worldwide [21].Numerous reasons can be attributed for this increase in incidence,morbidity, and mortality of Dengue viral illness. As we know, Dengue virus ispresent in four serotypes, DEN 1e4. Each of the four serotypes has beenevolved into multiple genotypes, which is one of the major factors contributingto the global burden of the disease. Infection with one strain of virus doesprovide subsequent immunity to that particular strain, but does not provideadequate immunity to other strains. In fact, it causes “sensitization” wheresubsequent infections with a different strain creates a more serious and criticalimpact in form of “Dengue hemorrhagic fever/Dengue shock syndrome”contributing to the morbidity and mortality. Dengue viral illness epidemicsoccur in those countries where clinical management of Dengue hemorrhagicfever is at times suboptimal that often leads to increased mortality. Partlybecause Dengue hemorrhagic fever has a very nonspecific constellation ofsymptoms developing rapidly leading to shock and ultimately death within fewhours. Global warming has also aided in the wider geographic distribution ofAedes mosquitoes, thereby increasing Dengue viral illness epidemic potentialin temperate regions. Increased globalization, high population density, rural tourban migration, and development of rapid transport systems and urbanizationhave also led to global spreading of the disease. Traveling has made a sig-nificant impact in the spread of the disease, with travelers harboring the virusgoing to nonendemic areas constituting the main source for triggering trans-mission. International travelers are most at risk of Dengue viral illness, withattack rates reported as high as 5$51 cases per 1000 travel-months. Dengueviral illness has now become the leading cause of fever in returning travelers,having overtaken malaria for travelers to Southeast Asia.Global economic impactDengue viral illness has also made a huge impact globally in terms of econ-omy. Although it is difficult to evaluate the true economic burden of thedisease on each nation, efforts have been made in the past to estimate theeconomic aspect of the disease. Limited availability, inadequate resources fordata collection in many countries, and poor availability of specific guidelinesand protocols in diagnosing the disease are the challenges facedby the nationsthat hinder estimating the amount of money invested for the disease. Inaddition to the cost borne by the health-care system, there is family economicloss to be considered. One study estimated 14.8 days lost in ambulatory casesand 18.9 days lost in hospitalized cases. The impact on the family by oneDengue viral illness episode can be estimated as high as three times the family44 Dengue Virus Diseasemonthly income. The disease leads to families selling their belongings/takingout a loan for repayment as 45% of health costs are borne by patients or theirfamily globally. Among those who do not seek health care, cost remainshidden. Most economic studies estimate the disease burden by looking atanywhere from 20 to 70 countries while Dengue viral illness transmission ispresent in almost 140 nations.Therefore studies were done where extrapolation of available data andestimate through various mathematical models help in estimating andanalyzing the disease somewhat accurately. One such study was done byShepard et al. [22] where the study used various factors to estimate the eco-nomic burden. It estimated the incidence of Dengue viral illness by taking intoaccount the reported episodes by the World Health Organization, Ministry ofNational Health Statistics and published literature, and estimated the under-reporting through various literature and formulated aDengue viral illness scorewhich comprised the probability of Dengue viral illness in an area, its popu-lation density, and death estimated. The study estimated that global averagecost per Dengue viral illness case is $333 (95% confidence interval 283e403)for cases admitted to hospital, while for ambulatory cases it is $60 (95%confidence interval 54e68). In case of death by Dengue viral illness, long-termcost was estimated to be $80,414 for death of a child while death of an adultwas estimated to be $75, 820 [22]. These figures show that the financial burdenof the Dengue viral illness ($8.9 billion) [22] is more than other major in-fectious disease such as rotavirus gastroenteritis ($2billion) [23], cholera ($3.1billion) [24], rabies ($8.6 billion) [25], and Chagas disease (7.2 billion) [26](Fig. 4.7, Table 4.3).Initial global responseDue to the concerns of the burden of the Dengue viral illness virus, historicallya unique mosquito control program was initiated in the 1940s. By 1960, effortsof the Pan American Health Organization, Aedes aegypti had been eradicatedfrom major South and Central American countries. Many programs werereduced after this achievement and their goal was directed to a differentproblem. Within 2 decades, the vector regained its former population.The World Health Organization in 2012 came up with a global strategyprimarily to reduce Dengue viral illnesserelated mortality by 50% andmorbidity by 25% by 2020 [6]. Primary focus was on preventive measures,early warning systems, and risk assessment in the form of early case detectionand appropriate identification and management of severe cases. Secondaryfocus was on capacity building, investment in health-care infrastructure, andresearch for better understanding and combating Dengue viral illness. The planalso called for estimating the true burden of the disease, which would help toknow the extent of the disease and to see if the policy is effective in reducingthe burden. Therefore it emphasizes the importance of locoregionalGlobal health-care perspective of Dengue viral disease Chapter | 4 45involvement where the ministries of health in endemic countries coordinatewith the World Health Organization for more accurate estimate of the diseaseand implementation of the strategies at the level of the public.In recent years, several new tools and strategies for Dengue viral illnesscontrol and prevention have been developed and are available to public healthpractitioners and clinicians. One of the important highlights is the developmentof rapid commercial diagnostic tests, which is used in many endemic countries.These tests have become very useful as studies in various countries show thatthe test is 60%e70% sensitive and >95% specific. The World Health Orga-nization has included dengue viral illness in its “pocket book of hospital care”,which health-care workers in endemic countries have been using for themanagement of dengue viral illness fever [27,28]. An audiovisual guide andtranscript for health-care workers responding to outbreaks has been an effectivetool in early recognition, diagnosis, and hospital management [28].012345678910RotaVirus GE Cholera Chagas Rabies DengueEconomic burden of various diseases globallyCost in Billions(in US Dollars) FIGURE 4.7 The cost of Dengue viral illness compared to other diseases globally.TABLE 4.3 The average cost of Dengue viral illness globally.Global average cost Value (in US dollars)Admission to hospital 333 (CI 283e403)Ambulatory cases 60 (CI 54e68)Death of child 80,414Death of adult 75,82046 Dengue Virus DiseaseThe World Health Organization created “Global Outbreak Alert andResponse Network”, which aims at responding to a developing outbreak veryswiftly to prevent the outbreak itself or limit the consequences in case of anoutbreak. The network developed an “integrated vector management approach”in 2004 where the main aim was to reduce or interrupt transmission of thedisease. The methodology emphasized on eliminating breeding grounds, use ofindoor residual sprays, and also use of an obligate intracellular bacteriumWolbachia which when injected intracellular into male Aedes mosquito isthought to bring a reproductive manipulation rendering offspring nonviablethereby reducing the mosquito burden [29,30]. .This method seems to have abeneficial impact on the rate of Dengue viral illness as evident in Brazil, whoreleased its first Wolbachia mosquito in 2014. Similar efforts have been seen inIndonesia under the “Eliminate Dengue viral illness Program”. Singaporeconducts a nation-based survey to identify the items around the house that arethe most proficient breeding sites such as flowerpots and ornamental containers.The World Health Organization has stressed the importance of research inthe field of dengue viral illness. Historically, Dengue viral illness research hasalways been underfunded due to underestimating of the disease burden and itsimpact on society. However, more and more research projects have been un-dertaken in the last few years. The US National Institute of Allergy and In-fectious disease has funded over 60 projects with emphasis on tetravalentvaccine development. Preventing outbreak, early detection, and to improvevaccine delivery remains a priority for the Bill and Melinda Gates Foundation.The European Commission provided 18 million Euros toward “Comprehen-sive control of Dengue viral illness Fever under changing climactic condition”.With the aim to generate awareness and improve research funding, the As-sociation of Southeast Asian Nations has designated June 15 to be “Dengueviral illness day” [31].Finally, most effective method to combat any disease is to generateawareness among people and Dengue viral illness is not an exception. Anotherreason for the rising epidemics is the difficulty to control mosquito breeding.Though government from endemic nations have invested a lot of money forinsecticide spraying, common people do not have the awareness of preventingbreeding grounds for mosquitoes that perpetuate their cycles. Efforts forawareness have been attempted on an international platform during the 2014World cup in Brazil and in the 2016 Summer Olympics. Social media can beused not only as a platform to increase awareness but also to report cases thatcould serve as a warning sign for potential outbreaks. The World Health Or-ganization has emphasized a lot on awareness and promoted community-based, “bottom-up” communication for behavioral impact [32]. It primarilyencourages active participation of community members in public healthmessaging. For example, in Thailand, there is one village health worker forevery 10 households with the responsibility toward generating awareness andwarning about outbreaks [32].Global health-care perspective of Dengue viral disease Chapter | 4 47Global innovations/interventionsNumerous efforts are being taken to prevent disease transmission, therebyreducing the impact on human lives. For adequate control over the disease,early identification of the epidemic is the first step. However, majority of thedetection of the cases nationally rely on hospital-based reporting. Ineffectivecommunication, untimely reporting, and frequent post hoc revisions limitidentification and optimization of necessary interventions.Therefore, an ideal tool should have the following characteristics: provideaccurate data to regional/national level, ability to detect outbreaks and swiftwarning, be updated in real time, and reduce resource related delays.Many epidemiological methods have been attempted to act as a supple-mental tool by reducing the limitations of the traditional system. Autore-gressive models like Seasonal Autoregressive Integrated Moving Average takeinto account seasonal patterns and help in determining useful incidence esti-mates [33e36]. Numerous and varied mechanistic models have been explored[37] and some long-term weather-driven models such as El Niño with Dengueviral levels in various countries [38].Real-time Internet searches for Dengue viral illness tracking has beendeveloping as an effective tool for disease identification and warning. Internetsearch is efficient, consistent, and gives a snapshot of real-time trends, therebyposing as a very strong supplemental tool. Studies have been done previouslyfor evaluating the role of using Internet search data to track Dengue viraldiseases [39,40]. Google Dengue Trends was started in 2011 and was one ofthe first tools to be used in quantification of Dengue viral illness in multipleregions of the world, thereby allowing a large section of population to accessthe data globally. Currently, live status and the trend of the disease are easilyavailable which help in early identification and taking appropriate measures toreduce the risk of the disease outbreak. One such method can be seen inFig. 4.8.It is imperative to evaluate the combination of traditional methods and real-time Internet searches for detection of cases thereby combining the respectivestrengths of the data source. One such attempt is made to combine theautoregressive models with real-time Google search queries to explore theeffectiveness of the combination [41]. The results are promising, but yet manyfacets have to be explored in order to create an efficient way of rapid, earlydetection with fast communication to the general population at risk to mini-mize risk and reduce burden of the disease.Global research and vaccine developmentDengue viral illness has emerged as a global public health problem in the last20 years. This has been caused by the expanding geographic distribution ofboth the viruses and the principal mosquito vectors subsequent to global48 Dengue Virus Diseasedemographic and societal changes. During this period, most tropical urbancenters of the world have become hyperendemic, thus increasing the risk ofepidemic transmission and the emergence of Dengue hemorrhagic fever. It isimportant to know the global impact and economic burden because such es-timates are needed by policy planners to help allocate limited resources forresearch, prevention, and control activities [42].The Scientific Working Group was organized by the United Nations In-ternational Children Emergency Funds/United Nation Development Program/World Bank/World Health Organization, a Special Program for Research andTraining in Tropical Diseases in Geneva. The priority of dengue viral illnessresearch areas are organized along four major research streams which willprovide evidence and information for policy-makers and control programmesleading to more cost-effective strategies which will reverse the epidemiolog-ical trend [27,28,43e46]. These streams are highlighted as shown in Table 4.4.As a result of the failure of vector control, the continuing spread andincreasing intensity of Dengue viral illness has renewed interest and investmentin Dengue viral illness vaccine development, making a safe, effective, andaffordable tetravalent dengue viral illness vaccine a global public health pri-ority. Dengue viral illness vaccine development has been in progress for severaldecades; however, the complex pathology of the illness, the need to control fourvirus serotypes simultaneously, and insufficient investment by vaccine de-velopers have hampered progress. The available data suggest that neutralizingantibodies are the major contributors to protective immunity; however, the roleof the cellular immune response requires further study. In this context, clinicalFIGURE 4.8 Trend of the Dengue viral illness in the last 3 months. Source: https://www.healthmap.org/dengue/en/.Global health-care perspective of Dengue viral disease Chapter | 4 49https://www.healthmap.org/dengue/en/https://www.healthmap.org/dengue/en/trials are crucial for vaccine development owing to the unique information theyprovide on immune responses and reactogenicity. Also, long-term observationsof vaccinated populations will be required to demonstrate the absence ofantibody dependent enhancement (ADE) or severe disease [47e55].An ideal vaccine for Dengue viral illness virus must be to tetravalentbecause each of these serotypes is present throughout the world and each cancause disease. The vaccine should include neutralizing antibody levelscompatible with those observed in wild-type virus infection to limit the risk ofadverse drug effect. Finally the vaccine should be available at a low cost forthe developing world. Multiple live attenuated vaccine candidates are pres-ently being evaluated in current clinical trials. Clinical safety and strongimmunogenicity have been observed for empirically derived vaccine stringentfor recombinant viruses using either genetically modified full-length vaccinestrains or antigenic chimeric viruses. Inactivated monovalent vaccines andrecombinant subunit vaccine consisting of purified and blood proteins arescheduled to be tested in clinical trial soon. Other approaches are beingevaluated pretty clinically. Considerable progress has been made in thedevelopment of Dengue viral illness vaccine candidates in the last few years.One or more of the strategies being currently perceived should be successful inreducing the disease burden caused by this emerging pathogen [56,57].Vaccine candidates should be evaluated in population-based efficacy trialsin several at-risk populations in different geographical settings, including Asiaand the Americas, which experience different patterns of Dengue viral illnesstransmission intensity and Dengue viral illness virus circulation. Vaccinedevelopers are working with the pediatric Dengue viral illness VaccineInitiative to establish suitable field sites. Developers are also working with theWorld Health Organization/Initiative for Vaccine Research to define theTABLE 4.4 Different streams and activity performed.Streams Activities performed Related informationStream 1 Research related to reducingdisease severity and case fatalityOptimization of clinicalmanagementStream 2 Research related to transmissioncontrol through improved vectormanagementDevelopment and evaluation ofvector control tools and strategiesStream 3 Research related to primary andsecondary preventionVaccinesStream 4 Health policy researchcontributing to adequate publichealth responseHealth policies50 Dengue Virus Diseaseimmunological correlates for protection and clinical trial design. Because ofthe important role of neutralizingantibodies as surrogates of protection, thevalidation of neutralization tests is a priority. Current approaches to vaccinedevelopment involve using live attenuated viruses, inactivated viruses, subunitvaccines, deoxyribonucleic acid vaccines, cloned engineered viruses, andchimeric viruses using yellow fever vaccine and attenuated Dengue viralillness viruses as backbones. Currently vaccines in different stages of clinicaltrials are shown in Table 4.5 [19,30,57e76].The United States Food and Drug Administration approved Dengvaxia, thefirst vaccine to be approved for the prevention of Dengue viral illness diseasecaused by all the dengue viral illness serotypes (1, 2, 3, and 4) in people ages 9through 16 years who have laboratory confirmed previous Dengue viral illnessinfection and who live in endemic areas. Dengvaxia is a live, attenuatedvaccine that is administered as three separate injections, with the initial dosefollowed by two additional shots given 6 and 12 months later. The safety andeffectiveness of the vaccine was determined in three randomized, placebo-controlled studies involving approximately 35,000 individuals living inendemic areas. The vaccine was determined to be approximately 76% effectivein preventing symptomatic laboratory-confirmed Dengue viral illness diseasein 9e16 years of age. Dengue vaccine has already been approved in 19 countriesTABLE 4.5 Dengue viral illness vaccines in research.Preclinical Subunit recombinant antigen (domain III) vaccine by IPK/CIGBLive attenuated chimeric yellow fever/Dengue virus vaccine (YF-DEN)by Oswald Cruz foundationTetravalent DNA vaccine by United States Naval Medical ResearchCenter (US NRMC) & GenPharPurified inactivated tetravalent vaccine by Walter Reed Army Institute ofResearch (WRAIR) and GlaxoSmithKline (GSK).PHASE I Live attenuated tetravalent vaccine comprising 30 deletion mutations andDEN-DEN chimeras by United States National Institute of HealthLaboratory of Infectious Disease (US NIH LID) and National Institute ofAllergy and Infectious Diseases (NIAID)Live attenuated chimeric DEN2-DEN vaccine by CDC & InviragenRecombinant E subunit vaccine by MerckPHASE II Live attenuated tetravalent chimeric yellow fever/Dengue virus vaccine(YF-DEN) by Sanofi PasteurLive attenuated tetravalent viral isolate vaccine byWalter Reed ArmyInstitute of Research (WRAIR) and GlaxoSmithKline (GSK).Global health-care perspective of Dengue viral disease Chapter | 4 51and the European Union. The most commonly reported side effects by thosewho received Dengvaxia are headache, muscle pain, joint pain, fatigue, injectionsite pain, and low-grade fever. The frequency of side effects was similar acrossdifferent populations who received Dengvaxia and these side effects intended todecrease after each subsequent dose of the vaccine [77].Dengvaxia is not approved for use in individuals who are not previouslyinfected with Dengue viral illness virus as it appears to act like a first-degreeinfection without actually infecting the person with wild-type by dengue viralillness virus such that a subsequent infection can result in severe degree ofdisease. Therefore, health-care professional should evaluate individuals forprior infections to avoid vaccinating individuals who have not been previouslyinfected by Dengue virus. This can be accessed through medical records forprevious laboratory confirmed negative infection or through serological testing(tests using blood samples from the patient) prior to vaccination [77].ConclusionDengue viral illness is now endemic in more than 125 countries globally.Reasons for the currently observed and predicted expansion are multifactorial.These reasons may include climate change, virus evolution, and societal fac-tors such as rapid urbanization, population growth and development, socio-economic factors, as well as global travel and trade. The known social,economic, and disease burden of Dengue viral illness internationally isalarming and it is evident that the wider impact of this disease is grosslyunderestimated. Dengue viral illness has been like a silent but potent weaponimpacting lives worldwide, with incidence rates climbing in the last decade.Multiple attempts and numerous measures to eliminate the disease have beenunsuccessful. The World Health Organization Global Strategy for Dengueviral illness Prevention and Control, 2012e20 highlights the need forimproved estimates of the true burden of Dengue viral illness globally due tothe currently presumed underrepresentation. Surveillance and reporting isparamount for effective Dengue viral illness control, and more accuratequantification of the impact of Dengue viral illness globally will allowimproved political, financial, and research prioritization as well as informeddecision-making and enhanced modeling. Active participation of nationsglobally along with political and economical resources will be a key toeradicate this disease in future [27,28,48,56].References[1] Dengue: guidelines for diagnosis, treatment, prevention and control. New Edition. Geneva:World Health Organization; 2009.[2] Halstead SB, Cohen SN. Dengue hemorrhagic fever at 60 years: early evolution of conceptsof causation and treatment. 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J Infect Dis 2010;201:370e7.[76] Parks W, Lloyd L. Planning social mobilization and communication for Dengue feverprevention and control: a step-by-step guide. Geneva, Switzerland: WHO; 2004.[77] Andre Sofair. FDA approved vaccine for prevention of Dengue disease in endemic regions.FDA news room; May 2019.56 Dengue Virus DiseaseChapter 5Mosquito-borne diseasesMuhammad A. Saleem1, Iryna Lobanova2,31Resident Physician, Family Medicine, Mercyhealth, Janesville, WI, United States; 2ProjectManager, Dengue virus disease project, Zeenat Qureshi Stroke Institute, St. Cloud, MN, UnitedStates; 3University of Missouri, Columbia, MO, United StatesThe dangerous insectsMosquitoes may lack in size but are by far the deadliest living being on earthbeating grizzly bears, tigers, cobras, and hippos with huge margins. Thesepesky insects are capable of killing more people in one day than sharks do in acentury. Unarguably, they are able to carry and spread more potentially fataldiseases than any other vector on the planet. The human lives these blood-thirsty creatures take and the resultant lost productivity accounts to billionsof dollars annually. Mosquitoes not only pose a deadlier threat to humans butalso to larger number of animals including amphibians, reptiles, squirrels,rabbits, and small mammals. The cross-infections caused by them haveresulted in novel epidemics in recent decades. Investigations have revealedtheir remarkable ability to adapt to new environments, quick and large scalereproduction, resistance to certain insecticides, and alteration in feeding habitsto survive against control methods.Life-cycle of mosquitoesThe mosquitoes lay plentiful eggs and the bloodsucking insects can grow froman egg to an adult in just five days. After the female mosquito obtains bloodmeal, she lays several hundred eggs directly on or near water, soil, and at thebase of some plants in places that may fill with water. The eggs can survive indesiccated conditions for a few months. The size of their nursery can be assmall as a jar top. The eggs hatch when exposed to water and the length of timeit takes for them to hatch also depends on water temperature and the type ofmosquito. The egg hatches into bristly aquatic larva about 8 mm long. Theselarvae swim with a jerking, wriggling movement thereby called “wrigglers.”Larva mostly feed on algae and organic debris from sewage. Larva moltsseveral times and most species surface to breathe air before developing intoDengue Virus Disease. https://doi.org/10.1016/B978-0-12-818270-3.00005-9Copyright © 2020 Elsevier Inc. All rights reserved. 57https://doi.org/10.1016/B978-0-12-818270-3.00005-9pupa. Mosquitoes in their pupal stage are called tumblers and are actively free-swimming thereby called “tumblers.” The pupa also lives in the water but nolonger feeds. After almost a week the pupa transforms into the adult mosquito(see Fig. 5.1). The duration of lifecycle may range from 4 days to a monthdepending on the species of mosquito. The adult mosquito emerges onto thewater’s surface and flies away, ready to begin its lifecycle [1].Mosquitoes can fly long distances; some more than 20 miles from the watersource that produced them. But they do not fly fast, only about 4 miles an hour.Since they typically fly into the wind to help detect host odors, fewermosquitoes are about on windy days.The sniffy navigationThe mosquitoes use scent to find humans. The chemistry of our perspirationespecially the lactate is attractive to them. They can also sense carbon dioxidein our exhalation sand follow the stream back to our faces. Mosquitoes canalso detect body heat and movements to identify the potential target [2]. Thefrequency of wing beats gives them their unique hum and may serve as ameans of gender recognition [3].Common genus of mosquitoesThere are thousands of different species of mosquitoes. While the deadliesttypes feed on our blood and spread horrible diseases, some of them areFIGURE 5.1 Lifecycle of mosquito.58 Dengue Virus Diseaseparasites on other animals and rare mosquito species do not feed on blood atall. Here we discuss the most common genus of mosquitoes.AedesThis group includes many species that transmit disease to humans. Theseinclude inland flood water mosquito (Aedes vexans), the Asian tiger mosquito(Aedes albopictus) and the tree hole mosquito (Ochlerotatus triseriatus)dallof which feed on the blood of mammals. Flood water mosquito lay its eggs onsoil which becomes flooded thereby allowing the life cycle to proceed. Asiantiger and Tree hole mosquitoes breed in containers, laying their eggs in smallwater filled cavities and thrive in urban areas.Inland flood water mosquitoes are brown with pale B-shapedmarks ontheir abdomens. Typical outbreaks are seen when river backwaters and low-lying places become flooded. They are usually the first crop noticed inspring and later after heavy rainfall. They can fly more than 10 miles fromtheir breeding places in search of blood meals. They are usually active in thedark and typically the infestations die in autumn with the first frosting.The Asian tiger mosquito is black with one white “racing” stripe on itsthorax. It is usually active during day time and is capable of carrying Zika,Dengue, Chikungunya, West Nile viruses and Yellow fever viruses. The biggestvector of La Crosse encephalitis is the Tree hole mosquito. It is a dark brownmosquito with silvery white bands on legs. It bites by day and lays its eggs insmall containers where water will pool, such as tree holes, discarded tires, cans,buckets, and barrels. They often are found in and around wooded areas.Aedes aegypti, the Yellow fever mosquito, is closely associated with andfeeds primarily on humans. It is a small container breeder and will be collectedaround and in homes. It is active primarily during dusk and dawn. It is oftennoticed flying about ankles looking for an opportunity to feed. It also wins thedistinction of main vector for Dengue virus, Chikungunya virus, and Zika virus.Mosquito-borne diseases Chapter | 5 59AnophelesAlso known as marsh mosquitoes, this genus has 460 different species. Themost prominent species in Anopheles gambiae which is well known for car-rying malaria in Africa and Anopheles freeborni in North America. They breedin natural water collections and therefore breeding increases in the rainyseason when water collects in bottles, tins, buckets, tyres etc. Constructionsites provide ample breeding places for the mosquito. Anopheles mosquitoesenter the house near sunset and again in early hours of morning. They startbiting by late evening and the peak of biting activity is at midnight and earlyhours of morning. Mosquitoes can fly up to several kilometers and they canreach far off places by taking shelter in motor vehicles, ships, and aircraft.CulexAlso known as the house mosquito, the Culex genus contains several speciesthat are vector of West Nile virus, West Nile encephalitis, and Rift Valleyfever. These include Culex pipiens, Culex quinquefasciatus, and Culex tarsalis.They typically bite at dusk and after dark. By day, they rest in and aroundstructures and vegetation. They lay eggs on still water in natural and manmadecontainers. Adult Culex mosquitoes do not fly far from where they develop aslarvae. Unlike other mosquitoes that die with the coming of the first hard frostin autumn, the house mosquito can “over-winter” in protected places likesewers, crawlspaces, and basements.CulisetaThese are larger mosquitoes which bear superficial resemblance to Culex.They have adapted to the cold, and are found everywhere except SouthAmerica. The larvae of most species are found in ground waters such as bogs,marshes, ponds, streams, ditches, and rock pools, but an African species occursin tree holes, a common eastern Palearctic species occurs in wells and rockpools, and several Australian species occur underground.MansoniaThese mosquitoes are bigger than most, and are black or brown with sparklingwings and legs. They are found in most parts of the world, and are known totransmit encephalitis. Mansonia mosquitoes prefer to bite in the evening. Theybreed in ponds and lakes containing certain aquatic plants, especially thefloating type like Pistia stratiotes and water hyacinth. The eggs are laid in star-shaped clusters on the undersurface of leaves of these plants. The larvae andpupae are found attached to the rootlets of these plants by their siphon tubes.They obtain their air supply from these rootlets. When about to become adult,these pupae come to the surface of water and the fully formed adults emergeand escape.60 Dengue Virus DiseasePsorophoraWith a realm of species, Psorophora mosquitoes vary in size, from small tovery large. They are mostly found in tropical areas of North and SouthAmerica, and are vectors for Ilheus virus and encephalitis. They are sometimesreferred to as flood water mosquitoes, since they like laying their eggs on mud.ToxorhynchitesAlso known as the elephant mosquito, this genus does not consume blood.Like their male counterparts, the females feed on plant nectar, and do not posea risk to humans. Interestingly, their larvae prey on the larvae of othermosquitoes; it is been suggested that Toxorhynchites could be introduced inareas they do not generally inhabit to help fight Dengue viral infection.WyeomyiaMostly found in Central and South America, this genus are not known to carrydiseases, so do not pose a risk to humans. There are 140 known species, andthey generally inhabit flowers, bamboo, tree holes, and containers. Adults areactive during the day, usually near larval habitats. Some species are found atcharacteristic elevations in the forest canopy, with others appearing to berestricted to ground level.Techniques for eradicationIntegrated pest management is a science-based, common-sense approach formanaging mosquitoes. It uses a variety of pest management techniques thatfocus on pest prevention, pest reduction, and the elimination of conditions thatlead to pest infestations [4]. Such pest management programs also rely heavilyon resident education and pest monitoring. Integrated pest management usesvarious ways to control mosquito populations with decision based on sur-veillance to keep track of infestations. This is backed with appropriate use ofinsecticides. The cornerstones of such a management strategy are elaboratedbelow:Remove mosquito habitatsSince most species of mosquitoes lay their eggs in or near water, it is animportant part of mosquito control strategy to make sure that there are nopotential reservoirs. The mosquitoes love standing water and would rush tomake it their home. The obvious ones are puddles and small ponds but smallwater bodies that are usually overlooked include landscape ponds, lawn oryard ornaments, puddles, bird baths, clogged gutters, and downspouts [5]. It isimportant to drain rain gutters, old tires, plastic covers, and toys whereMosquito-borne diseases Chapter | 5 61mosquitoes may be hiding. Draining temporary pools of water or filling themwith dirt and keeping swimming pool water treated are also cornerstone ap-proaches to handling potential mosquito breeding sites in a modern household.Control mosquitoes at the larval stageAlthough the most natural solution to eliminate mosquito reproduction is todrain their breeding grounds, sometimes it is very difficult. Mosquitoes havebeen demonstrated to maintain their colonies in as much as half an inch bodiesof water. Areas like runoff drains and landscaping are inherently designed tohold water. The greatest impact on mosquito occurs when they are immobile,accessible, concentrated, and fragile. Larviciding typically involves applyingpesticides containing methoprene or Bacillus thuringiensis israelensis orB. sphaericus bacteria, to water where mosquito larvae develop. As larvae feedon Bacillus, a bacterial toxin is released which perforates mosquito’s gut.Methoprene is a larvae growth regulator and works by disrupting larvaemetamorphosis [6]. The toxicity of both of these methods is very low and theyhave been deemed safe to be used in waters containing fish. They can be usedeffectively where it is undesirable or impractical to empty the water in con-tainers, such as water in decorative pools or horse watering tanks. Other typesof larvicides include those that cover the surface of the water with thin films ofliquid designed to prevent larvae from obtaining oxygen at the water’s surface.Control adult mosquitoesAdult mosquito control involves application of fine droplets of pesticidesreleased as an ultralow volume treatment from a specializedtruck or aerialequipment. This technique is usually used to combat an outbreak of mosquito-borne diseases being transmitted by adult mosquitoes. In contrast to larvi-ciding, adulticiding is a broad spectrum application that can kill beneficialinsects as well and is much more expensive. Adulticiding should be seen as asupplement to larviciding, to be used when mosquitoes become too numerousor when high levels of virus activity in mosquitoes threaten populated areaswith disease [7].Use of structural barriersSince the mosquitoes frequently bite indoors, using structural barriers is animportant way to reduce the incidence of bites. It is important to make surethat the windows and door are bug tight by maintaining screens integrity andsealing all gaps through which mosquitoes might enter. Completely coveringbaby carriers and beds with netting. Nets can be especially important forprotecting a sick person from getting more mosquito bites, which couldtransmit the disease to other people. Repellents like N,N-diethyl-meta-62 Dengue Virus Diseasetoluamide, picaridin and lemon oil of eucalyptus provide fair benefit whenused appropriately [8].Mosquitoes in AmericaIn the 17th century, the trade ships arriving at the Southern ports in the UnitedStates are believed to have brought Aedes aegypti mosquito and resultantlyYellow fever and Dengue fever to the country [9,10]. Initially, the epidemicswere confined to the temperate and tropical zones in south and the west [11],until the 19th century numerous outbreaks of malaria have been recorded asfar north as Massachusetts [12]. Malaria spread quickly in the Midwest statesalong the Mississippi valley during the American Revolutionary War and theCivil War. Malaria emerged as a major catastrophe during 4 years of the CivilWar claiming thousands of lives [13]. The transmission of disease causingorganism was proven in 1889 and since then the US government has under-taken multidisciplinary efforts to control mosquitoes [14]. With changes in thelifestyle and population spending more time indoors than outdoors, frequentuse of door and window screens, depopulation of the rural south, and improvedsocioeconomic conditions malaria was no longer endemic in any area ofcontinental United States by the early 1940s [15].During the 18th and beginning of 19th century, Yellow fever epidemicswere common in Northeastern states [16]. It is believed that the Yellow feverepidemic in Philadelphia in 1793 was a contributing factor in the decision tomove the United States capital to Washington [17]. Between 1693 and 1905,approximately 10 million deaths are attributed to Yellow fever along theMississippi River, from the Gulf of Mexico to Memphis, Tennessee, St. Louis,Missouri, and New Orleans [18]. The epidemics that claimed most of the livesoccurred during summer in port cities with active trade with the CaribbeanIslands. After identification of Aedes aegypti as the primary vector of yellowfever in 1901, the US companies that took over the construction of PanamaCanal participated actively in the mosquito eradication drives by draining oroiling the larva breeding sites and fumigation [19]. Later on, the programsimplemented by Pan American Health Organization eradicated the Aedesaegypti mosquito and consequently Dengue viral infection until 1970. Becauseof the questions raised about the safety of Dichlorodiphenyltrichloroethane usein environment and the huge success of elimination program, the eliminationprograms fell on the back foot leading to reemergence of mosquito, escalationin its population and ultimately failure to eradicate Dengue viral infection[20].The socioeconomic changes in the United States spanning on 18th and 19thcentury have substantially affected the transmission of those pathogens forwhich humans are the primary amplifying host. The availability of potablewater has eliminated the need to store water in the household and therebyreduced the breeding grounds of mosquitoes. Likewise, improved sanitationMosquito-borne diseases Chapter | 5 63and sewage disposal processing have shrunk the vector nurseries as well. Afterthe second world war, the boom in the US economy improved the livingstandards and aided widespread use of television and air-conditioning [21].These commodities encouraged general population to spend more time inindoor screened areas thereby reducing potential mosquito targets [22].The improvement in socioeconomic conditions has lead to substantialreduction in prevalence and transmission of anthroponotic pathogens likedengue and malaria viruses but it does not affect the transmission of zoonoticpathogens such as West Nile virus, St. Louis encephalitis, Eastern equineencephalitis, Western equine encephalitis, and La Crosse encephalitis [23].Since animals other than humans play a major role in their transmission, theseare maintained in natural transmission cycles without being affected byimproved accommodation for humans. Therefore, zoonotic viruses like easternequine encephalitis and La Crosse viruses continue to cause diseasethroughout the continental United States, Southern Canada, and SouthAmerica [24].West Nile virusThis virus first appeared in the United States in 1999 and spread by 2001 toFlorida. This disease is primarily an infection found in birds, but can also betransferred to humans, dogs, horses, and other animals. Humans, while alsosusceptible, often show no symptoms (less than 1% of those infected showsymptoms). The mortality rate of those who become ill ranges from 3% to15%, and is highest among the elderly.West Nile virus is an arbovirus of the Flavivirus kind in the family Fla-viviridae. Mosquitoes spread from West Nile to 48 of the 50 US states, Africa,Europe, the Middle East, and West and Central Asia. In 2012, the US expe-rienced one of its worst epidemics where 286 people died, with the state ofTexas being hit hardest [25]. As of 2014, there have been 36,437 cases of WestNile virus. Of these, 15,774 have resulted in meningitis/encephalitis and 1538were fatal. There have been at least 1.5 million infections (82% are asymp-tomatic) and over 350,000 cases of West Nile fever, but the disease isunderreported due to its similarity to other viral infections. This virus usuallycirculates between mosquitoes and birds in Africa and Europe. However, in1999 an outbreak of West Nile encephalitis was reported in New York City.Since then, the virus has spread to 48 states and the District of Columbia[26e30].Most people who get West Nile virus do not have any symptoms. About 1in 5 will have a fever and other flu-like symptoms. A few people (less than 1%)get more serious infection West Nile neuroinvasive disease, which causesmeningitis, encephalitis, meningoencephalitis, and poliomyelitis-like syn-drome. People of advanced age, the very young, or those with immunosup-pression are most susceptible. Many patients with WNND have normal64 Dengue Virus Diseaseneuroimaging studies, although abnormalities may be present in various ce-rebral areas including the basal ganglia, thalamus, cerebellum, and brainstem.West Nile virus encephalitis is the most common neuroinvasive manifes-tation of West Nile neuroinvasive disease. West Nile virus encephalitis pre-sents with similar symptoms to others viral encephalitis with fever, headaches,and altered mental states. A prominent finding in West Nile virus encephalitisis the muscular weakness (30%e50% of patients with encephalitis), often withlower motor neuron symptoms, flaccid paralysis, and hyporeflexia with nosensory abnormalities. West Nile meningitis usually involves fever, headache,and stiff neck. Pleocytosis, an increase of white blood cells in cerebrospinalfluid, is also present. Changes in consciousness are not usually seen and aremild when present. West Nile poliomyelitis, an acute flaccid paralysis syn-drome associated with infection, is less common than West Nile meningitis orWestNile virus encephalitis. This syndrome is generally characterized by theacute onset of asymmetric limb weakness or paralysis in the absence of sen-sory loss. The pain sometimes precedes the paralysis. The paralysis can occurin the absence of fever, headache, or other common symptoms associated withWest Nile virus infection. Involvement of respiratory muscles, leading to acuterespiratory failure, can sometimes occur. West Nile reversible paralysis, likeWest Nile poliomyelitis, the weakness or paralysis is asymmetric [31e36].Reported cases have been noted to have an initial preservation of deep tendonreflexes, which is not expected for a pure anterior horn involvement. Thedisconnect of the upper motor neuron influences on the anterior horn cellspossibly by myelitis or glutamate excitotoxicity have been suggested. Theprognosis for recovery is excellent [37].Nonneurologic complications of West Nile virus include fulminant hepa-titis, pancreatitis [27], myocarditis, rhabdomyolysis [34], orchitis [38], opticneuritis [31], cardiac dysrhythmias, and hemorrhagic fever with coagulopathy[38], chorioretinitis [39,40]. The real danger may be for pregnant women andtheir babies. It is linked to a birth defect called microcephaly [32]. There is nospecific treatment for West Nile virus [41,42].Chikungunya virusChikungunya is another arbovirus transmitted through the bite of an infectedfemale mosquito Ae. aegypti and, to a lesser extent, the Ae. albopictus species.Chikungunya virus has recently appeared in such places as India, Sri Lanka,Mauritius, and countries in Europe involved in frequent tourism to thesedestinations. Concern has arisen recently that will soon be increasing the rangein Europe over the spread of the Asian Tiger mosquitoes (Aedes albopictus),that can act as significant vectors for this infection. The traditional range forthis virus also includes Africa and South East Asia. Infection with chi-kungunya can be severe and temporarily debilitating, but is generally not life-threatening in otherwise healthy people. No vaccine or curative drug treatmentMosquito-borne diseases Chapter | 5 65is currently available. Prevention must rely entirely on measures that reduceexposure to mosquito bites [43e49].Typical symptoms of infection by Chikungunya virus are:- Abrupt onset of fever above 38�C lasting 2e3 days- Moderate headache- Moderate-to-intense joint and tendon pain and swelling- Intense muscle pain- Fatigue- Rash (between the 2nd and 5th day in 50% of the cases)- Conjunctivitis/eye redness (30% of the cases)- Very rare cases include neurological manifestations in the form of en-cephalitis, Guillain-Barre syndrome, and myelitis among othercomplications.No specific treatment for Chikungunya viral fever is available at this stage.The infected person should take analgesics to treat fever (except acetylsalicylicacid) and antiinflammatory for the joint pain, drink plenty of fluids, rest andeat normally, and take precautions against mosquito biting to prevent spread ofdisease [43e49].Symptoms usually appear 3e7 days after the bit of an infected mosquito.The most common symptoms of chikungunya virus infection are fever andjoint pain. Other symptoms may include a headache, muscle pain, jointswelling, or rash. Chikungunya viral disease does not often result in death, butthe symptoms can be severe and disabling. Most patients recover fully, but insome cases, joint pain may persist for several months or even years. Occa-sional cases of eye, neurological, and heart complications have been reported,as well as gastrointestinal complaints. Serious complications are not commonexcept in older people where the disease can cause of death. Often symptomsin infected individuals are weak and the infection may go unrecognized or bemisdiagnosed in areas where Dengue viral illness occurs [43e49]. There is novaccine, and primary treatment is limited to pain medication [50].Eastern equine encephalitis virusEastern equine encephalitis virus is transmitted to humans by the bite of aninfected mosquito. Most cases occur in the Atlantic and Gulf Coast states.Most persons infected with Eastern equine encephalitis virus have no apparentillness. Severe cases of Eastern equine encephalitis begin with the suddenonset of a headache, high fever, chills, and vomiting. The illness may thenprogress into disorientation, seizures, or coma. Eastern equine encephalitis isone of the most severe mosquito-transmitted diseases in the United States withapproximately 33% mortality and significant brain damage in most survivors.There is no specific treatment for Eastern equine encephalitis; care is based onsymptoms [51,52].66 Dengue Virus DiseaseThis is a disease found in horses, but there is a vaccine available. The virusis maintained in birds, which are not affected by the disease. Humans dosometimes contract the disease, but humans are not a preferred target of themosquitoes carrying this virus and are considered a “dead end” host. Thismeans that, while a human can contract Eastern equine encephalitis,mosquitoes have not been shown to become infected by a human host andspread it to other organisms. There are normally a few cases of Eastern equineencephalitis in Florida each year and the disease has shown to have a relativelyhigh mortality rate of approximately 35%. Many of those who survive Easternequine encephalitis will have mild to severe brain damage resulting from highfever.Japanese encephalitis virusCountries with the proven epidemics of Japanese encephalitis virus are India,Pakistan, Nepal, Sri Lanka, Burma, Laos, Vietnam, Malaysia, Singapore,Philippines, Indonesia, China, maritime Siberia, Korea, and Japan. The virus istransmitted to humans by the bite of an infected mosquito, which serves as adead end host due to its short duration and low viremia in man. Most importantmosquito vector in Asia is Culex tritaeniorhynchus, which breeds in thestagnant water like paddy fields or drainage ditches. Other species are Culexvishnui (India), C. gelides, C. fusco cephalea (India, Malaysia, Thailand), andC. pipiens. About 50%e60% of the survivors suffer from serious long-termneurologic complications manifesting as convulsions, tremors, paralysis,ataxia, memory loss, impaired cognition, behavioral disturbance, and othersuch symptoms. There is an incubation period of 4e14 days in humans duringJapanese encephalitis virus infection and patients are presented with few daysof fever including coryza, diarrhea, and rigors. Convulsions occur 10% morefrequently in children (85% of cases) than in adult patients (75% of cases).Vector control alone cannot be relied upon to prevent Japanese encephalitisvirus since it is practically almost impossible to control mosquito density inthe rural areas which are the worst affected areas due to poor socioeconomicconditions. Three types of Japanese encephalitis virus vaccine are currently inuse: mouse-brain derived inactivated, cell-culture-derived inactivated, andcell-culture-derived live attenuated Japanese encephalitis virus vaccine.Formalin-inactivated vaccines are safe and effective against Japanese en-cephalitis virus for at least 30 years [53e55].Japanese encephalitis virus is the leading cause of vaccine-preventableencephalitis in Asia and the Western Pacific [53e55]. Pigs are an importantmaintenance host for this virus, which is mainly transmitted by night-bitingmosquitoes in the Culex tritaeniorhynchus group. Japanese encephalitis vi-rus is found in India and South East Asia upto Japan and has a similar risk forfatality and permanent debilitation as Eastern Equine Encephalitis, exceptJapanese encephalitis virus, affects a wider range of age groups. It has recentlyMosquito-borne diseases Chapter | 5 67expanded its range to reach northern Australia. A total of 30% of those whoshow Japanese encephalitis virus symptoms die and another 30% developserious and permanent neurological damage.Between 30,000e50,000 clinicalinfections are reported each year across Asia. The majority of infections,however, are asymptomatic [56e60]. Steps to prevent Japanese encephalitisvirus include using personal protective measures to prevent mosquito bites. Aspreviously mentioned, an effective vaccine is available for preventing thisinfection [61].Murray valley encephalitis virusMurray Valley encephalitis is an uncommon disease caused by the MVE en-cephalitis virus. Murray Valley encephalitis virus is a flavivirus endemic tonorthern Australia and Papua New Guinea [62]. The most important species ofmosquito to carry the virus is the common banded mosquito, Culex annulir-ostris. Most people with this infection remain completely well while othersmay only develop a mild illness with fever. A small proportion of thoseinfected develop encephalitis.Symptoms of Murray Valley encephalitis usually appear 5e28 days(average 14 days) after the infected mosquito bite. The early symptomsinclude the following: a headache, fever, nausea, vomiting, and muscle aches.Symptoms may also include drowsiness, confusion, seizures or fits (especiallyin infants), and in severe cases delirium, coma, and death. Some who recoverare left with ongoing problems such as deafness or epilepsy. There is nospecific treatment for Murray Valley encephalitis [63e65].La Crosse encephalitis virusLa Crosse encephalitis was discovered in 1965, after the virus was isolatedfrom preserved brain tissue and spinal cord of a child who died from theunknown infection in La Crosse, Wisconsin in 1960. It occurs in the Appa-lachian and Midwestern regions of the United States. Recently there has beenan increase of cases in the South East of the United States. An explanation tothis may be that the mosquito Aedes albopictus is also an efficient vector of LaCrosse virus [66]. Between 2004 and 2013 there were 787 total cases of LaCrosse encephalitis and 11 deaths in the U.S [67].Many people infected with La Crosse encephalitis virus have no visiblesymptoms. Among people who become ill, initial symptoms include fever,headache, nausea, vomiting, and tiredness. Some of those who become illdevelop the severe neuroinvasive disease. The incubation period for La Crosseencephalitis virus disease ranges from 5 to 15 days. La Crosse encephalitisvirus disease is usually characterized by fever (usually lasting 2e3 days),headache, nausea, vomiting, fatigue, and lethargy. The severe La Crosse en-cephalitis virus disease often involves encephalitis and can include seizures,68 Dengue Virus Diseasecoma, and paralysis. Severe disease occurs most often in children under theage of 16 years. In rare cases, long-term disability or death can result from LaCrosse encephalitis. No vaccine against La Crosse encephalitis virus infectionor specific antiviral treatment for clinical La Crosse encephalitis virus infec-tion is available. Patients with suspected La Crosse encephalitis virus en-cephalitis should be hospitalized, appropriate serologic and other diagnostictests performed, and supportive treatment (including seizure control) provided[68e70].MalariaMalaria is a disease transmitted by Anopheles mosquitoes in the genusdistributed throughout the world as a mosquito-borne disease caused by aparasite. Malaria is present in more than 100 countries and imposes aneconomically significant burden on the populations of at least 80 million.Malaria kills at least 1.1 million people per year, and probably more due toincomplete reporting in many of the countries on which it imposes the greatestburdens. Four species of parasites affect humans, but two of them, Plasmo-dium falciparum and P. vivax account for more than 95% of malaria cases.P. falciparum, the maximum dangerous of the pair which exists throughout thedeep tropics from Africa to Asia and South America. In 2015, an estimated214 million cases of malaria occurred worldwide and 438,000 people died,mostly children in the African Region. Malaria was endemic in the UnitedStates in the 19th and 20th centuries. About 1500 cases of malaria are diag-nosed in the United States each year. The vast majority of cases in the UnitedStates are in travelers and immigrants returning from countries where malariatransmission occurs, many from sub-Saharan Africa and South Asia [71e76].The immune evasiveness of malaria parasites prevents complete immunityfrom developing, but older children and adults who have experienced multipleinfections, enjoy some level of protection from the most severe manifestationsof the illness.Certain complications, such as cerebral malaria, strike quickly, cloggingsmall blood vessels in the brain to produce coma. Stories of expatriates fallingill on a Friday, putting off treatment till Monday, and dying over the weekendare not uncommon. Thus, malaria prevention requires serious attention whenvisiting areas where it is transmitted. Although no vaccine is currently avail-able, prophylactic drugs and measures that reduce exposure to night-bitingAnopheles mosquitoes, such as bed nets and repellents can be very effective.Unlike some infections, the victims of malaria often never get a secondchance.The causative agent of tropical malaria-infected red blood cells, especiallymature trophozoites, adhere to the vascular endothelium of venular bloodvessel walls and do not freely circulate in the blood. When this sequestrationof infected erythrocytes occurs in the vessels of the brain, it is believed to be aMosquito-borne diseases Chapter | 5 69factor in causing the severe disease syndrome known as cerebral malaria,which is associated with high mortality [77e80].Uncomplicated malariaThe classical (but rarely observed) malaria attack lasts 6e10 h. It consists of acold stage (sensation of cold, shivering), a hot stage (fever, headaches, vom-iting; seizures in young children), and finally a sweating stage (sweats, returnto normal temperature, tiredness). Classically (but infrequently observed) theattacks occur every second day with the “tertian” parasites (P. falciparum,P. vivax, and P. ovale) and every third day with the “quartan” parasite(P. malaria). More commonly, the patient presents with a combination of thefollowing symptoms: fever, chills, sweats, headaches, nausea, vomiting, bodyaches, and general malaise.The diagnosis of malaria depends on the demonstration of parasites in theblood, usually by microscopy. Additional laboratory findings may include mildanemia, the slight decrease in blood platelets (thrombocytopenia), elevation ofbilirubin, and elevation of aminotransferases [81e89].Severe malariaSevere malaria occurs when infections are complicated by serious organfailures or abnormalities in the patient’s blood or metabolism. The manifes-tations of severe malaria include the following:l Cerebral malaria, with abnormal behavior, impairment of consciousness,seizures, coma, or other neurologic abnormalitiesl Severe anemia due to hemolysisl Hemoglobinuria due to hemolysisl Acute respiratory distress syndrome, an inflammatory reaction in the lungsthat inhibits oxygen exchange, which may occur even after the parasitecounts have decreased in response to treatmentl Abnormalities in blood coagulationl Low blood pressure caused by cardiovascular collapsel Acute kidney failurel Hyperparasitemia, where more than 5% of the red blood cells are infectedby malaria parasitesl Metabolic acidosis, often in association with hypoglycemial Hypoglycemia, which may also occur in pregnant women with uncom-plicated malaria, or after treatment with quininel Neurologic defects may occasionally persist following cerebral malaria,especially in children. Such defects include ataxia, palsies, speech diffi-culties, deafness, and blindness [90e95].Quinine and other antimalarial drugs cure patients by attacking the para-sites in the blood. Despite the need, no effective vaccine exists. The70 Dengue Virus Diseaserecommendedtreatment for malaria is the combination of antimalarial med-ications that includes an artemisinin. The second medication may be eithermefloquine, lumefantrine, or sulfadoxine pyrimethamine. Quinine along withdoxycycline may be used if artemisinin is not available. There are a number ofmedicines that can help prevent or interrupt malaria in travelers to areas wherethe infection is common. Many of these drugs are also used in treatment.Chloroquine may be used where chloroquine-resistant parasites are not com-mon. In places where Plasmodium is resistant to one or more medications,three medications:Mefloquine (Lariam), doxycycline (available generically),or the combination of atovaquone and proguanil hydrochloride(Malarone)dare frequently used when prophylaxis is needed. Doxycyclineand the atovaquone plus proguanil combination are the best tolerated; mef-loquine is associated with death, suicide, and neurological and psychiatricsymptoms [96e100]. Drug resistance is now common against all classes ofantimalarial drugs apart from artemisinins [101].St. Louis encephalitis virusThis viral disease is maintained in birds and transmitted to both humans andhorses by the mosquito Culex nigrapalpus. While the virus is present in thewild every year, it occurs as a public health outbreak only periodically. Thisirregular outbreak pattern is the result of the number of susceptible individualsin the bird population coupled with large populations of the transmittingmosquitoes. These mosquito population increases are triggered by a combi-nation of climactic conditions including drought and rain cycles and individualrain events.Unfortunately, an accurate prediction model has yet to be developed.Currently, the best method of determining the presence of active viral trans-mission is the use of sentinel chicken flocks. This information coupled withmosquito population surveillance provides a basis for treating to limit thisdisease. Humans exposed to the virus often exhibit no symptoms. Thoseexhibiting symptoms will often suffer long-term neurological impairment.This disease has a mortality rate of 3%e30% and is highest in the elderlypopulation.Saint. Louis encephalitis is transmitted from birds to humans and othermammals by infected mosquitoes (mainly some Culex species). Saint. Louisencephalitis is found throughout the United States, but most often along theGulf of Mexico, especially Florida. Symptoms are similar to those seen inEastern Equine encephalitis and like Eastern Equine encephalitis, and there isno vaccine. Mississippi’s first case of SLE since 1994 was confirmed in June2003. Previously the last outbreak of Saint. Louis encephalitis in Mississippiwas in 1975 with over 300 reported cases. It was the first confirmed mosquito-borne virus in the United States in 2003. Another occurance turned up inOctober 2003 in California Riverside County in sentinel chickens. The lastMosquito-borne diseases Chapter | 5 71Saint. Louis encephalitis human case in California occurred in 1997[102e105].Less than 1% of Saint. Louis encephalitis virus infections are clinicallyapparent and the vast majority of infections remain undiagnosed. The incu-bation period for Saint. Louis encephalitis ranges from 5 to 15 days. The onsetof illness is usually abrupt, with fever, headache, dizziness, nausea, andmalaise. Signs and symptoms intensify over a period of several days to a week.Some patients spontaneously recover after this period, and others developsigns of central nervous system infections, including stiff neck, confusion,disorientation, dizziness, tremors, and unsteadiness. Coma can develop insevere cases. The disease is generally milder in children than in older adults.About 40% of children and young adults with Saint. Louis encephalitis virusdisease develop only fever and headache or aseptic meningitis. Almost 90% ofelderly persons with Saint. Louis encephalitis virus disease develop enceph-alitis. The overall case-fatality ratio is 5%e15%. The risk of fatal disease alsoincreases with age [103,106]. There is no vaccine or any other treatmentsspecifically for Saint Louis encephalitis virus, although one study showed thatearly use of interferon-alpha 2b may decrease the severity of complications[107].Yellow fever virusYellow fever virus, which has a 400-year history, is found in tropical andsubtropical areas in South America and Africa. Every year about 200,000cases occur with 30,000 deaths in 33 countries. In 2002, one fatal yellow feverdeath occurred in the United States in an unvaccinated traveler returning froma fishing trip to the Amazon. Like Dengue viral illness, it is transmitted byAedes mosquitoes, especially Aedes aegypti, the Yellow Fever mosquito[108,109].Most people infected with the virus of Yellow Fever have no illness or onlymild illness. In persons who develop symptoms, the incubation period (timefrom infection until illness) is typically 3e6 days. The initial symptomsinclude sudden onset of fever, chills, severe headache, back pain, general bodyaches, nausea, vomiting, fatigue, and weakness. Most people improve after theinitial presentation. After a brief remission of hours to a day, roughly 15% ofcases progress to develop a more severe form of the disease. The severe formis characterized by high fever, jaundice, bleeding, and eventually shock andfailure of multiple organs.Yellow fever disease is diagnosed based on symptoms, physical findings,laboratory testing, and travel history, including the possibility of exposure toinfected mosquitoes [110e113]. There is no specific treatment for Yellowfever. Vaccination is highly recommended as a preventive measure for trav-elers to and people living in endemic countries [114].72 Dengue Virus DiseaseRift valley fever virusThe disease was first reported among livestock in Rift Valley of Kenya in theearly 1900s, and the virus was first isolated in 1931. Outbreaks usually occurduring periods of increased rain, which increase the number of mosquitoes.The virus is transmitted through mosquito vectors, as well as through contactwith the tissue of infected animals. Two speciesdCulex tritaeniorhynchus andAedes vexansdare known to transmit the virus. The mild symptoms mayinclude fever, muscle pains, and headaches which often last for up to a week.The severe symptoms may include the following: loss of the ability to see(beginning 3 weeks after the infection), infections of the brain that cause se-vere headaches and confusion, and bleeding together with liver problems,which may occur within the first few days. Those who have bleeding symp-toms have a chance of death as high as 50%. Diagnosis relies on viral isolationfrom tissues or serological testing with an enzyme-linked immunosorbentassay. Once infected there is no specific treatment.Infected mosquitoes can give the disease to people and animals. It iscommon in parts of Africa. People have also contracted the virus in SaudiArabia and Yemen [115,116].In humans, the virus can cause several syndromes. Usually, sufferers haveeither no symptoms or only a mild illness with fever, headache, muscle pains,and liver abnormalities. In a small percentage of cases (<2%), the illness canprogress to hemorrhagic fever syndrome, meningoencephalitis (inflammationof the brain and tissues lining the brain), or affect the eye. Patients whobecome ill usually experience fever, generalized weakness, back pain, dizzi-ness, and weight loss at the onset of the illness. Typically, people recoverwithin two to 7 days after onset. About 1% of people with the disease die ofthe disease. In livestock, the fatality level is significantly higher. Other signs inlivestock include vomiting and diarrhea, respiratory disease, fever, lethargy,anorexia, and sudden death in young animals [117e119].There is a human vaccine; but, as of 2010, it is not widely available. Thereis no specific treatment and medical efforts are supportive [118,120].KunjinvirusThe main mosquito associated with the spread of Kunjin virus is Culexannulirostris, which is geographically widespread and is associated withfreshwater habitats. The kunjin virus is a zoonotic virus from the familyFlaviviridae and the genus Flavivirus. The virus was isolated in mosquitoes inSouth East Asia but in humans, only in Australia [121,122]. Symptoms ofKunjin virus disease vary. The vast majority of infected people do not developany symptoms. A small number of people may experience a mild illness withsymptoms including fever, headache, muscle pain, swollen lymph nodes, fa-tigue, and rash. Some of those people may experience encephalitis. SymptomsMosquito-borne diseases Chapter | 5 73of encephalitis may include confusion, drowsiness, and seizures. There is nospecific treatment for the disease [121,123,124].Ross river virusRoss River virus is endemic to Australia, Papua New Guinea, and other islandsin the South Pacific [125]. Ross River virus is transmitted from animals tohumans by a number of different types of mosquitoes with Culex annulirostris,Aedes vigilax (salt marsh mosquito) and Aedes notoscriptus being mostcommon. Ross River virus causes epidemic polyarthritis. The symptoms mayinclude fever with joint pain and swelling which may then be followed in 1 to10 days by a raised red rash affecting mainly the trunk and limbs. The rashusually lasts for 1 to 10 days and may or may not be accompanied by a fever.The joint pain can be severe and usually lasts 2 to 6 weeks [126e128].Barmah forest virusBarmah Forest virus is an Alphavirus. This disease was named after the area inNorthern Victoria where it was first isolated in 1974. As of 2015, it has beenfound only in Australia [129e131]. Barmah Forest virus causes inflammationand joint pain and has similar symptoms to Ross River virus infection(epidemic polyarthritis), but usually, lasts for a shorter duration. The symp-toms may include fever, headache, tiredness, painful joints, joint swelling,muscle tenderness, and skin rashes. Some people, especially children, maybecome infected without showing any symptoms. The initial fever anddiscomfort only last a few days but some people may experience joint pain,tiredness, and muscle tenderness for up to 6 months. There is no specific drugtreatment for Barmah Forest virus infection [130,132e134].Jamestown canyon virusJamestown Canyon virus, which can be transmitted to several different speciesof mosquitoes throughout Minnesota, is a rarely reported cause of illness inhumans [135]. The virus is closely related to La Crosse virus, although thedisease is reported less frequently and any age group may be affected.About 2 days to 2 weeks after the bite from an infected mosquito, diseasesymptoms are nonspecific summertime illness with a sore throat, runny nose,and cough, followed by fever, headache, nausea, and vomiting may develop.The neuroinvasive disease occurs in two-thirds of reported cases and ischaracterized by a severe headache and neck stiffness as in meningitis orincreasing lethargy and altered mental status up to coma as in meningoen-cephalitis. No specific therapy exists for arboviral infections; treatment islimited to supportive care and managing complications, such as relievingincreased intracranial pressure [136].74 Dengue Virus DiseaseEastern equine encephalitis virusEastern equine encephalitis (EEE) is a rare illness in humans, and only a fewcases are reported in the United States each year. It is among the most seriousof a group of mosquito-borne arboviruses that can affect the central nervoussystem and cause severe complications and even death. EEE is found infreshwater hardwood swampland in the Atlantic and Gulf Coast states in theeastern part of North America, Central and South America, and the Caribbean.It has a complex life cycle involving birds and a specific type of mosquitoesincluding several Culex species and Culiseta melanura [137].Symptoms can range from none at all to a mild flu-like illness fever,headache, and sore throat. More serious infections of the central nervoussystem lead to a sudden fever and severe headache followed quickly by sei-zures and coma. About half of these patients die from the disease. Of thosewho survive, many suffer permanent brain damage and require lifetimeinstitutional care. There is no specific treatment. A vaccine is available forhorses, but not humans [51,52,138].Zika virus diseaseZika virus is a mosquito-borne flavivirus, which was first isolated in Africa in1947 [139]. The first documented outbreak among people occurred in 2007in the Federated States of Micronesia [140]. An outbreak started in Brazil in2015, and spread to the Americas, Pacific, Asia, and Africa [141]. This led tothe World Health Organization declaring it a Public Health Emergency ofInternational Concern in February 2016 [141]. The emergency was lifted inNovember 2016, but 84 countries still reported cases as of March 2017 [142].The most common signs and symptoms of zika fever are fever, rash,conjunctivitis, muscle and joint pain, and headache [143]. The time from amosquito bite to developing symptoms is a few days to a week [144]. Thedisease lasts for several days to a week and is usually mild enough that peopledo not have to go to a hospital [145].Zika virus can be identified by reverse transcriptase PCR (RT-PCR) inacutely ill patients. However, the period of viremia can be short [146] and theWorld Health Organization (WHO) recommends RT-PCR testing be done onserum collected within 1e3 days of symptom onset or on saliva samplescollected during the first 3e5 days [147]. Later on, serology for the detectionof specific IgM and IgG antibodies to Zika virus can be used. IgM antibodiescan be detectable within 3 days of the onset of illness [148].There is currently no specific treatment for zika virus infection. Care issupportive with treatment of pain, fever, and itching [147]. Zika virus had beenrelatively little studied until the major outbreak in 2015, and no specificantiviral treatments are available as yet [145]. 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MMWR (Morb Mortal Wkly Rep)2016;65(2):30e3.Mosquito-borne diseases Chapter | 5 83Chapter 6Viral genetics and structureIqra Naveed AkhtarClinical Research Fellow, Department of Neurology, University of Missouri, Columbia, MO,United States; Zeenat Qureshi Stroke Institute, St.Cloud, MN, United StatesGenomic organization of the Dengue virus: anintroductionThe viral genome of the Dengue virus is a linear, single-stranded, positivesense RNA molecule (z11 kb) translated as a single open reading frame(ORF), bordered by 50 and 30 untranslated regions (UTRs) on each side [1][summarized in Fig. 6.1]. Through the combination of cryoelectron micro-scopy, X-ray crystallography, and imaging reconstruction, the structure of theDengue virus particle was deciphered as a spherical 50 nm virion [2]. Multiplecopies of the capsid (C) protein encapsulates the ssRNA genome, creating theFIGURE 6.1 An Illustration of the Dengue Viral Genome. Brief functions of each Structuraland Nonstructural protein of the Dengue Virus are mentioned in the outlined box below theprotein. Illustration adapted from two combined resources: Guzman, M. G et al. Dengue: Acontinuing global threat. Nature Reviews Microbiology (2010) and Khetarpal N et al. DengueFever: Causes, Complications, and Vaccine Strategies. J Immunol Res. (2016)Dengue Virus Disease. https://doi.org/10.1016/B978-0-12-818270-3.00006-0Copyright © 2020 Elsevier Inc. All rights reserved. 85https://doi.org/10.1016/B978-0-12-818270-3.00006-0nucleocapsid, which forms the electron-dense core of the Dengue virus par-ticle. The core is further covered by a lipid bilayer into which two trans-membrane proteins are positioned, forming an outer glycoprotein protectivecasing [3]. This protective armor is composed of 180 copies of envelope(E) and membrane (prM/M) proteins, with varying configuration depending onthe stage of maturity the virus has met [3]. Upon entering the cell and releasefrom the capsid, the positive ssRNA genome is translated to a polyprotein of3400 amino acids in length, which is then subsequently cleaved by viralencoded proteases and host proteases to yield 10 proteins: three structuralproteins (C, E, prM) and seven nonstructural (NS) proteins, labeled as NS1,NS2A, NS2B, NS3, NS4A, NS4B, and NS5) [1]. The structural proteins arevital in virion assembly, release, maturation, and infectivity; while thenonstructural proteins play a major role in viral replication and in eluding thehost’s immune response. There are four main serotypes of the Dengue virus(numbered 1e4), each sharing approximately 60%e70% amino acid sequencehomology, with a further subdivision of serotypes into genotypes where thesimilarities begin to wane (at 3%) [4]. While these serotypes can causewavering presentations in the host, ranging from mild symptoms to life-threatening Dengue hemorrhagic fever, a common feature among all ofthem, and among all the flaviviruses are the production of these 10 matureproteins, all of which have a unique role to play in the life cycle of Denguevirus.Life cycle of Dengue virus: A brief overviewDengue virus is transmitted by the Aedes mosquito (Aedes aegypti or Aedesalbopictus) and introduced into the host by the infected vector during its bloodmeal [4]. Flavivirus adheres to target cells, predominately monocytes, mac-rophages, and dendritic cells, by the envelope protein (E), after which inter-nalization of the virus occurs through clathrin-dependent receptor-mediatedendocytosis into endosomes [4]. The acidic environment within the endosomethen causes structural rearrangement of the E protein, a necessary featresulting in fusion between the viral and host cellular membranes. Ubiquiti-nation of the capsid ultimately releases the viral genome into the cytosol [5].The viral RNA serves as the mRNA template for translation (earlyresponse) into a precursor polypeptide, which is then subsequently co- andposttranslated by host and viral proteases into structural and nonstructuralproteins. After translation into protein components, an RNA-dependent-RNApolymerase (RdRp) derived from the NS5 protein synthesizes a complemen-tary minus-strand from the positive-strand RNA that makes up the Dengueviral genome [6]. This minus strand then serves as a template for synthesis of anew positive strand that will be used as the Dengue virus genome for futurevirions. This viral replication process occurs in the cytoplasm. Immature,noninfectious virions then assemble within the endoplasmic reticulum, with86 Dengue Virus Diseasethe newly synthesized RNA bound with the capsid protein, forming thenucleocapsid. The virion will then bud off the membrane of the endoplasmicreticulum into the lumen, acquiring the E protein and the prM/M proteins en-route through the lipid membranes [illustrated in Fig. 6.3]. In the trans-Golginetwork, the prM will be cleaved by furin, catapulting the release of themature infectious virions from the cell via exocytosis [6].FIGURE 6.2 Dengue Life Cycle: An Overview. Dengue virus E protein binds to the target cell(2), initiating receptor-mediated endocytosis (3) of the Dengue virus into endosomes (4). Theacidic environment in the endosomes causes uncoating of the virion, with the release of thenucleocapsid (NC) into the cytosol. The viral RNA undergoes an early response, in which aprecursor polypeptide is made and cleaved into structural and nonstructural proteins (5). An RNA-dependent RNA polymerase derived from the NS5 protein (nonstructural) initiates viral replicationin the cytosol (6). This newly synthesized RNAwill then bind to capsid protein in the endoplasmicreticulum, forming a nucleocapsid (7). Assembly of the virion will occur within the roughendoplasmic reticulum (8). The virion then buds off the membrane of the endoplasmic reticulum,acquiring the E protein and prM/M proteins and makes its way to the trans-Golgi network. Theacidic environment of the trans-Golgi network will cause a change in structure, yielding a matureDengue virion (9). Furin cleavage of the prM from the E protein will allow for the release of themature Dengue virion into the extracellular space, allowing them to infect new cells (10). Illus-tration from Screaton, Gavin et al. New insights into the immunopathology and control of denguevirus infection. Nature Reviews Immunology (2015).Viral genetics and structure Chapter | 6 87The role of structural proteinsDengue viral capsid protein (C)Capsid structureThe C protein is the first polypeptide synthesized during RNA translation ofthe Dengue virus: a highly basic moiety (rich in arginine and lysine) of 12 kDa[6]. The basic nature of this 100-residue protein (with 26 basic amino acidscompared to only three acidic amino acids) is thought to be the reason that itcan cooperate so readily with virion RNA to form a nucleocapsid, the core ofthe mature Dengue virus particle [6]. Newer nuclear magnetic resonancetechniques have allowed detailed stereographic visualization of the Dengueviral capsid protein. This homodimer shows that the basic residues amasspredominately on one end of the protein, with an apolar surface on the otherend. Each monomer has four a helices. The N-terminal domain, lacking anN-terminal hydrophobic signal sequence, is composed highly of basic aminoacids, and shows flexibility, and a more disordered structure, especially insolution [7]. This unveils the fact that although the C protein is conservedfairly well structurally among the flaviviruses, it is also yields the mostFIGURE 6.3 Dengue Viral Assembly: A portrayal. An anchor binds both the C protein and theprM protein to the membrane of the endoplasmic reticulum. The C protein anchor junction is firstcleaved by the NS3 protein, which is a serine protease, using NS2b as a cofactor. Occurring rightafter this cleavage is a second cleavage, at the prM-anchor site performed by a host-encodedsignalase. Once the C protein is released from the anchor, it is considered a mature capsid pro-tein, and readily available to bind to dengue viral RNA, forming the nucleocapsid. The Denguevirion buds off the membrane of the endoplasmic reticulum into the lumen acquiring the E proteinand the prM/M proteins in the process.NS2B-3: Nonstructural protein 2B-3; prM: pre-membrane;M: membrane; E: envelope; C: capsid; ER: endoplasmic reticulum. Illustration adapted from BykLA et al. Properties and functions of the dengue virus capsid protein. Annu Rev Virol. (2016)88 Dengue Virus Diseasestructural variability among the structural proteins, owing to this uniqueN-terminal region of the protein.The a-helices are situated in the homodimer in the following manner: a2and a4 of one monomer are antiparallel to the a2 and a4 of the othermonomer, forming the crux of the dimer surface [illustrated in Fig. 6.4]. Thecore of the monomer is formed by the a1, a2, and a3 helices, while the longesthelix, the a4, is predominately basic in amino acids, with the a4-a40 inter-action among the monomers vital for dimer formation, production of infec-tious Dengue virus particles, and protein stability [7,8]. In research studyingthe specifics of the Dengue virus serotype 4 (DENV4) spearheaded in 1997,researchers found a hydrophobic sequence of residues (from 45 to 65 posi-tions). They concluded that this hydrophobic region was highly conservedamong flaviviruses and is responsible for the anchoring of the mature Denguevirus capsid protein to the membrane of the endoplasmic reticulum, thecytoplasmic side serving as the site for viral replication [9]. The matureDengue virus capsid has a distinctive duality to it: it can bind to both the viralRNA and the lipid membranes (such as the membrane of the endoplasmicFIGURE 6.4 The alpha-helical structure of the Dengue virus. a1: blue, a2: green, a3: yellow,a4: red. Illustration by Ma Lixin et al. Solution structure of Dengue virus capsid protein revealsanother fold. PNAS (2004). Copyright (2004) National Academy of Sciences, U.S.A.Viral genetics and structure Chapter | 6 89reticulum). This duality can be traced back to the a-helical structure of thehomodimer, with the highly basic a4-a40 interaction interacting with the viralRNA, and the uncharged, apolar a2-a20 region making up the hydrophobicportion of the capsid, interacting with the membrane of the endoplasmic re-ticulum [7]. The basic residues composing the N-terminal region are alsofound to play a part in RNA binding to the capsid protein as well as maturevirus particle production.Chaperoning role of the capsidThe capsid proteins of Dengue virus can serve as chaperones, safeguarding theviral RNA to which they are bound. Studies investigating the West Nile Viruscapsid in vitro are used as a model for this, due to the fact that the Denguevirus capsid aggregates when bound to viral RNA, possibly due to the fact thatthe positive-sense RNA causes aggregation of the hydrophobic zones of theDengue virus capsid protein [10]. The capsid protein can prevent misfolding ofthe viral RNA or even correct misfolded RNA all the while without utilizingATP. The chaperone activity lies in the key basic domains of the C protein,those of which were shown previously to be highly flexible [11].Maturation of the capsid proteinThe capsid protein is transiently bound to the membrane of the endoplasmicreticulum on the cytoplasmic side by an “anchor”, a hydrophobic signalsequence that spans the membrane, which also binds to the premembrane(prM) protein, a protein that is translated just following the capsid protein, onthe luminal side of the endoplasmic reticulum membrane [6]. Cleavage of thecapsid protein from the anchor site is a requisite to the maturation of the capsidprotein. This cleavage at the capsid-anchor site is accomplished by way of aviral encoded protease, NS3 (using NS2B as a cofactor). At the prM-anchorjunction in the lumen of the endoplasmic reticulum, a separate host signalpeptidase (“signalase”) separates the prM and the anchor from each other(illustrated in Fig. 6.2). It is important that this two-step cleavage at both sites,occurs in this order: with cleavage of the capsid from the anchor occurringfirst, followed by the cleavage of the prM from the anchor [12,13]. Studieshave shown in other flaviviruses, when the order of the sequential cleavage isaltered, it can lead to particles being released extracellularly that had no in-fectious nucleocapsid, while retaining the E and prM proteins on the surface,rendering the “empty” particles [14]. Only when the capsid protein is cleavedfrom the anchor, is the viral capsid considered mature and ready to bind toviral RNA.Capsid binding to RNA: nucleocapsid formationAssembly of Dengue virus within cells starts with the cardinal step of thenucleocapsid formation. After cleavage by the viral protease NS3, the mature90 Dengue Virus Diseasecapsid protein is then able to bind to viral RNA especially at the a4-a40 helicalsite on the capsid protein. Viral replication occurs in isolated “vesiclepackets”, structures derived from endoplasmic reticulum outpouchings andsurrounded by membrane. Upon completion of replication, the newly syn-thesized RNA is then extruded from an opening in the vesicle packets into thecytoplasm, where it can bind to the mature capsid protein [15]. Encapsidationby the viral capsid is mediated by viral encoded signals, of which are poorlyunderstood, and may be a result of immeasurable factors in the noncodingregions of the Dengue viral genome. A proposed prototype showing thetransportation of the viral genome to the site of nucleocapsid assembly at themembranes of the endoplasmic reticulum, also illustrates the “buds” that formoff the endoplasmic reticulum membrane, acquiring both the E and prMproteins in the process [15e17]. The replication of the RNA genome in thesereplication pockets (or vesicle packets) is a prerequisite for encapsidation later,as it has been shown that for RNA replicated out of these pockets, encapsi-dation does not occur [18].The budding of the Dengue virus nucleocapsid and the acquiring of the Eand prM proteins is proposed to both be independent processes, as emptyviruses containing surface E and prM but without the nucleocapsid can beproduced and extruded from the cell, if the E and prM proteins are overex-pressed artificially [19]. The NS3 protease that is responsible for capsidmaturation (by cleaving the capsid protein from the anchor) may play a role innucleocapsid incorporation into buds off the endoplasmic reticulum membranetoward the lumen. This highlights the fact that not only is the NS3 (along withNS2B) required for capsid maturation, but that it may also be significant fornucleocapsid recruitment and viral assembly. However, it is still not knownhow changes in NS2B or NS3 impair particle formation, whether it is due tobinding to the C protein, viral RNA, or other proteins or membranes in the cell.Other nonstructural proteins may also play noteworthy roles in viral assembly,with recent studies pointing toward NS2A and NS1, showing that mutations inthese can lead to abhorrent host protein interactions and fewer infectiousparticles released [20]. Nucleocapsid assembly and the acquiring of the E andprM proteins is a complex process, one that is still not fully uncovered,and may be a result of the interplay between many parts of the viral genomeand host proteins, warranting further studies.Mutations or deletions in the capsidThe complete Dengue viral capsid-coding RNA sequence was reported by awidespread analysis using biochemical probes [21]. The first 160 of the 300total nucleotides that make up the RNA encoding the capsid, were conservedRNA structures, shared among other viruses in its genome. These cis-actingRNA functional units include: (A) RNA cyclization signal (50CS) vitalfor RNA synthesis, (B) a hairpin loop (cHP) also essential for RNA synthesis,Viral genetics and structure Chapter | 6 91and (C) a pseudoknot included in the region C1 which augments cyclization ofthe genome due to its sequence being complementary to a region in the 30 endof the genome, thus also increasing RNA synthesis [21e23].Understanding that there are highlyconserved sequences in the capsidcoding RNA sequence, allows for mutational studies to segregate theseconserved cis-sequences from purely capsid-coding sequences, to assess thefunction of various areas of the capsid RNA sequence [24,25]. Alterations inspecific portions of the viral capsid sequence were of significance, while othersshowed no effect on viral RNA synthesis and nucleocapsid assembly. De-letions in the highly basic N-terminal region of the capsid were shown tosignificantly alter the formation of Dengue viral particles, indicating that theyultimately affect viral assembly. A mutational analysis of this region indicatedthat at least two positive charges in each of the two main basic clusters in thisregion (R5K6K7R9 and K17R18R20R22) are necessary, showing that positivecharges might play a larger role in viral assembly rather than just the place-ment of residues in specific positions [24].In the a-helices of the Dengue virus capsid, mutations yielded a wide arrayof results. Mutations in the a1 and the a1-a2 connecting bend showed noeffect on Dengue viral assembly and propagation, while those in the morehydrophobic regions such as the a2 helix showed marked suppression ofDengue virus propagation [25]. This importance of the hydrophobic region ofthe a2 helix is a prevailing feature in other flaviviruses, suggesting that eventhough the capsid structure varies among them, any alterations in the hydro-phobic sequences can severely affect nucleocapsid assembly and viral prop-agation [26e29]. This phenomenon is further reiterated by studies thatdemonstrated that large deletions or mutations affecting as many as 36 nu-cleotides was better stomached than smaller mutations/deletions in the Nterminal hydrophobic coding sequence [30].Capsid localizationThe Dengue viral capsid is at an ideal location being associated with themembranes of the endoplasmic reticulum: in close vicinity to (A) the vesiclepacket exit sites that release newly synthesized viral RNA, and (B) near thebudding particles sites that are made by the protrusion of the membrane of theendoplasmic reticulum [15e17,31,32]. While large mutations or deletionsinvolving the capsid protein structure have a negligible effect on viral prop-agation (in previous section), newer studies have shown that the mutationsaffecting the localization of the capsid protein are much more consequential,significantly altering viral propagation [25]. There are two main locationswhere the Dengue viral capsid can be found in infected cells: in the cytoplasmand in the nucleus. In the nucleus, the Dengue viral capsid is found specificallyin the nucleolus, while in the cytoplasm it is concentrated both in the mem-branes of the endoplasmic reticulum and lipid droplet surfaces [6].92 Dengue Virus DiseaseLipid dropletsLipid droplets are organelles derived from the ER, with a core of neutral lipidsand an outer layer of phospholipids and protein [33]. In a study investigatingthe subcellular localization of cytoplasmic C protein, BHK cells were infectedwith the Dengue virus serotype 2. Visualization of the dengue virus particlesafter fixation by paraformaldehyde and Triton X-100 showed that the C proteingathered in spherical shapes, with the C protein in a ring-like pattern [25]. ERand Golgi markers showed that it was not accumulated in these regions. Thering-like structure of the C protein resembled the structure of hepatitis C(HCV) which is known to accumulate on the surface of the lipid droplets[34e36]. This led researchers to believe that like HCV, the Dengue viruscapsid accumulation on the surface of lipid droplets plays a role in patho-genesis. Dengue viral infections were shown to also increase the size andquantity of lipid droplets in infected cells (as much as a 3-fold increase) [25].Cytoplasmic C protein is directed to lipid droplets by particular amino acids(spanning residues 46e66) on the hydrophobic a2 helix [26]. In the study,substitution of two specific hydrophobic residues (L50S and L54S) in the a2helix resulted in a C protein that although was distributed in the cytoplasm, didnot accumulate on the lipid droplet surfaces, highlighting that they are key toC protein-lipid droplet association [25].Although viral translation, RNA replication, and encapsidation all occur inthe cytoplasm of infected cells, the understanding of the coordination betweenthe three processes is still murky. Artificial substitutions in L50S or L54Saltered C protein targeting to the LDs, which resulted in normal RNA repli-cation, but faulty production of an infectious particle, suggesting that viralassembly is affected [25]. The E protein, responsible for infectivity, was foundto be reduced in these altered cells. When both L50S and L54S were mutated(a double mutant), also impaired infectious viral particle production, but alsoimpaired amplification of viral RNA. This demonstrates that the when the Cprotein accumulates in the cytoplasm instead of being sequestered in the lipiddroplets, it may prematurely interact with viral RNA, halting amplification ofthe viral RNA. A proposed biological function of the lipid droplet is that it actsas temporary storage site, sequestering the C protein, thereby controlling theamount of C protein available to interact with the viral RNA [37]. The un-derstanding of the mechanism employed by the C protein to recruit viral RNAto form the nucleocapsid, as well as the location in which this occurs, is stilluncertain. Two mechanisms have been proposed: (1) C protein is stored in thelipid droplet early in infection, and then deployed to the membrane of theendoplasmic reticulum where it will assemble with viral RNA to form nu-cleocapsids or (2) Nucleocapsids are produced at the site of C protein on thesurface of the lipid droplets, and is then deployed to the membrane of theendoplasmic reticulum for further infectious viral assembly [25,33]. Never-theless, localization of the C protein to the LDs has shown to be an integralViral genetics and structure Chapter | 6 93part in infectious viral production, and thus can be a target for future eradi-cation strategies for the Dengue virus. A study has shown that when a fattyacid synthase inhibitor was given, the amount of lipid droplets were reduced,leading to a substantial reduction in infectious viral particles produced [25].Capsid localization in the nucleusIn the nucleus, the C protein is primarily localized within nucleolus [38e40].The role of the C protein in the nucleus is poorly understood, as its mainfunction in nucleocapsid formation and virion assembly takes place in thecytoplasm. Recent studies have shown that within the nucleus of the infectedcell, the C protein interacts with mostly nuclear host proteins. One suchprotein, the cellular protein nucleolin, is found in a larger extent in thenucleolus, with smaller amounts in the cytoplasm and plasma membrane.Nucleolin has a role in many cellular processes, including ribosome synthesis,protein transport, chromatin remodeling, control of translation, and RNAprocessing [41e44]. A study from 2013 demonstrated that the C protein-nucleolin interaction plays a significant role in formation of infectious viralparticles (much like the lipid droplets). Researchers manipulated interactionsbetween nucleolin and the C-protein, either through siRNA silencing ofnucleolin, or with a nucleolin binding aptamer (AS1411), resulting in bluntedinteractions between the two. What was observed was a discernible reductionin viral titers, with no change in the intracellular Dengue viral RNA, C protein,or the E protein, indicating that viral RNA replication and synthesis of viralproteins were unaffected [45]. The C-protein-nucleolin interaction plays alarger role in virion morphogenesis, perhaps by the nucleolin protein acting asa chaperone for the C protein, aiding the formation of stable nucleocapsidparticles. Other proteins that the C protein can interact withinthe nucleolus areDAXX and core histones. Binding of the C protein to DAXX was proposed toinduce apoptosis via Fas, whereas binding to the core histones was proposed todisturb transcription by affecting the assembling of nucleosomes [46].Premembrane/membrane protein (prM/M) and envelope protein (E)The precursor membrane-envelope protein complexThe premembrane (prM) and the envelope (E) protein are acquired by thevirion during budding off from the ER after nucleocapsid formation and alongwith lipids, forms the glycoprotein shell of the dengue viral particle. The Eprotein is the primary protein that is exposed to the immune system, to whichantibodies are produced against during a Dengue viral infection. The E proteinconsists of an ectodomain (soluble E protein), a stem, and a transmembranedomain [47]. Both proteins have varying conformations depending on thematurity of the Dengue virus particle. In an immature Dengue virus particle,the prM and E together give a “spiky” appearance from the particle surface,94 Dengue Virus Diseasemade by 60 trimeric projections from 90 heterodimers [3]. During the tran-sition through the Trans-Golgi Network, the spiky immature structure un-dergoes a conformational change into a mature, “smooth” appearance, ametamorphosis initiated by a low pH (5.8e6) [48e50] [illustrated in Fig. 6.5].The E protein has a natural flexibility that allows the Dengue viral particle toundergo many conformational changes with changes in the environment.Not only do the EDI-III regions differ in structure, they also differ greatlyin procuring an immune response. The EDIII region generates robustneutralizing antibodies, which are usually serotype specific [51]. Serotype-specific antibodies are also generated against the EDI/EDII hinge zones andother complex epitopes on the E protein dimer [52]. These serotype-specificneutralizing antibodies are superior in preventing infection compared tocross-reactive nonneutralizing antibodies, which have been linked to causingantibody dependent enhancement (ADE) which leads to a more severe sec-ondary infection with a different Dengue virus serotype [53]. The EDI andEDII regions also elicit antibodies, as does the prM protein, but these anti-bodies are weaker, nonneutralizing and frequently cross-reacting (especiallyanti prM antibodies). When the mature virus infects a new cell throughclathrin-dependent receptor-mediated endocytosis, the E protein will undergoreconstruction to a trimer configuration in the endosome, and will protrudefrom the virus surface, allowing membrane fusion, allowing uncoating of thevirion which will enable the release of viral RNA into the cytoplasm forsubsequent translation. Therefore, the E protein can therefore undergoswitching between three different oligomeric states: prM-E heterodimers in theimmature Dengue virus particle, a dimer in the mature Dengue virus particle,and a trimer when the Dengue virus particle fuses with a host cell [47][illustrated in Fig. 6.5].“SPIKY” immature virusIn the immature Dengue virus, the E and prM proteins together give a spikyappearance to the surface. This is made of 60 trimeric projections and 90heterodimers. The E protein is capable of transforming from this immatureconfiguration to a mature one due to its three domains that allow flexibility:EI: the central-most region of the E protein. It consists of an 8-strandedb-barrel. Is responsible for maintaining the structure of the E proteinEII: EDII is dimer with 12 b strands, two a helices, and a fusion loop (FL).EII forms a hinge motion with EI.EIII: contains an immunoglobulin-like domain with 10 b-strands andfunctions in receptor binding to target cells. EIII also allows a hinge motionwith its junction with EI. Also EIII generates the most robust antibodies.The prM protein is unaffected by changes in pH, unlike the E protein. Afterthe endoplasmic reticulum, within the trans-Golgi network the prM proteinViral genetics and structure Chapter | 6 95FIGURE 6.5 Conformational changes in the Dengue viral particle prM and E proteins. prM,premembrane; M, membrane; E, envelope; EI, Envelope domain one; EII, Envelope domain two;EIII, Envelope domain three; FL, Fusion loop; ER, Endoplasmic reticulum; RNA, Ribonucleicacid. Illustration redrawn and adapted from Khetarpal N, Khanna I. Dengue Fever: Causes,Complications, and Vaccine Strategies. J Immunol Res. (2016).96 Dengue Virus Diseasewill be cleaved by furin which will yield a pr portion and an M portion. Aftercleavage, the mature virion with the E and the M proteins is excretedextracellularly.“SMOOTH” immature virusDuring the transition of the Dengue virus through the trans-Golgi network,there is a conformational change from a spiky appearance to a smoothappearance, a process initiated by the low pH within the trans-Golgi network.The M and E complexes are now lying flat along the surface, as 90 homo-dimers. In the E homodimer, the monomers are arranged facing each other. Ifthe pH rises at any point after this, the Dengue virion can once again switch toa spiky configuration, due to the flexibility of the E protein domains.In contrast to the E protein, the tertiary structure of prM is unaffected bychanges in pH. The prM protein consists of 166 amino acids, with sevenantiparallel b-strands all stabilized with three disulfide bonds. While in thetrans-Golgi network, the prM protein is cleaved by a host-encoded proteasecalled furin that cleaves the prM protein into an N-terminal 91 amino acid “pr”peptide and an M protein. After cleavage, the “pr” peptide remains bound tothe E protein forming a “cap,” covering the hydrophobic fusion loops of all 3 Eproteins, making the immature particle more hydrophilic. The pr peptideforming a cap on the E protein protects the fusion peptide on E from causingpremature fusion of membranes, prior to mature virion release [50]. Followingfurin cleavage, the mature virion consisting of E and M are secreted extra-cellularly, and the pr peptide dissociates from the mature particle. Virionmaturation is irreversible; after maturation the virion cannot revert back to itsimmature form, independently of pH. The average area of contact between thepr peptide and the E protein in an immature virus at a neutral pH was shown tobe larger than the area of contact between the two in an immature virus at alower pH (such as within the trans-Golgi network) [47]. This indicates that thepr protein has a greater affinity toward the E protein spike of an immature viruscompared to the smooth surface of the mature virus. This reduction in affinitymakes it easier for the pr protein to be released from the mature E structure at aneutral pH after cleavage by furin [47].Role of nonstructural proteins in viral replication andimmune evasionThe NS proteins are synthesized after the structural proteins, following the Eprotein in the following order: NS1, NS2a, NS3, NS4a, NS4b, and NS5.NS1 is the first NS protein to be synthesized. A 45kDA glycoprotein, it ishighly conserved region, the structure differing only minutely among theflaviviruses. It contains two signals of Asn-X-Ser/Thr (where X is any aminoacid) that is used for the insertion of an N-linked oligosaccharide [54]. It isViral genetics and structure Chapter | 6 97synthesized initially as a hydrophilic monomeric glycoprotein on the roughendoplasmic reticulum, then shortly after switches to a homodimer that ismore hydrophobic. Following dimer formation, the NS1 protein is thentransferred to the lumen of the endoplasmic reticulum, followed by the Golgiapparatus, where its N-linked oligosaccharides are modified. This modificationby the Golgi apparatus makes NS1 able to be transported to the cell mem-brane, remain intracellular, or secreted from the cell, although secretion of theNS1 is limited to mammalian cells and not seen in mosquitos [55]. It isinvolved in viral RNA replication, although the exact mechanism of howitdoes this is still unclear. Its role in viral defense is better understood, as itinhibits complement activation, thereby successfully evading cytolysis ofinfected cells [56].NS2A/B are the next synthesized NS proteins. Unlike NS1 which is mostlysecreted from the cell, both NS2A and NS2B remain predominately astransmembrane proteins. NS2A is 22 kDa primarily hydrophobic protein thatis essential for proteolytic processing of the C terminus of the NS1 protein andis necessary for viral production [57]. Host signalases in the lumen of theendoplasmic reticulum cleave the transmembrane junction of NS2B-NS3,which causes the NS2A protein to split into two forms: NS2A and NS2Aa,both of which are necessary for proper viral replication and assembly. NS2B,after cleavage by host signalases, lies in the cytoplasm and is an essentialcofactor for NS3, which is a viral serine protease, an RNA helicase, and anRTPase/NTPase [3]. When examining the Dengue viral two serotype, it wasshown that residues of NS2B (40e80) are included in the NS3 protein, whichwere linked via a Gly-Ser linker to the N terminus of the full NS3 protein [3].It was later discovered that the residues 67e80 specifically were crucial for theprotease activity of the NS3 protein, due to NS2B encasing the proteasedomain of NS3, forming a fundamental portion of the protease active site ofNS3 [58]. Hydrophobic parts of the NS2B protein also interact with NS3, suchas the hydrophobic flanking regions that can anchor the NS2B-NS3 complex tothe ER membranes [59]. This places the NS2B/NS3 protease complex in closeproximity to the transmembrane portions of the polypeptide they are taskedwith cleaving: The C-protein from the transmembrane anchor on the cyto-plasmic side of the endoplasmic reticulum.NS3 is a large, hydrophilic protein with a molecular weight of approxi-mately 70,000 [54]. As stated previously, the NS3 complexes with the NS2B atthe N-terminal region which functions as a serine protease which acts to cleavethe polyprotein at the C protein-anchor site. The C-terminal site of NS3 re-sembles nucleoside-triphosphate binding protein sequences, therefore, playinga role in Dengue viral RNA replication. The NS3 protein is one of the mostimportant proteins involved in Dengue pathogenesis, with three main func-tions: (1) Protease activity which cleaves the viral polyprotein, (2) RNAhelicase activity and RTPase/NTPase-viral RNA replication, and (3) inducesapoptosis in virally infected Dengue cells [31]. The helicase portion of the98 Dengue Virus DiseaseNS3 protein (residues 180e618), has three subdomains very similar instructure to others flaviviruses. The subdomains I and II (residues 181e326,residues 327e481, respectively), has six parallel b-pleated sheets, with foura-helices on the ends of the sheets, a configuration very similar to the hepatitisC virus (HCV) helix structure [60,61]. The last subdomain, subdomain III(residues 482e618) is more unique in dengue virus, composed of four parallela-helices, enclosed by three a-helices and two antiparallel b strands. Thehelicase activity of the NS3 protein is thought to be influenced by the proteaseactivity of NS3, as helicase activity was found to be significantly enhanced(about 30-fold) in the full-length NS3 protein compared to the solitary helicasedomain of the NS3 protein [62]. This suggests that the protease domain islinked to the helicase domain via a linker molecule that allows the proteasedomain to influence and augment helicase activity. The NS3 helicase domainis also suggested to interact with the NS5 protein, which is the RNA poly-merase responsible for viral RNA replication [63]. The NS3 protein also playsa role in inducing apoptosis, and recently has shown to also play a significantrole in viral assembly that takes place after viral RNA replication [64,65].Overall, the NS3 protein is a multifaceted, involved in multiple processeswithin the infected cell, thereby playing a vital role in viral morphogenesis.NS4A and NS4B, 16 kDa and 27 kDA, respectively, are membrane pro-teins, and are responsible for inducing membrane alterations necessary for thereplicative process of the Dengue virus. The NS4A protein predominatelyinduces membrane alterations and enhances autophagy (destruction of cellularcomponents in vacuoles), both of which augment viral RNA replication[66,67]. NS4B assists in viral replication by its interaction with the NS3protein, a protein vital for viral replication via helicase activity [68]. NS4Balso plays a role in immune evasion, by blocking IFN a/b signal transductionvia the JAK/STAT pathway, which allows the virus to circumvent the host’sinnate immune response to viral infections [69].The last NS protein, NS5, is the largest of the NS proteins, with a mo-lecular weight of 104,000 and composed of 900 amino acid residues. It is alsothe most conserved of the NS proteins among the Dengue serotypes, with a67% sequence homology among the four serotypes [3]. Residues 1e296 at theN-terminus make up the methyltransferase domain of the protein, while the C-terminus consists of an RNA-dependent RNA-polymerase (RdRp), which iscomposed of residues 320e900. The RdRP domain is similar in structureamong the flaviviruses, with a canonical conformation with palm, fingers, andthumb subdomains [70]. It contains a GDD motif, common among flavivi-ruses, which is responsible for catalytic activity responsible for adding nu-cleotides using metal ions. “Finger tips” connect the finger and thumb domainsto form an active site, a feature that differentiates the RdRps from DNA-dependent RNA polymerases [3]. Between residues 320e405, RdRps have anuclear localization signal (NLS), as NS5 protein is predominately localizedwithin the nucleus in Dengue viral infections, a feature not shared byViral genetics and structure Chapter | 6 99flaviviruses [71,72]. Residues 320e368 specifically are suggested to play a rolein the interaction of the NS5 protein with the NS3 protein (the helicase domain)[63]. The localization of the NS5 protein within the nucleus is an oddity, as itsmain function (RdRp) is required for viral replication within the cytoplasm.The reasoning behind this is unclear, but suggests that in addition to itsenzymatic activities, the NS5 protein perhaps interacts with host proteins tomodulate viral morphogenesis [73]. The methyltransferase domain at theN-terminal end of the NS5 protein is composed of a core consisting of S-adenosyl-methionine-dependent MTase (SAM-dependent MTAse), that is fol-ded into an a/b/b structure between the N and C terminal regions of NS5 [3].The methyltransferase activity of this region is responsible for the synthesis ofthe methyl-guanosine cap at the 50 end of the newly synthesized viral RNAduring viral replication. The SAM-binding site of the NS5 region has shown tohave a more open configuration, when compared to the West Nile Virus[74,75]. This increase openness suggests that the byproduct of methylation, theS-adenosyl-homocysteine (SAH) is more readily released from the cell.Cis-acting RNA structures that influence viral replicationThe highly structured 50 and 30 untranslated regions (UTRs) as well as 50 and 30coding regions contain RNA elements that are responsible for the initiation oftranslation and can act to enhance, suppress, or mediate replication of theDengue viral genome.50 UTR and 50 coding regionsThe viral 50 UTR of the Dengue virus genome, around 100 nucleotides long, isvital for proper viral replication, with a cap-like structure on the 50 end that ishighly conserved among the flaviviruses [9]. The 50 UTR region contains threeessential regions for viral replication: (1) a promoter region called the stem-loop A (SLA) that binds and activates viral RNA polymerase [76], (2) stem-loop B (SLB), which has a sequence known as 50 upstream of the AUG(start codon) region (50 UAR) that is complementary to the 30 end of the viralgenome, responsiblefor the long RNAeRNA interactions causing cyclization,and lastly (3) A region rich in U nucleotides between SLA and SLB that actsas an enhancer of replication [77]. Additionally, there are regions in the 50coding region, especially in the coding sequence of the viral capsid protein,that play a significant role in mediating long range RNAeRNA interactions.These include: (1) a 50 cyclization sequence (50CS) and (2) a 50 downstream ofAUG region (50DAR) both of which are located in the capsid coding region,and (3) a hairpin located between the 50CS and the 50DAR called cHP [76].Cyclization of the flavivirus genome is proposed to be essential for viral RNAreplication, possible by complementary sequences present at the 50 and 30 endsof the genome (depicted in Fig. 6.6].100 Dengue Virus Disease30 UTR regionThe 30 UTR region is a 450-nucleotide-long region that lacks a poly-A tail.Instead, it ends in a highly conserved 30 stem loop (30SL), a structureconserved not only among different Dengue virus serotypes, but also amongthe flaviviruses [78]. This 30SL region is essential for viral replication. Up-stream of the 30SL is a region known as the conserved sequence (CS1), whichcontains a cyclization sequence (30CS). Both these regions of the 30UTR havecomplementary sequences to areas in the 50 UTR and 50 coding sequences,which allows for cyclization of the viral genome, bringing the 30 end closer tothe RNA polymerase at the promoter site of the 50 end [79]. Cyclization of theflavivirus genome is proposed to be essential for viral RNA replication. Thereis also a variable region (VR) downstream of the ORF. It has been suggestedthat this VR sequence may have a similar function to the poly-A tail, due tostudies that have shown similar efficiencies in translation between mRNAswith a VR sequence and mRNAs with poly-A tails [78]. Deletions in the VRFIGURE 6.6 Sequences located at the 50 and 30 ends of the Dengue Virus genome. SLA, stemloop A; SLB, stem loop B; UAR, upstream of the AUG region. Illustration from Villordo S M et al.Genome Cyclization as Strategy for Flavivirus RNA Replication. Virus Res (2017).Viral genetics and structure Chapter | 6 101region can lead to a reduction in viral replication in vivo [80]. In one studystudying the Dengue virus 2 serotype, it was shown that a 10-nucleotidedeletion was linked to dengue clinical presentation, resulting in a milderclinical picture [81]. Located just downstream to the VR region and just up-stream of the 30SL, are the A2 and A3 regions of the 30UTR. These regionscontain highly conserved sequences CS2 within the A3 region and repeatedCS2 (RCS2) within the A2. While deletions in either A2 or A3 had no sig-nificant effect on viral translation, concomitant deletions in both A2 and A3decreased viral translation by 30%e40%, suggesting A2-A3 may play a role instabilizing RNA. Individual deletions in either A2 or A3 while showing onlytrivial reductions in viral translation, showed large reductions in viral RNAsynthesis, an effect that was augmented by a deletion in both [82].Cyclization of the viral genomeCyclization of the viral genome is mediated by complementary sequences inthe 50 end of the viral genome (both 50 UTR and 50 coding regions) and the 30UTR. There are two main complementary sequences that hybridize requiredfor cyclization: (1) between the 30SL and the 50 UAR located in the 50 UTR and(2) between the 30CS to a region in the coding sequence for capsid protein inthe 5’ [21]. Mutational studies in these regions demonstrated that these in-teractions are essential for viral RNA synthesis, as there was a notablereduction in viral RNA synthesis (as much as 200-300-fold) when these areaswere mutated or deleted [77]. Single mutations that disturbed the 50-30 UARhybridization, were shown to drastically reduce synthesis of viral RNA, insome cases to subdetectable levels. Mutations that restored the base pairings inthese regions, were shown to “recover” the base pairings, and thus RNAsynthesis occurred normally [77]. Single nucleotide mutations in the 50 UARregion that did not affect the complementary sequences, were also shown tobetter tolerated, highlighting the importance of these complementary se-quences for effective viral replication. The 30SL region was suggested to alterin structure upon binding to the 50 UAR region. One such proposed mechanismsuggested that terminal nucleotides in the 30 end were structurally changed(“melted”), and that triggered the initiation of RNA polymerase activity[83e85]. Further studies are warranted to determine if the hybridization of the50UAR region and the 30SL region is essential in only bringing the 30 end inclose proximity to the polymerase at the 50 end, or if it is necessary for trig-gering a conformational change in the 30SL that initiates viral RNA poly-merase activity. Alterations in the SLB stem region of the 50 UTR notincluding the 50 UAR region were shown to have no effect of viral RNAsynthesis [77]. The stem of the SLB region was thought to assist in main-taining UAR complementarity, but the unaffected viral translation after mu-tations, suggest that the SLB stem can play a role in other parts of viralmorphogenesis, such as viral assembly.102 Dengue Virus DiseaseHost specification by RNA duplication regions in the 30UTRRNA structures that arise from duplications within the 30 UTR known asdumbbells (DB1 and DB2) have shown to play a role in host switching be-tween mosquito and human cells. These dumbbells contain sequences thatoverlap with the essential RNA sequences in the 30 UTR responsible forcyclization, and DB1 and DB2 are thought to have differing functions regu-lating genome cyclization [86]. The dumbbell regions were initially thought toenhance viral replication in both mosquitos and humans, but recent researchhas discovered that these regions greatly enhance or diminish viral replicationin mosquito cells only, while having a negligible effect on viral replication inhumans [87]. This demonstrates that these RNA structures in the 30 UTR areunder different pressures depending on the host infected by the Dengue virus.Not only are there two dumbbell elements (DB1 and DB2), there is also nearlyidentical stem-loop structures (SLI and SLII), elements not to be confusedwith the 30 stem loop (30SL) [86]. These pairs of duplicated RNA elements(SLI-SLII and DB1-DB2) interact with an area called the pseudoknot (PK),which is able to hinder degradation of the viral genome. In the mosquito,mutations in the DB2 sequence, enhanced viral replication. Sequences in theD1 region hybridize with a sequence in the 50 capsid coding sequence,enhancing genomic cyclization. The DBs have contrasting effects: DB2complexes with PK forming a DB2-PK complex that reduces long-rangeRNAeRNA interactions responsible for cyclization, and therefore reduceviral replication, while DB1 promoted RNAeRNA interactions and cycliza-tion [88]. This contrasting nature shows that mutations in DB1 or DB2 yielddifferent effects on viral RNA production. Complete deletions of DB2 resultedin an increase in viral replication in mosquito cells [86].Understanding these DB structures and their effect on viral replication ininfected mosquito cells, suggests that they can be targets for future therapies toreduce Dengue viral transmission to human hosts. Another structure, a smallhairpin-like structure (sHP) in the 30 UTR, lying just upstream form the ter-minal 30SL, is an essential region for Dengue viral replication in both hosts. Asequence in the loop of the sHP was found in recent studies to play a vital rolein replication, but only in mosquito cells, while being expendable in humanhosts [86].Subgenomic flavivirus RNAs (sfRNAs)Subgenomic flavivirus RNAs (sfRNAs) are derived from the 30 UTR and arecapable of blunting the host antiviral response (innate immunity) in bothmammalian cells as well as mosquito cells, increasing the epidemiologicalfitness of the Dengue virus [89]. During Dengue viral replication, a cellular 50-30 exoribonuclease (XRN1) “trims” the viral genomic RNA, digesting it untilthe conserved stem loop-II (SL-II) structure, which stalls exoribonucleaseactivity. SL-II is a structure in the 30 UTR that interacts with the pseudoknotViral genetics and structure Chapter | 6 103(PK) also in the 30UTR. This results in the production of a subgenomic fla-vivirus RNA (sfRNA) (z0.5 kb) [90]. XRN1 also stalls at SL-IV and DB1RNA structures in the 30UTR, forming a smaller sfRNA2 and sfRNA3 [90].One of the initial responses to a viral infection involves the production ofinterferons (IFNs), especially IFN-a and IFN-b, which are responsible for thecytolysis of Dengue virus infected cells. sfRNAs interact with host proteins,functioning to suppress translation of the IFN genes [91].The innate immune response against flaviviruses is initially triggered byretinoic acid-inducible gene-I-like receptors (RIG-I, MDA5) sensing viralRNA [90]. The binding of the RIG-I receptors to the viral RNA will prompttranscription of the interferon response factor (IRF) which will ultimately leadto the induction of interferon (IFN). A recent study exploring the functionalintricacies of sfRNAs demonstrated a binding of sfRNA from the Dengue virus2 serotype to the ubiquitin ligase tripartite motif protein 25 (TRIM25) [90].TRIM25 functions normally to polyubiquitynate the RIG-I receptor, which isessential for RIG-I signaling a vigorous response when encountering viralRNA. The strong binding of sfRNA to TRIM25 prevents the function ofTRIM25, preventing ubiquitination of RIG-I [90]. sfRNA also represses innateimmune responses indirectly. It was shown to interact with stress granules (inthe ER), especially components G3BP1/2 and Caprin, which subsequentlydecreased IFN-stimulated gene (ISG) translation [90].ER stress and the unfolded protein response (UPR):implications in viral pathogenesisDengue virus utilizes the host machinery for the synthesis of viral proteins thatcontribute to its pathogenicity. Dengue virus infections cause significant stressin the endoplasmic reticulum which triggers a cellular reaction in infectedcells called the unfolded protein response, which is activated to relieve stressin the endoplasmic reticulum [92]. In Dengue virus, this response allows forcellular survival and expedites removal of the virus. Dengue virus can adapt toalter this host response to its own advantage, interacting with complex cellularreactions such as autophagy, apoptosis, innate immune responses, and proin-flammatory reactions [93,94]. Its modifications of these host cellular processesallows Dengue virus to escape immune surveillance and continue viral repli-cation within cells. The overproduction of unfolded or misfolded proteins are anormal consequence during viral/bacterial infections and overwhelm theability of the endoplasmic reticulum to handle them [94,95]. This resultingstress in the endoplasmic reticulum caused by the unfolded/misfolded proteinstriggers the onset of the unfolded protein response for cell survival. There arethree separate pathways activated by the unfolded protein response (depictedin Fig. 6.4) that are induced by Dengue infection. The pathways ultimatelyresult in: (1) suppression of mRNA translation (halts protein synthesis), (2) anincrease in endoplasmic reticulum protein folding capacities, (3) endoplasmic104 Dengue Virus Diseasereticulum-associated degradation (ERAD) of misfolded proteins that haveaccumulated in the endoplasmic reticulum, (4) increased synthesis of chap-erones in the endoplasmic reticulum, (5) inducing a proinflammatory statewithin the cell, and (6) inducing apoptosis of the cell [92].Pathways utilized by the unfolded protein responseInositol requiring kinase 1 (IRE1) pathwayWhen IRE1 is intrinsically autophoshorylated, it will result in mRNA andmiRNA degradation in a manner called regulated IRE1-dependent decay(RIDD). mRNAs produced will also then induce ER-associated degradation(ERAD) genes to degrade misfolded proteins. Dengue virus activates thispathway, and further activates cellular caspases (3 and 9) to trigger cellularapoptosis [92] [Summarized in Fig. 6.7].Protein kinase R-like ER kinase (PERK) pathwayPhosphorylated PERK will inhibit the eukaryotic translation initiator factor 2a(eIF2a), resulting in a blockage of protein translation. eIF2a also activates atranscription factor ATF-4, which will further activate a C/EBP protein(CHOP) and a protein called the growth arrest and DNA damage-inducibleprotein (GADD34), both of which will trigger apoptosis [92]. [Summarizedin Fig. 6.7].Activating transcription factor-6 (ATF6) pathwayATF6 when activated, will result in the uncovering of a Golgi localizationsignal. Cleavage of the ATF6 in the Golgi apparatus, will yield an activatedtranscription factor, ATF6p50, which upon translocating to the nucleus, willresult in the increased transcription of genes related to ERAD [92] [Summa-rized in Fig. 6.7].Manipulation of the unfolded protein response by the Dengue virusViral replication enhancementThe modulation of the unfolded protein response by the Dengue virus iscomplex, with the PERK arm of the pathway activated early in disease, fol-lowed by the IRE1-XBP1, and lastly the ATF6 late in the infection [96]. Thisallows the Dengue virus to overcome the translation attenuation created by theunfolded protein response, allowing it to continuously produce viral proteinsnecessary for viral replication (such as NS5 which functions as the RNApolymerase). Dengue virus is also able to inhibit apoptosis by blocking thedownstream signaling of IRE1-XBP1 that normally activates caspases,allowing for uninterrupted dengue viral replication [96].Viral genetics and structure Chapter | 6 105Autophagy modulationIn viral infections, autophagy plays a significant role in removing viral com-ponents and allows for viral antigen presentation to augment an immuneresponse. Dengue virus manipulates this process by inducing autophagy in thecell, allowing sequestration of Dengue virus within the double membraneFIGURE 6.7 Unfolded protein response and ER stress: Modulation by the Dengue Virus. (A)IRE1 pathway: Dengue virus will activate IRE1 phosphorylation which will result in mRNAdegradation. Dengue virus also activates caspases (caspase three and 9), (B) PERK pathway:Dengue virus phosphorylates eIF2a and activates GADD34 to trigger apoptosis, (C) ATF6pathway: Dengue virus causes activation of ATF6p50, a transcription factor that increases tran-scription of genes related to ER stress pathways. ER: endoplasmic reticulum. Illustration fromPerera N, Miller JL et al. The role of the unfolded protein response in dengue virus pathogenesis.Cell Microbio (2017).106 Dengue Virus Diseasevesicles that are normally produced in the autophagic process. Isolation ofDengue virus in these vesicles allows for not only viral replication goingunhindered, but also evades the antibody-dependent neutralization againstviral antigens [97,98]. Another advantage to activating autophagy is thatb-oxidation of lipids occur, which provides ATP necessary for viral replication[99].Inflammation induced by Dengue virusDengue viral infections normally trigger activation of the unfolded proteinresponse, which will then activate innate proinflammatory immune mediatorssuch as PKR, IRF3, IL-1b, and NF-kB, which function to eradicate Denguevirus [93]. It is a double-edged sword however, as too much of a robust in-flammatory response can cause harm to the host, as seen with the phenomenonof increased vascular permeability in severe Dengue infections. The type I IFNresponse is a vital antiviral mechanism, to which dengue virus has adaptedstrategies to evade. Dengue virus not only inhibits the induction of IFN, butalso its downstream JAK-STAT signaling to ensure its survival within infectedcells[100]. While evading the innate immune response (IFN), Dengue virusadditionally increases the production of proinflammatory cytokines, such asTNF-a. This results in essentially a “cytokine storm” that can cause the graveclinical presentations seen in severe Dengue infections [92].ConclusionStructural analysis of the Dengue virus has certainly come a long way in therecent years and has allowed for a much-needed perceptivity regarding thecomplex pathogenesis of Dengue viral infections. While great strides havebeen made in understanding the Dengue virus genomic structure, the presenceand functions of viral structural and NS proteins, and the Dengue virus lifecycle, Dengue virus is very much still on the rise today, posing a significantglobal health risk. Dengue virus is an astute one, capable of manipulating thehost’s response in its favor, making mechanisms to eradicate or control it quitechallenging. 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J Mol Biol 2014;426(6):1148e60. https://doi.org/10.1016/j.jmb.2013.11.023.Viral genetics and structure Chapter | 6 113https://doi.org/10.1126/science.aab3369https://doi.org/10.1016/j.chom.2015.07.009https://doi.org/10.1371/journal.ppat.1004242https://doi.org/10.1371/journal.ppat.1004242https://doi.org/10.1111/cmi.12734https://doi.org/10.1093/abbs/gmv108https://doi.org/10.3389/fmicb.2014.00222https://doi.org/10.1038/nrmicro3393https://doi.org/10.1038/nrmicro3393https://doi.org/10.1074/jbc.M111.222703https://doi.org/10.1074/jbc.M111.222703https://doi.org/10.1016/j.micpath.2014.03.004https://doi.org/10.1016/j.micpath.2014.03.004https://doi.org/10.1038/srep32243https://doi.org/10.1038/srep32243https://doi.org/10.3390/v3081332https://doi.org/10.3390/v3081332https://doi.org/10.1016/j.jmb.2013.11.023https://doi.org/10.1016/j.jmb.2013.11.023Chapter 7Clinical manifestations andlaboratory diagnosisSargun Singh Walia1,2, Mohammad A. Arif3,4, Jahanzeb Liaqat51Clinical Research Fellow, Zeenat Qureshi Stroke Institute, St. Cloud, MN, United States;2Department of Neurology, University of Missouri, Columbia, MO, United States; 3InternalMedicine Shaheed Zulfiqar Ali Bhutto Medical University/Pakistan Institute of Medical Sciences,Islamabad, Punjab, Pakistan; 4Internal Medicine, Ali Medical Centre, Islamabad, Punjab,Pakistan; 5Lt. Colonel, Pak Emirates, Military Hospital, Rawalpindi, PakistanClinical features of dengue viral illnessThe full spectrum of illness and severity can be produced by any of the fourdengue viral serotypes. In most cases, Dengue viral illness is self-limiting,however the febrile illness is often debilitating. The signs and symptoms canvary ranging from a mild, nonspecific febrile illness, to the more severe formsof the disease [1].ClassificationIn 1997, the World Health Organization classified Dengue viral illness wasclassified into three types on the basis of symptoms. It was divided into thefollowing:1. Dengue viral fever2. Dengue viral hemorrhagic fever3. Dengue viral shock syndromeThe initial case definitions of Dengue viral illness were given by WorldHealth Organization in 1997. The classification was very complex and hadvaried applicability in the clinicalsetting. These definitions were not veryefficient in guiding therapy for patients suffering from severe Dengue viralillness, but the disease did not qualifying to be categorized as Dengue viralhemorrhaging fever. A multicenter study was conducted in seven countries ofAsia and Latin America in 2009 demonstrated gaps in existing case definition.Dengue Virus Disease. https://doi.org/10.1016/B978-0-12-818270-3.00007-2Copyright © 2020 Elsevier Inc. All rights reserved. 115https://doi.org/10.1016/B978-0-12-818270-3.00007-2The World Health Organization reclassified Dengue viral illness in 2009 on thebasis of severity into:I. Dengue viral illness without warning signsII. Dengue viral illness with warning signsIII. Severe Dengue viral illnessAccording to World Health Organization, the prevalence of Dengue viralillness is increasing at an alarming rate. The reported case number increasedfrom 2.2 million in 2010 to more than 3.34 million in 2016. Although, threeWorld Health Organization member states regularly report the annual numberof cases, global estimates of Dengue viral illness prevalence still remain un-certain. A study done to analyze the prevalence of Dengue viral illness cases in128 countries has estimated that 3.9 billion people are at risk of Dengue viralillness [2]. Another study estimates that 390 million cases occur each year outof which 96 million show manifestation [3].World Health Organization classification 19971) Dengue viral fever2) Dengue viralhemorrhagic fever3) Dengue viral shocksyndromel Also called break bonefever.l Criteria: Fever þ two ofthe following -o retro-orbital pain or ocularpaino Headacheo Muscle paino Bone/joint paino Rasho Low white blood cellcounto Hemorrhagicmanifestation:petechiae, positivetourniquet test,epistaxis, vaginalbleeding, hematuria,rectal bleeding, orhematemesis.l Plasma leakagel Hematocrit increasefrom baselineby �20%l Dengue viralhemorrhagic fevercriteria must include:o Fever: biphasiclasting at least 2e7 dayso Hemorrhagicmanifestationsincluding at leastone of thefollowing:l Positivetourniquet testl Bleeding frommucosal Melena orhematemesiso Thrombocytopenia:Plateletlevels �,100,000cells/mm3o Pleural effusion orascites due toplasma leakagel Dengue hemorrhagicfever leading to acirculating collapse dueto plasma leakagel Early signdNarrowingpulse pressurel Late signdHypotensionl Rapid and weak pulsel Cold clammy skin116 Dengue Virus DiseaseSigns and symptomsAdult patients with Dengue viral illness are more likely to present with clinicalsymptoms, whereas in children the infection is mostly asymptomatic. Inendemic areas, history of prior infection with Dengue virus is very common.Secondary infection by a different serotype of Dengue virus can lead to moresevere symptoms than the primary episode [3,4]. Clinical manifestation canstart around day 4 after an infected mosquito bites a person. Dengue virusincubation period in humans ranges from 3 to 14 days [5].The initial clinical manifestations of Dengue viral illness without warningsigns and severe Dengue viral illness are similar, and the course of infection isshort. Identifying patients that may develop severe Dengue viral illness is achallenging task. Severe Dengue viral illness may be distinguishable byclinical course that it passes through three stages of pathophysiology. Theseare the febrile phase, with high fever driven by viremia; the critical/plasmaleak phase, which is manifested by sudden onset of varying degrees of plasmaleak into the pleural and abdominal cavities; and convalescence/recovery/reabsorption phase, the hallmark of which is a sudden arrest of plasma leakwith concomitant reabsorption of extravasated plasma and fluids Figs. 7.1 andWorld Health Organization classification 20091) Dengue viralillness withoutwarning signs2) Dengue viral illnesswith warning signs3) Severe Dengue viralillnessFever þ two of thefollowing:l Nausea, vomitingl Rashl Aches and painsl Leukopenial Positive tourniquettestDengue viral illness feverwith any of thefollowing:l Abdominal pain ortendernessl Persistent vomitingl Clinical fluidaccumulation (ascites,pleural effusion)l Mucosal bleedingl Lethargy, restlessnessl Liverenlargement >2 cml Laboratory: Increasein HCT concurrentwith rapid decrease inplatelet countl (Requires strictobservation andmedical intervention)Dengue viral illness fever withatleast one of the followingcriterial Severe plasma leakageleading to:a) Dengue shock syndromeb) Fluid accumulation withrespiratory distressl Severe bleeding as evaluatedby clinicianl Severe organ involvementa) Liver: Aspartatetransaminase and Alaninetransaminase �1000b) Neurologic: impairedconsciousnessc) Failure of heart and otherorgansClinical manifestations and laboratory diagnosis Chapter | 7 1177.2. Figure 7.1 depicts the symptoms of Dengue viral illness divided intoasymptomatic and symptomatic.According to World Health Organization 1997 classification, Denguehemorrhagic fever (DHF) and Dengue shock syndrome (DSS) includes allphases of infection i.e., febrile phase, critical phase, and the recovery phase. InDengue fever (DF), there is no critical phase.According to World Health Organization 2009 classification, severedengue viral illness and dengue viral illness with warning signs include allphases of infection i.e., febrile phase, critical phase, and recovery phase. Indengue viral illness without warning signs, there is no critical phase.Figure 7.2 depicts the different phases of dengue viral infection classified bythe World Health Organization in 1997 and reclassified in 2009.Febrile phaseFever is one of the first symptoms to be seen in Dengue viral illness. Thenonspecific nature of fever makes it challenging to distinguish from otherfebrile illnesses [6,7]. Other diseases that are mostly common in endemic areasof Dengue viral illness may include malaria, typhoid fever, and leptospirosis.In febrile phase, there is a sudden onset high grade fever (�101.3 F). The feverpresents in a biphasic pattern in which the fever subsides and then reoccurs in2 days. Fever can last anywhere from 2 to 7 days. Fever is associated withnonspecific symptoms including cranial, musculoskeletal, and gastrointestinalmanifestations.Dengue virus infec�onAsymptoma�c Symptoma�cViral syndrome -undiffern�ated syndrome Dengue viral fever syndromeNo hemorrhagic manifesta�onHemorrhagic manifesta�onDengue viral hemorrhagic feverNo shock Dengue viral shock syndromeFIGURE 7.1 Adapted from Dengue hemorrhagic fever: diagnosis, treatment, prevention andcontrol. World Health Organization 1997.118 Dengue Virus DiseaseSevere headaches and retro-orbital eye pain are the major manifestations ofcranial symptoms. Retro-orbital eye pain occurs due to various pathologies.The most accepted pathway is due to bleeding in the ophthalmic region sec-ondary to thrombocytopenia. The bleeding in the macula and retinal peripherymay lead to episodes of retro-orbital pain [8,9]. The patients top describe thepain as retro bulbar due to hemorrhage in the sub conjunctival region [10].Others report the pain to be diffuse.The musculoskeletal features include arthralgia and myalgia [11]. Dengueviral illness is also known as the break bone fever. Diffuse muscle and jointpain is one of the key symptoms of dengue viral illness. It is also known asDengue associated muscle dysfunction (DAMD). The pathogenesis behindWorld Health Organiza�on 1997 classifica�onDengue feverFebrile phaseRecovery phaseDengue hemorrhagic fever Febrile phaseCri�cal phaseRecovery phaseDengue shockFebrile phaseCri�cal phaseRecovery phaseWorld Health Organiza�on 2009 classifica�onDengue without warning signsFebrile phaseRecovery phaseDengue with warning signsFebrile phaseCri�calphaseRecovery phaseSevere DengueFebrile phaseCri�cal phaseRecovery phaseFIGURE 7.2 Different phases of infection in World Health Organization classifications ofdengue viral illness from 1997 to 2009.Clinical manifestations and laboratory diagnosis Chapter | 7 119these pains is believed to be due to interstitial hemorrhage, necrosis, andedema. Occasionally myophagocytosis has also been seen in patients sufferingfrom Dengue viral illness. Most of the patients improve within 7e14 days ofinfection [12]. Serum creatinine kinase levels may be raised due to musclebreakdown. In rare cases, mortality due to cardiomyopathy has also been re-ported [13,14]. Gastrointestinal symptoms can occur varying from intermittentnausea and vomiting to anorexia.A characteristic rash occurs in majority of patients suffering from feversecondary to Dengue viral illness. Primary rash occurs within 1e2 days ofsymptom onset and typically starts from the face. The rash is transient,flushing, and erythemic in nature occurring due to dilation of capillaries. After3e6 days of fever onset, a transient second rash appears. Mobiliform ormaculopapular eruptions are seen which are typically asymptomatic Fig. 7.3.In a minority of patients, this rash may be pruritic in nature [15,16].Hemorrhagic manifestations such as petechiae and bleeding from mucosalmembrane may be seen. Minor, subtle petechial hemorrhages are often foundon the lower extremities, but may also be encountered on the hard and softpalates, buccal mucosa, and the subconjunctivae. Petechial rash generallystarts from lower extremities and then spreads to the thorax and other bodyparts. In some rare cases, vaginal hemorrhage has been reported in pregnantFIGURE 7.3 Early dengue fever rash. Source: commons.wikimedia.org. Attribution: RanjanPremaratna. Professor in medicine. Department of medicine. University of Kelaniya. Sri Lanka[CC BY-SA 4.0].120 Dengue Virus Diseasehttp://commons.wikimedia.orgwomen. A positive tourniquet test can be used as to test the fragility of cap-illaries [17]. In a tourniquet test, a blood pressure cuff is placed around theupper arm. The tourniquet is kept inflated for 5 min up to the middle pressurebetween systolic and diastolic pressures. A positive test is identified by morethan 20 petechiae per 2.5 cm2 area Figs. 7.4e7.7.These clinical features do not predict the severity of Dengue viral illness.Therefore, it is crucial to monitor for warning signs and other clinical pa-rameters in order to recognize progression to the critical phase. Dengue viralillness fever is often misdiagnosed as influenza or other viral diseases in theabsence of a rash especially if the other symptoms are mild.Critical phasePatients with subsidence of fever will improve and not transition into thecritical phase. Patients that do transition into the critical phase present withwarning signs and symptoms of severe Dengue viral illness [18]. Gastroin-testinal symptoms worsen in the form of persistent vomiting and severeabdominal pain. Bleeding can be seen from previous venipuncture sites.Dyspnea and lethargy is seen in majority of cases. Patients with increasedcapillary permeability transition from febrile phase into the critical phase.Critical phase lasts between 24 and 48 h. This phase will present withsymptoms indicative of plasma leakage. This can manifest as a pulmonarysyndrome and/or renal syndrome [19]. The patients present with worsening ofsymptoms which typically occurs around day 3e7. Young adults and childrendevelop a systemic vascular leak syndrome. This typically presents as shock,bleeding episodes, plasma leakage, and organ impairment or failure. Initially itpresents with a hypotensive phase with a significant decrease in blood pres-sure. The hypotension is accompanied by hemoconcentration [20,21]. Capil-lary leak can be first evidenced with the presence of increased hematocrit but adecrease in albumin levels [22]. If left untreated, the disease can lead to 20%mortality rate. Proper management with intravenous hydration can reduce thismortality rate to less than 1% [23]. Systemic vascular leak syndrome is pre-ceded by progressive leukopenia (�5000 cells/mm3) with a rapid decline inplatelet count to about 100,000 cells/mm3. Rise in hematocrit is one of theearliest signs of plasma leakage and can reflect the severity of plasma leakage.High volume plasma leak can result in pleural effusion causing increasingrespiratory distress, ascites, gastrointestinal bleeding, and hypovolemic shock.Hypoperfusion of tissues can cause metabolic acidosis, progressive organfailure, and disseminated intravascular hypoperfusion. Disseminated intra-vascular hypoperfusion can result in severe hemorrhage leading to a decreasein hematocrit level. White cell count is often increased in patients with severehemorrhage. Acute kidney injury can rarely occur in Dengue viral illness.Hematuria and proteinuria can be seen along with glomerular abnormalities[24e26]. Initially the patient might appear normal, but immediate fluidClinical manifestations and laboratory diagnosis Chapter | 7 121resuscitation is necessary to prevent the complications of fluid leakage. Theinitial increase in hematocrit levels go back to normal or subnormal levels afterfluid administration. Frequent hematocrit level checks are used to guideintravenous fluid administration to counter the plasma leak [27]. Plasma leakcan also be seen in Ebola virus, Marburg virus, and Hantavirus illnesses.FIGURE 7.4 A positive tourniquet test on the left side of the image in a person with denguefever. Source: https://commons.wikimedia.org/wiki/File:Positive-tourniquet-test.gif; Center ofdisease control and prevention. Attribution: Center for disease control and prevention [Publicdomain].FIGURE 7.5 “Isles of white in sea of red.” Source: https://commons.wikimedia.org/wiki/File:Dengue_recovery_rash_(White_islands_in_red_sea).jpg Attribution: Ranjan Premaratna. Profes-sor in medicine. Department of medicine. University of kelaniya. Sri Lanka. [CC BY-SA 4.0].122 Dengue Virus Diseasehttps://commons.wikimedia.org/wiki/File:Positive-tourniquet-test.gifhttps://commons.wikimedia.org/wiki/File:Dengue_recovery_rash_(White_islands_in_red_sea).jpghttps://commons.wikimedia.org/wiki/File:Dengue_recovery_rash_(White_islands_in_red_sea).jpgFIGURE 7.6 Course of Dengue viral illness. Source: Adapted from Dengue: guidelines fordiagnosis, treatment, prevention and control. © World Health Organization 2009.FIGURE 7.7 Chest X-ray showing pleural effusion. Arrow A shows fluid accumulation andarrow B shows the normal width of the lung. Centers for disease control and prevention.Clinical manifestations and laboratory diagnosis Chapter | 7 123Recovery phaseAfter the critical phase subsides, slow reabsorption of fluid starts from theextravascular compartment. This takes approximately 48e72 h. During thisphase hemodynamic status improves, vital signs stabilize and gastrointestinalsymptoms start improving. In rare cases, an erythematous or petechial rashmay be seen. There are small patches of normal skin in between the rash and isreferred as “isles of white in the sea of red” Figure 7.5.Electrocardiographic changes and bradycardia are common during thisstage. Hematocrit decreases to normal levels or a little below baseline due tohemodilution. The leukocyte count starts rising after fever subsides. Use ofintravenous fluid should be cautious as excessive use can lead to complicationssuch as respiratory distress from massive pleural effusion and ascites, pul-monary edema, or congestive heart failure. Figure 7.6 depicts the course ofDengue viral illness in various phases of infection.ComplicationsVarious complications can arise during the different phases of infection, whichshould be carefully screened for include the following:1) Febrile phase:a) Dehydrationb) Neurological symptoms: encephalopathy and seizures2) Critical phase:a) Shockb) Hemorrhagec) Organ impairment: Acute kidney injury [28].3) Recovery phasea) Excessive intravenous fluid administration leading to hypervolemiab) Acute pulmonary edema124 Dengue Virus DiseaseDiagnosisSince there is no therapeutic agent available for Dengue viral illness treatment,successful management depends upon timely and judicious use of supportivecare, including administration of isotonic intravenous fluids or colloids, andclose monitoring of vital signs and hemodynamic status, fluid balance, andhematologic parameters. Dengue viral illness may be routinely diagnosedbased on clinical manifestation. Laboratory test can help in diagnosing aDengue viral illness. These tests can be broadly divided into nonspecific testsand definitive tests. Example of a nonspecific test includes a complete bloodcount panel, while an example of a definitive test is a nonstructural protein 1(NS1) antigen assay and serology testing. A study done in 2011 published thatapproximately 50% of primary care physicians use definitive tests to diagnoseDengue viral illness [29].Use of diagnostic techniques vary based on the stage of infection. Basiclaboratory test can help in the detection and management of Dengue viralillness. In the early stages of the infection, isolation of virus, nucleic acid, orantigen serves as the best method of detection. In the later stage or at the endof the acute phase of infection, routine laboratory diagnostics and a clinicalexamination often do not lead to a definitive diagnosis. The diagnosis ofDengue viral illness will remain elusive unless serological or molecular testsfor identification of Dengue virus are undertaken. The diagnosis is confirmedby serological tests for antidengue virus IgM antibodies or dengue virusribonucleic acid by molecular testing.Definite diagnosis of Dengue viral illness requires isolation of virus but isnot practical as it takes several weeks for results to be available [30]. If lessthan 5 days have passed since the onset of fever then definitive and serotypespecific identification tests are used. For rapid diagnosis, viral nucleic acids aredetected by reverse transcriptase-polymerase chain reaction and secreted anonstructural protein 1 antigen is capture by enzyme-linked immunosorbentassays [31]. These tests can help in managing cases especially in primary caresetting due to clinical uncertainties. Nonstructural protein 1 antigen testing canbe done at a very lower cost and recommended by the Ministry of Health inSingapore to be done in less than 7 days from symptom onset [32].Nonstructural protein 1 antigen detection is a highly sensitive and specific testfor Dengue viral illness [33,34]. Sensitivity is of a nonstructural protein 1 hasbeen found to be as high as 90% in primary dengue viral illness and 60%e80% in secondary Dengue viral illness [35]. Reverse transcriptase-polymerase chain reaction becomes positive within 5 days of illness and isvery sensitive and specific if performed in laboratory with specializedequipment and trained staff. In clinical practice most of the tests are now doneusing commercially available kits which are less reliable due to the lack ofstandardization and quality control [36].Clinical manifestations and laboratory diagnosis Chapter | 7 125Nonstructural protein 1 viral protein is secreted from infected cells, whichcan be detected in blood of infected individual within 7 days of illness [37].The level of a nonstructural protein 1 seems to correlate with viral titers andcan be viewed as a surrogate marker for viremia [38,39]. Now most of thehospitals are using a nonstructural protein 1 capture by enzyme-linkedimmunosorbent assay and rapid strip test for early diagnosis of Dengue viralillness especially in primary infection. In secondary infection, preformed an-tibodies against nonstructural protein 1 antigen sequester nonstructural protein1 in immune complexes thus interfering with detection by assay.Serological diagnosis of Dengue viral illness can be made by detection ofIgM antibodies as early as four days after the onset of illness. In a patientpresenting with clinical features of Dengue viral illness, presence of IgM anti-bodies in a single serum sample has been widely used to get a preliminarydiagnosis. The time for the patient to form antibodies against the Dengue viralillness is called seroconversion. The seroconversion is seen in seven days inprimary infection and in four days in secondary infection. Definite diagnosisrequires seroconversion and a fourfold rise in titers of antibodies between pairedand convalescent phase samples taken 2 weeks apart [40]. A hemagglutinationinhibition assay antibody titer of 1280 or higher is diagnostic of a probabledengue viral illness. Both probable and confirmed dengue viral illness casesshould be reported to health authorities. Serological test are not reliable in pa-tients who have been vaccinated [41] or had recent infection with antigenicallyrelated viruses like yellow fever virus, Japanese encephalitis virus or zika virus.Viral proteins are detected by immunohistochemical staining of tissue samples[42]. Liver biopsy has the highest yield in this regard and is usually done forpostmortem diagnosis of a suspected case of Dengue viral illness.Viral isolationSpecimens are usually collected before day 5 in the early phase of infection inthe period of viremia [43]. Samples are collected from serum, peripheralblood, or biopsy tissue. Most commonly used method to isolate Dengue virusis cell culture. Host cells used are mosquito cell AP61 (cell line from Ae.pseudoscutellaris) or line C6/36 (cloned from Ae. albopictus) [44e46]. Thespecimen should be properly stored and transported to preserve the viability[47]. It takes 1e2 weeks to isolate the virus and confirm using immunofluo-rescence assay. Suckling mice can also serve as clinical specimens and areused to inoculate via intracranial route. Virus antigens can then be detectedusing anti-Dengue antibody staining in mouse brain samples.Nucleic acid detectionRibonucleic acid (RNA) is very unstable and heat labile and requires properhandling. Similar methods are used for storage and transport as used in nucleic126 Dengue Virus Diseaseacid detection methods. Reverse transcriptase-polymerase chain reactions havebeen used since 1990s to detect nucleic acids [48]. This offers better sensitivity(80%e100%) than viral isolation methods and the detection time is also lesser.Principal steps involved are the following: nucleic acid extraction and purifi-cation; amplification of the nucleic acid; and detection and characterization ofthe amplified product. Real time reverse transcriptase-polymerase chain re-action is a one-step system that can be used to detect and quantify viral RNAspecific to different Dengue serotypes [49]. This test comes in two types of kitsthat are either singleplex type or multiplex type [50]. A singleplex type kit candetect one serotype at a time whereas a multiplex type kit can detect all fourserotypes in a single sample. Other methods used for nucleic acid detectioninclude nucleic acid sequenceebased amplification assay [51e53]. Nucleicacid sequenceebased amplification assay has been adapted to be used to studyDengue viral illness in the field. Loop mediated amplification method isanother way to detect nucleic acid [54e56].Antigen detectionAntigen detection in patients suffering from secondary infection is difficult inan acute-phase serum sample as these patients have preexisting virus Immu-noglobulin G antibodies. However with the developments in enzyme-linkedimmunosorbent assay and dot blot assays such diagnosis has been madeeasy. High concentrations of nonstructural protein 1 and envelop/membraneantigen immune complexes can be detected in both primary and secondarydengue viral illness. This can be done within 9 days after the onset ofsymptoms. Nonstructural protein 1 kitsare available commercially which canhelp in early diagnosis of dengue viral illness [57,58]. But such kits cannotdifferentiate between different serotypes of infection. Immunoperoxidase,avidinebiotin enzyme, and fluorescent antibody assays can be used in autopsybiopsy samples.Immunoglobulin M antibody-capture enzyme-linkedimmunosorbent assay (MAC-ELISA)Immunoglobulin M antibody-capture enzyme-linked immunosorbent assay(MAC-ELISA) is a technique to test antibodies against Dengue virus [59]. Amicroplate is coated with anti-m chainespecific antibodies which then capturestotal Immunoglobulin M in the serum sample. Antigens specific to one of theserotypes of Dengue virus attach to the captured dengue virus antibodies.These are then detected via directly or indirectly conjugated enzyme whichtransforms into colored substrate. Spectrophotometer is used to measure op-tical density. Serum samples are collected after 5 days or more from symptomonset. Immunoglobulin M antibody-capture enzyme-linked immunosorbentassay is highly sensitive and specific if used after 5 days of symptom onset.Clinical manifestations and laboratory diagnosis Chapter | 7 127Rapid commercial kits in the form of enzyme-linked immunosorbent assay areavailable [60]. Cross reactivity has been reported with malaria and secondaryDengue infection [61]. The disadvantage of Immunoglobulin M antibody-capture enzyme-linked immunosorbent assay is that it cannot differentiateamong different serotypes of Dengue virus [62].Immunoglobulin G of enzyme-linked immunosorbent assay(Immunoglobulin G ELISA)Immunoglobulin G enzyme-linked immunosorbent assay uses the same type ofantigens used in Immunoglobulin M antibody-capture enzyme-linked immu-nosorbent assay. The advantages of using Immunoglobulin G enzyme-linkedimmunosorbent assay is that it can detect both recent and past Dengue viralillness. It can even detect Immunoglobulin G antibodies after 10 months ofDengue viral illness using E/M-specific capture Immunoglobulin G enzyme-linked immunosorbent assay (GAC). Immunoglobulin G antibodies can bedetected for life but a 4x increase in number can be used to detect a recentinfection. This test can also be used for the surveillance and serologicaldiagnosis of Dengue viral illness cases. This can also be used to differentiatebetween a primary and a secondary infection.Immunoglobulin M/Immunoglobulin G ratioImmunoglobulin M/Immunoglobulin G can be used to distinguish primaryfrom secondary dengue viral illness. If the immunoglobulin M/immunoglob-ulin G optical density ratio is �1.2 or 1.4 (depending on the dilution level), itis termed as primary infection. If immunoglobulin M/immunoglobulin G op-tical density ratio is �1.2 or 1.4 the dengue viral illness is classified as asecondary infection.Immunoglobulin AAntidengue virus immunoglobulin A capture enzyme-linked immunosorbentassay is used to detect antidengue immunoglobulin A antibodies in the patientsera. It usually becomes positive 1 day after Immunoglobulin M. Peak levelscan be recorded around day 8 of symptom onset.Hemagglutinationeinhibition testPrincipal of this test is that Dengue virus antigens agglutinate red blood cells(RBCs). This agglutination process is inhibited by anti-Dengue antibodieswhich can be measured in a hemagglutinationeinhibition test. Paired serumsamples should be collected on admission and on discharge or the samplesshould be spaced 7 days. In a primary dengue viral illness low level of128 Dengue Virus Diseaseantibodies in samples taken before 5 days which starts elevating slowlythereafter. In secondary infection, hemagglutinationeinhibition antibody titerlevels rise at a much rapid rate exceeding 1:1280.Other laboratory diagnostic modalitiesDengue fever is suspected in endemic areas when a patient with acute febrileillness develops thrombocytopenia and bleeding complications. Other diseasesthat can lead to thrombocytopenia besides Dengue viral illness are malaria,rickettsial infections, scrub typhus, leptospira, and meningococci infections.The decrease in platelet count occurs due to reduction in platelet productionand increased destruction of platelets [63]. Sepsis by gram positive and gramnegative bacterial infections can lead to thrombocytopenia. Platelet specificimmunoglobulin G antibodies can cause the platelets to attach to damagedvascular surfaces. World Health Organization suggests using tourniquet testfor early diagnosis of a suspected case of dengue fever [64] as definite diag-nosis in acute settings has little impact in overall management of the patient.Basic laboratory tests that are routinely used in the management of sus-pected Dengue viral illness cases are as follows:1) Complete blood count [65,66].l Leukopenia: White cell count less than equal to 5000 cells/mm3 in theearly stages of infection. It later normalizes after defervescence.Lymphocytosis may be seen in the presence of shock.l Thrombocytopenia: Platelet count is less than 100,000 cells/mm3.Platelet count recovery is typically slow even in recovery stages ofinfection. Platelet levels should be reassessed every 24 h to detectconversion to Dengue hemorrhagic fever.l Hematocrit increase by more than equal to 20% above the baseline. Thismay vary with the level of fluid administration (lower due to dilutionaleffect) and hemorrhage from the gastrointestinal tract. A study done inThailand in 2004 found that hematocrit levels done at the time ofadmission cannot predict the future outcome of Dengue hemorrhagicfever and dengue shock syndrome [67]. It emphasized that repeatedlevels are necessary to better predict outcome. Hematocrit levels shouldbe rechecked every 24 h to detect dengue hemorrhagic fever in the earlystages. If Dengue shock syndrome is suspected then the hematocritlevels should be repeated every 3e4 h.2) Metabolic panell Hypoproteinemial Metabolic acidosisClinical manifestations and laboratory diagnosis Chapter | 7 129l Electrolyte disturbance: Hyponatremia may be seen in dengue hemor-rhagic fever or dengue shock syndrome.l Blood urea nitrogen (BUN): Levels may be increased in shock.3) Liver panel [65,68].l Serum aspartate transaminase (AST) is usually elevated to 2 to 5 timesthe upper limits but marked elevation up to 15 times can also occur.l Jaundice and acute liver failure are uncommon due to direct viral effectbut have been described in dengue shock syndrome. Prolonged hypo-perfusion and hypoxia are presumed to be the contributory factors [69].4) Coagulation profile and disseminated intravascular coagulation (DIC)panel.l Prolonged prothrombin timel Prolonged activated partial thromboplastin timel Decreased fibrinogenl Increased amount of fibrin split productsl Signs of early coagulopathy may be very subtle.5) Guaiac testl Should be used in all patients suspected with Dengue viral illness.l Positive for occult blood in the stool.6) Urinalysisl Used to identify hematuria7) Culturesl Blood, urine and, cerebrospinal fluid cultures may be performed toexclude other pathological causes.8) Arterial blood gasOther than the basic information, medical professionals should also recordtravel history and history of onset of symptoms. Capillary leak syndrome cancause development of bodily effusions, which can be detected as early as3 days after the infection with the help of ultrasound of chest and abdomen[27]. Right lateral decubitus chest X-ray is useful for detection of developmentof pleural effusion in places where ultrasound facility is not availableFigure 7.7. In most centers, imaging is done on daily basis after the first fewdays of infection so that capillary leak is detected very early on and appro-priate management can be instituted Table 7.1.130 Dengue Virus DiseaseTABLE 7.1 Summary of Dengue viral illness diagnostic methods.DengueviralillnessDetectionmethoddELISA Interpretation Sample collectionTime of collection afteronset of symptomsConfirmed Virus Virus isolated l Serum (day 1e5)l Necropsy tissue1e2 weeksGenome detection þ ve RT-PCR or þ ve real time RT-PCR 1 or 2 daysAntigen detection þ ve NS1 antigen >1 dayþ Immunohistochemistry l Necropsy tissueImmunoglobulinM seroconversionConversion from eve Immunoglobulin Mto þ ve Immunoglobulin M in paired serumsamplesl Serum (day 1e5)l Convalescent serum (15e21 days after 1st sample)Rapid test 30 min/ELISA 1e2 daysImmunoglobulin GseroconversionConversion from eve Immunoglobulin Gto þ ve Immunoglobulin G in paired serumsamples/4� increase in Immunoglobulin Glevels in paired samples7 days or moreProbable ImmunoglobulinMþve titers l Serum after day 5 1e2 daysImmunoglobulin G High serum Immunoglobulin G titers onhemagglutination inhibition assay (�1280)7 days or moreAdapted from Dengue and Control (DENCO) Study and.ELISA (Enzyme-linked immune sorbent assay); NS1 Antigen (nonstructural protein 1 antigen); Immunoglobulin M;Immunoglobulin G; RT-PCR (reverse transcriptase-polymerase chain reaction).ClinicalmanifestationsandlaboratorydiagnosisChapter|7131Future diagnostics methods under developmentMicrosphere-based immunoassays (MIA)It is a highly advanced serology diagnostic technique which can be used forlaboratory testing for variety of diseases [70]. In this, antibody or antigens arecovalently bonded to microspheres or beads. With the help of lasers, fluorescenceof varying wavelengths can be elicited. One of the advantages of microsphere-based immunoassays over IgM antibody capture enzyme-linked immunosorbentassay is faster diagnostic time [71]. It also has the ability to identify antibodyresponse to different viruses by multiplexing serological tests [72,73].Biosensor technologyThis technology is developing very rapidly [74]. It uses mass spectrometry and ishelpful in discriminating biological components in a complex mixture. It pro-duces mass spectra and serves as a fingerprint of the bacteria or virus making amolecular profile. The software used by the device has a vast database [75]. Ituses this database to compare the mass spectra produced to identify the path-ogen. It can also identify a pathogen which does not exist in the database bycomparing the similarities with the other pathogens. This test can be very helpfulin identifying different serotypes of Dengue virus in an outbreak. Identificationkits are being developed to take samples which can later be processed for DNA,mass spectrometry, and identification analysis [76].Microarray technologyThis test is an advanced diagnostic tool that can screen a sample for multipleviruses by analyzing their nucleic acid fragments [77]. The sample needs to beamplified and then hybridization is done on a microarray. This can analyzeboth random sequences and conserved sequence [78]. The advantage of thistechnology is that it can detect divergent strains of viruses. The DNA frag-ments are labeled with fluorescent dyes. This test is then analyzed using a laserbased scanner. It can be used to test patients in a region which is endemic fordengue viral illness and other arboviruses than can mimic symptoms of adengue viral illness [79].Luminescence technologyThe test is still under early development. Advantage of this system will be itsinexpensive instruments.Differential diagnosisMany different etiologies can present in a similar way as Dengue viral illness.Some of the infection that may present in a similar way as Dengue viral illnessare as follows:132 Dengue Virus DiseaseChikungunya virus infectionChikungunya and Dengue are both transmitted by Aedes aegypti and Aedesalbopictus mosquitoes. On comparison both may present with a febrile illnessalong with a rash. Both lead to arthralgias but in chikungunya joint swelling(inflammatory arthritis) is a hallmark symptom whereas thrombocytopenia andbleeding symptoms are more specific to dengue [80]. These can also be differ-entiated using reverse transcriptase-polymerase chain reaction (RT-PCR) [81].Zika virusZika and Dengue viral illness are both transmitted by Aedes aegypti and Aedesalbopictus mosquitoes. Zika virus infection leads to conjunctivitis in patientsinvariably, which is not seen in dengue viral illness. Reverse transcriptase-polymerase chain reaction can be used to definitely differentiate between them.In rare cases, a coinfection between Dengue, Zika, and Chikungunya has beenreported [82].TyphoidBoth typhoid and dengue may present with fever, abdominal pain, and rash.An easy way to distinguish them is by using blood or stool culture. Ramanspectroscopy has also been used to differentiate between them [83].Rickettsial infectionAlso known as African tick bite fever, is most commonly seen in people travelingto Africa and Caribbean islands. The infection presents with similar symptoms asdengue that include fever, headache, and myalgias. The differentiating featuresinclude the presence of solitary or multiple eschars that are associated withregional lymphadenopathy. It can be differentiated from dengue with the use ofdirect smear and the use of Polymerase Chain Reaction techniques [84].MalariaMalaria infection can be differentiated from dngue by visualizing parasites inperipheral blood smears [85].Hemorrhagic feverMultiple viruses can lead to hemorrhagic fever like Dengue viral illness. Theseinclude Ebola virus, Lassa virus, Hantavirus, Crimean-congo virus, Yellowfever virus, and Marburg virus. The easiest way to distinguish among them isby epidemiological exposure. Confirmatory tests include polymerase chainreaction and viral serology.Clinical manifestations and laboratory diagnosis Chapter | 7 133Bacterial sepsisBacterial sepsis may also present with fever and even shock. Blood culturesare an easy way to distinguish it from dengue viral illness.References[1] Whitehorn J, Simmons CP. The pathogenesis of dengue. Vaccine 2011;29(42):7221e8.[2] Brady OJ, et al. 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MAC-ELISA for the detection of IgM antibodies to dengue type I virus(rapid diagnosis of dengue type I virus infection). Zhonghua Min Guo Wei Sheng Wu JiMian Yi Xue Za Zhi 1989;22(4):278e85.[60] Sathish N, et al. Comparison of IgM capture ELISA with a commercial rapid immuno-chromatographic card test & IgM microwell ELISA for the detection of antibodies todengue viruses. Indian J Med Res 2002;115:31e6.[61] Hunsperger EA, et al. Evaluation of commercially available anti-dengue virus immuno-globulin M tests. Emerg Infect Dis 2009;15(3):436e40.[62] Vázquez S, et al. Diagnosis of dengue virus infection by the visual and simple AuBioDOTimmunoglobulin M capture system. Clin Diagn Lab Immunol 2003;10(6):1074e7.[63] Parikh F. Infections and thrombocytopenia. J Assoc Physicians India 2016;64(2):11e2.136 Dengue Virus Disease[64] Cao XT, et al. Evaluation of the World Health Organization standard tourniquet test and amodified tourniquet test in the diagnosis of dengue infection in Viet Nam. Trop Med IntHealth 2002;7(2):125e32.[65] Potts JA, Rothman AL. Clinical and laboratory features that distinguishdengue from otherfebrile illnesses in endemic populations. Trop Med Int Health 2008;13(11):1328e40.[66] Ralapanawa U, et al. Value of peripheral blood count for dengue severity prediction. BMCRes Notes 2018;11(1). 400-400.[67] Wiwanitkit V, Manusvanich P. Can hematocrit and platelet determination on admissionpredict shock in hospitalized children with dengue hemorrhagic fever? A clinical obser-vation from a small outbreak. Clin Appl Thromb Hemost 2004;10(1):65e7.[68] Trung DT, et al. Liver involvement associated with dengue infection in adults in Vietnam.Am J Trop Med Hyg 2010;83(4):774e80.[69] Neeraja M, et al. Unusual and rare manifestations of dengue during a dengue outbreak in atertiary care hospital in South India. Arch Virol 2014;159(7):1567e73.[70] Johnson AJ, et al. 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Biosens Bioelectron 2018;99:122e35.[76] Mehrotra P. Biosensors and their applications e a review. J Oral Biol Craniofac Res2016;6(2):153e9.[77] Govindarajan R, et al. Microarray and its applications. J Pharm BioAllied Sci 2012;4(Suppl.2):S310e2.[78] Raghavachari N. Microarray technology: basic methodology and application in clinicalresearch for biomarker discovery in vascular diseases. Methods Mol Biol 2013;1027:47e84.[79] Dı́az-Badillo A, et al. A DNA microarray-based assay to detect dual infection with twodengue virus serotypes. Sensors 2014;14(5):7580e601.[80] Taraphdar D, et al. A comparative study of clinical features between monotypic and dualinfection cases with chikungunya virus and dengue virus in West Bengal, India. Am J TropMed Hyg 2012;86(4):720e3.[81] Lee VJ, et al. Simple clinical and laboratory predictors of Chikungunya versus dengueinfections in adults. PLoS Negl Trop Dis 2012;6(9):e1786.[82] Waggoner JJ, et al. Viremia and clinical presentation in Nicaraguan patients infected withzika virus, chikungunya virus, and dengue virus. Clin Infect Dis 2016;63(12):1584e90.[83] Naseer K, et al. Raman spectroscopy based differentiation of typhoid and dengue fever ininfected human sera. Spectrochim Acta A Mol Biomol Spectrosc 2019;206:197e201.[84] Jensenius M, et al. African tick bite fever. Lancet Infect Dis 2003;3(9):557e64.[85] Tangpukdee N, et al. Malaria diagnosis: a brief review. Korean J Parasitol2009;47(2):93e102.Clinical manifestations and laboratory diagnosis Chapter | 7 137Chapter 8Psychological and socialaspects of Dengue virus illnessvirus infectionIhtesham Qureshi1, Arbaab Qureshi21Resident physician, Department of Neurology, Texas Tech University of Health Sciences Center,Paul L. Foster School Of Medicine, El Paso, TX, United States; 2Clinical research fellow,Department of Neurology, Texas Tech University of Health Sciences, El Paso, TX, United StatesIf you thought Dengue Virus Illness was about fever and falling platelet count,think again. There’s a mental hazard associated with this affliction, one that isserious enough to make the government sit up and take notice———— News Reporter highlighting the substantial increase in the number ofdengue virus illness patients suffering from major psychiatric conditions in Indiaafter dengue virus illness outbreak [1].Denguevirus illness infection is a viral disease transmitted byAedesmosquitoand is endemic in tropical and subtropical regions of the world. Dengue virusillness infection is considered a global public health threat accounting for about50% of world’s population at a potential risk of having the infection [2].Within the last couple of years, there have been collective reports of Denguevirus illness presenting with atypical features, predominantly psychiatric symp-toms. Several studies have established that there is a strong linkage between theonset of Dengue virus illness and the appearance of manic symptoms [3].There are several case reports highlighting the emergence of psychiatricsymptoms ofDenguevirus illness,which presents early onset ofmanic symptoms,observed during febrile and convalescence phase of Dengue virus illness. Initialmanifestations include talkativeness, authoritative, and short-tempered conduct.In addition, patients may exhibit grandiosity, increased sexual behavior, anddecreased need to sleep. To regulate these symptoms, mood stabilizers and anti-psychotics are prescribed, which usually cause almost complete disappearance ofsymptoms within a short period of week to a month [4].There is a strong association between physical and mental health. Peopleliving with mental issues have a higher chance of suffering from physical healthissues, the same goes vice-versa, where people living with long lasting physicalDengue Virus Disease. https://doi.org/10.1016/B978-0-12-818270-3.00008-4Copyright © 2020 Elsevier Inc. All rights reserved. 139https://doi.org/10.1016/B978-0-12-818270-3.00008-4health issues face widespread psychological conditions of which depression andanxiety are most commonly reported in comparison to general population. Thisis validated by a study performed by Jahnjee et al. [5] reported substantialpsychiatric morbidity among patients affected with Dengue virus illness, evenamong them, majority of symptoms were observed in the early phase of Denguevirus illness which includes fear of death followed by anxiety. Of those patientsrecovered from Dengue virus illness, 35% exhibited persistent insect phobia and7% reported death phobia at the end of sixth week [5].Similar studies performed by Hashmi et al. and Gill et al. [6,7] also reportedsimilar findings especially in the early phase of dengue virus illness where pa-tients reported thanatophobia (fear of death) among nearly 90% of patients.Panic attacks in 15%e23% and approximately 80% had anxiety-related symp-toms. As the physical health improved, the intensity and regularity of the psy-chiatric symptoms improved concurrently. Of those who recovered, about 50%developed insect phobia. Also, depressive symptoms were initially seen in 50%e60% of patients with 5%e20% of patients ending up with persistent symp-toms. These psychiatric symptoms were most predominantly observed amongwomen [6,7]. In a study performed by Gill et al. to assess the psychologicaleffects of Dengue virus illness reported a finding that is worth mentioning thatmedia, both print and electronic played a major role in the creation of psy-chological distress particularly fear of death. When questioned the real causebehind their extreme fear, they attributed this to the “breaking news” appearingon television constantly, along with scrolls and highlighting the deaths caused byDengue virus illness in a dreadful way almost every day has created this fearamong the people. This fear further multiplied in leaps and bounds when theyfound out that they have been affected by Dengue virus illness [7].To understand this issue in a better way, there was an interesting studyperformed by Wong et al. on a focused group study in Malaysian citizens tounderstand their health benefits and practices related to Denguevirus illnessprevention. The study reported that participants mainly fell into two differentattitude themes in regards to Dengue virus illness: serious or highly deadly,and not a threat. The first category are participants that comprise about 33% oftotal participants, of which most knew of neighbors, friends, or colleagues whopassed away from Dengue virus illness considered the infection as a dreadfuland dangerous disease. These first category participants regarded Dengue virusillness as a disease that can kill people really fast as they heard from peoplewho they know died in a short time soon after getting admitted to hospital. Therest of the participants (second category) that comprise about 66% regardedDengue virus illness as not a hazard. These second category participants weremostly younger persons between 18 and 35 years old who believe that Denguevirus illness is not a dreadful disease and if diagnosed and treated early peoplecan survive the disease.Most participants consider Dengue virus illness a common and widelyprevalent disease in most areas of Malaysia. A small portion of these140 Dengue Virus Diseaseparticipants believe that the chance of contracting the disease is next tonegligible and those people who get affected from the disease are mainly frombad luck, ill fate, or chance. These participants further rationalize that onlyAedes mosquito can transmit the infection and all other kinds of species arenot capable for disease transmission. Furthermore, even among the Aedesmosquitoes, only those infected by dengue virus has the potential to causeDengue virus illness. Few participants think their risk of attaining Denguevirus illness is extremely low as they are almost certain that the mosquitoes intheir area were not Aedes species.The mosquitoes here are from the jungle, they are not dangerous. Every eveningthey come, we are used to it, mosquito bites are common, we turn on the fanevery night.Malay female, 50 years old, housewife.Additionally, there are a lot of participants, majority of them are youngmen, who has a perception that, if someone has a strong immune system, theycannot be affected by Dengue virus illness. Only those people with decreasedimmunity will be susceptible for contracting the Dengue virus illness fever.The place I stay . very likely .. because there is a lake, water stagnant. Manycases, I may get Dengue Virus Illness, but I think I am at low risk, because mybody defense is strong and I am healthy.Indian male, 21 years old, college student.One of the participants voiced that mosquitoes mostly favor young popu-lation over elderly, as the presence of thickened skin layer among elderly, iswhat will serve as a protection against mosquito bites.The mosquitoes prefer to bite small children than old people. Our skin thick andhard, cannot go through.Malay male, 69 years old [8].Social implications of dengue virus illness virus diseaseThe influence of the Dengue virus epidemic has profound and grave impli-cations on the functionality of multiple organizations at various levels.Effect on health care infrastructure and managementIt was scary. Both public and private service centers were overflowing. Theurgent care centers in private hospitals were also over capacityThe response above clearly indicates the anxiety and panic the patientsaffected by the Dengue virus illness epidemic experienced during their visit tothe health care facilities.Psychological and social aspects of Dengue Chapter | 8 141Lack of required space and equipment to accommodate and treat has forcedthe health care facilities to shift the patients or refer them to other hospitalsfurther away from their homes, causing further inconvenience.The environment created by the outbreak made the case notifications andfollow-up processes more difficult.To notify a certain pathology, you need to have time. Many times, the number ofpatients that you see is so extensive that you do not have the time to fill out amandatory notification form for each patient. There is a huge demand and I thinkthat it’s because of it that the number of Dengue Virus Illness cases are notalways notified properlyThe physicians and the hospital authorities reported a strong spike in theinformal complaints and protests expressed by the patients and communitymembers, which were related to procedural changes and longer waitingperiods.Effect on householdsCommunity leaders reported the dire consequences were much more apparentif the infected member of the household were women head as opposed to men.The impact when a female head of household is diagnosed with Dengue VirusIllness is greater than a male head of household is diagnosed with Dengue VirusIllness because she is usually responsible for running the household.Overall, financial impact of dengue virus illness outbreaks on householdswas limited, but its effect was greatest for low-income families. Communitymembers reported purchasing a range of mosquito control methods during anoutbreak, such as citronella candles, insect swatters, special plants, sand to fillempty receptacles and insect repellants. It has been reported that the higherincome households used insect repellant as the method of prevention, whereasnothing substantial was reported among the lower income households. Eventhough, majority of the health care facilities provided free treatment to theinfected individuals, most of the patients tried self-medicating to alleviate thesymptoms during the beginning of the outbreak. However, they werecompelled to seek medical attention once the symptoms failed to subside orhad gotten worse.Effect on schoolsSchool authorities reported higher number of absenteeism during Dengue virusillness outbreaks. Increased absenteeism was recorded among students andteachers with the percentage ranging from 10% to 15%, respectively.School administrators noted that schools lacked additional funding andnecessary support for Dengue virus illness prevention and control measures.142 Dengue Virus DiseaseNo additional funding were received by the schools for Dengue virus illnessprevention and control measures. To address the need for community sensi-tization during a Dengue virus illness outbreak, schools adapted new curric-ulums. Educational materials and lessons on Dengue virus illness preventionand control were introduced into the curriculum, where the focus was pri-marily on identification and removal of mosquito breeding sites and personalcare. Several meetings were organized where parents were educated and givenan insight about prevention of Dengue Virus Illness. They were also reassuredthat appropriate measures were being taken to control the spread of theinfection.Effect on government and role of mediaA Dengue Virus Illness outbreak can lead to a political breakdown, despite thebest efforts of leadersIn response to the growing demand for care and treatment during anoutbreak and the resulting community dissatisfaction with government denguevirus illness prevention and control efforts, governments intensified their ef-forts. In order to accommodate the increasing demands for care and treatment,and the deepening dissatisfaction among the community, the governmentsreinforced and intensified their efforts to prevent and control the Dengue virusillness. States of emergencies were declared during Dengue virus illnessoutbreaks and additional powers were given to the governments to takenecessary actions aiming toward vector control activities and policies.The political structures were significantly impacted due to the growing fearand anxiety of contracting the Dengue virus illness. Community discontentand apprehension for government’s efforts in the prevention and control effortsheightened and, incited a high level of community disengagement with theseefforts. The media played a prominent role in spreading the news and thereaction of the community inresponse to the outbreak.The International media primarily focused on the consequences of theoutbreak and the repercussions on the growing political tensions. The nationaland local media reported the government announcements, release of newepidemiological bulletins, and the direct consequences of the outbreak amongthe communities.The leaders of the governments recognized the impact of the Dengue virusillness outbreaks and the serious implications this could cause which perhapsmay lead to consequential political disintegration [9].ConclusionEducating the population about how Dengue virus illness spreads is the keyto reducing stress and anxiety. Dengue virus illness escalated to a socialPsychological and social aspects of Dengue Chapter | 8 143frenzy due to the neglect of the community leaders and governmentagencies to educate the public. The government and community leadersneed to take responsibility and aid in the proper outflow of information tothe public. It is therefore essential that leaders from the community andgovernment postings participate in dissemination of information includingthe right/left wing activists, religious scholars, and government agencies.Without a combined effort to properly dispel the misinformation, the publicwill continue to live in a frenzy without seeking proper aid or treatment.Studies demonstrate that the sections of the public population thatresponded best to the Dengue virus illness outbreak and followed appro-priate practices for dengue virus illness control were those that werewell-organized, having active leaders, participated with government/Nongovernmental Organization (NGO) in awareness campaign for Denguevirus illness eradication. These efforts include educating the public aboutthe appropriate use of insecticides, sharing information regarding Denguevirus illness, and keeping mutual coordination with health department/NGOs or other agencies in Dengue virus illness control [10].Ideally, community participation is the strongest in countries that havestable political systems. Mobilization of the community needs to occurfrom the national level as well as the grassroots level to effectively pre-vent and control Dengue virus illness. One manner in which the localauthorities can contribute is by eliminating breeding places of Denguevirus illness mosquitoes. This is the only cost-effective and sustainableway of ensuring control in any Dengue virus illness-affected country,especially those deficient in resources [11]. One such success story is the“Thai National Dengue Virus Illness Prevention and Control Plan” thatwas helpful in guiding the public protect and prevent against Dengue virusillness [12]. Successful community mobilization cannot occur withoutdecentralization of resources and power. It also requires a high level ofcoordination between the agencies and the public. The first step should beto understand the daily problems of the community and then delegateproper prevention techniques. Without proper coordination, all efforts tocontrol Dengue virus illness may be ineffective and costly. Studiesdemonstrate the need for proper planning and management in watersupply, the drainage system and discarding broken items, are effectivesteps for successful Dengue virus illness control [13].A similar success story was witnessed in Singapore by “DO THEMOZZIE”campaign launched on 28 April 2013 and since then, the annualDengue virus illness prevention campaign calls for the community toactively check for and get rid go stagnant water in their homes byparticipating the 5-STEP MOZZIE WIPEOUT. The campaign is supportedby local grassroots advisors and the community, with the mobilization ofgrass root leaders and the Dengue virus illness prevention volunteers144 Dengue Virus Disease(DPVs) to conduct house visits and organize Dengue virus illness outreachevents (Fig. 8.1) [14].The spread of Dengue virus illness can be controlled by the communitythrough eliminating the Dengue virus vectors. The Dengue virus illnessprevention campaign insists that the community keeps their living envi-ronment clean, removes standing water and burry discard broken items toeradicate the Dengue virus illness virus vectors [10]. These findings aresimilar to Spiegel et al. [15] who states that Dengue virus illness vectorscan be eliminated through the hard work of the community people, leadersand government agencies. This study demonstrates the importance of theDengue virus illness campaign and how being informed about Denguevirus illness fever play a role in maintaining control of the Dengue virusillness outbreak [15].To deal with the issue in a better way and gain confidence of the localcommunity members especially with regards to vector control, guidelinesprecisely intended for each environment should be developed in specific. Thebest practices must be swiftly executed at the commencement of the outbreakto achieve maximum effect [16]. Preparation should include novel approachesto educate community people with regards to Dengue virus illness, increasingthe accessibility of various resources before outbreaks and sustaining essentialresources and amenities in between the outbreaks. Essentially during theoutbreaks, real emphasis should be placed on eliminating mosquito breedingFIGURE 8.1 Pamphlets distributed in 2013 by the new (National Environment agency)Singapore as part of the “do the Mozzie Wipeout” campaign.Psychological and social aspects of Dengue Chapter | 8 145sites, which may require mobilizing and educating the local communitymembers (Fig. 8.2).The best way to manage and increase confidence of community membersduring an outbreak is to have precise guidelines toward vector control, suitedfor different types of environment, ready for rapid implementation [16].Planning should include educating community members and leaders aboutDengue virus illness, increasing the availability of Dengue virus illness re-sources before outbreaks and maintaining necessary resources and servicesbetween outbreaks.During outbreaks, local sensitization campaigns must focus on communitymobilization activities to eradicate mosquito breeding sites (Fig. 8.3).Multiple factors need to fall in place to properly control a Dengue viralIllness outbreak. Adequate knowledge or awareness is a major factor in thesuccessful implementation of approaches used to eliminate Dengue virusillness. However, knowledge of Dengue Virus Illness prevention is notenough. Policy makers and planners need to get involved and run sensiti-zation campaigns on lifestyle changes the public must adopt to encouragepreventative measures. Strong prevention campaigns that are implemented atthe right time will help the public gain confidence in government responsesto Dengue viral illness outbreaks and decrease the chances of politicalbreakdown [9,17].FIGURE 8.2 The community leaders in Hadhramaut, Yemen getting educated about mosquitobreeding sites and the importance of preventing disease transmission during the investigation ofdengue virus illness outbreak.146 Dengue Virus DiseaseThe World Health Organization (WHO) has therefore come up with newstrategies, in order for countries to achieve zero mortality from dengue virusillness and for this they recommend, a country must.Improve case management and diagnosis to prevent deaths from Denguevirus illness by the following:- Improving early clinical case detection, especially for Dengue virus illnesswith warning signs and severe Dengue virus illness;- Improving management of severe cases with appropriate interventionsespecially careful intravenous rehydration and a greater evidence base forinterventions.Improve capacities to facilitate a reduction in the burden of the disease:- Improve health service organization, including access and triage inendemic countries to prevent dengue virus illness related deaths;- Improve health service reorganization for managingoutbreak situations;- Build capacity and establish quality assurance in both private and publicsector;- Develop evidence-based informed training material, including Dengue viralillness courses;- Prepare for the arrival of vaccines and their public health implications,bearing in mind the challenges of widespread implementation [18].FIGURE 8.3 The residents of Yemen conduct a Dengue virus illness fever investigation byexamining stagnant water for aides mosquito larvae which is responsible for transmitting theDengue virus illness fever.Psychological and social aspects of Dengue Chapter | 8 147If the above-mentioned strategies and protocols are efficiently imple-mented by the organizations, health officials, governmental agencies andcommunity people at large at various levels, the psychological stress andanxiety and social implications surrounding dengue virus illness virus diseasecan be contained to a larger extent.References[1] Chandra N. Patients to get mandatory counselling as figures reveal almost eighty percentsuffer from anxiety. 2013. http://www.dailymail.co.uk/indiahome/indianews/article-2419416/Dengue Virus Illnesss-hidden-toll-mental-health-Patients-mandatory-counselling-figures-reveal-EIGHTY-cent-suffer-anxiety.html.[2] Gunathilaka N, Chandradasa M, Champika L, Siriwardana S, Wijesooriya L. Delayedanxiety and depressive morbidity among Dengue Virus Illness patients in a multi-ethnicurban setting: first report from Sri Lanka. Int J Ment Health Syst 2018;12:20. https://doi.org/10.1186/s13033-018-0202-6.[3] Jhanjee A, Bhatia MS, Srivastava S. Mania in dengue virus illness fever. Ind Psychiatry J2011;20(1):56e7. https://doi.org/10.4103/0972-6748.98418.[4] Bhatia MS, Saha R. Neuropsychiatric manifestations in dengue virus illness fever. Med JDY Patil Univ 2017;10:204e6. http://www.mjdrdypu.org/text.asp?2017/10/2/204/202111.[5] Jhanjee A, Bhatia MS, Srivastava S, Rathi A. A study of psychiatric symptomatology indengue virus illness patients. Delhi Psychiatry J 2013;16:21e3.[6] Hashmi AM, Butt Z, Idrees Z, Niazi M, Yousaf Z, Haider SF, et al. Anxiety and depressionsymptoms in patients with dengue virus illness fever and their correlation with symptomsseverity. Int J Psychiatry Med 2012;44:199e210.[7] Gill KU, Ahmad W, Irfan M. A clinical study to see the psychological effects of denguevirus illness fever. Pak J Med Health Sci 2011;5:101e4.[8] Wong LP, AbuBakar S. Health beliefs and practices related to dengue virus illness fever: afocus group study. PLoS Neglected Trop Dis 2013;7(7):e2310. https://doi.org/10.1371/journal.pntd.0002310.[9] Ladner J, Rodrigues M, Davis B, Besson M-H, Audureau E, Saba J. Societal impact ofDengue Virus Illness outbreaks: stakeholder perceptions and related implications. A qual-itative study in Brazil, 2015. PLoS Neglected Trop Dis 2017;11(3):e0005366. https://doi.org/10.1371/journal.pntd.0005366.[10] Zahir A, Ullah A, Shah M, Mussawar A. Community participation, dengue virus illnessfever prevention and control practices in swat, Pakistan. Int J Mch AIDS 2016;5(1):39e45.[11] Khun S, Manderson L. Community participation and social engagement in the preventionand control of Dengue Virus Illness fever in rural Cambodia. Dengue Virus Illness Bulletin2008;32:145e55.[12] Kantachuvessiri A. Dengue Virus Illness hemorrhagic fever in Thai society. Southeast AsianJ Trop Med Public Health 2002;33:56e62. Bangkok Thailand: faculty of public health,Mahidol university.[13] Claro LB, Kawa H, Cavalini LT, Rosa MLG. Community participation in dengue virusillness control in Brazil. Dengue Virus Illness Bulletin 2006;30:214e22.[14] National Environment Agency. 2018. https://www.nea.gov.sg/programmes-grants/cam-paigns/do-the-mozzie-wipeout-campaign.148 Dengue Virus Diseasehttp://www.dailymail.co.uk/indiahome/indianews/article-2419416/Dengue%20Virus%20Illnesss-hidden-toll-mental-health-Patients-mandatory-counselling-figures-reveal-EIGHTY-cent-suffer-anxiety.htmlhttp://www.dailymail.co.uk/indiahome/indianews/article-2419416/Dengue%20Virus%20Illnesss-hidden-toll-mental-health-Patients-mandatory-counselling-figures-reveal-EIGHTY-cent-suffer-anxiety.htmlhttp://www.dailymail.co.uk/indiahome/indianews/article-2419416/Dengue%20Virus%20Illnesss-hidden-toll-mental-health-Patients-mandatory-counselling-figures-reveal-EIGHTY-cent-suffer-anxiety.htmlhttps://doi.org/10.1186/s13033-018-0202-6https://doi.org/10.1186/s13033-018-0202-6https://doi.org/10.4103/0972-6748.98418http://www.mjdrdypu.org/text.asp?2017/10/2/204/202111https://doi.org/10.1371/journal.pntd.0002310https://doi.org/10.1371/journal.pntd.0002310https://doi.org/10.1371/journal.pntd.0005366https://doi.org/10.1371/journal.pntd.0005366https://www.nea.gov.sg/programmes-grants/campaigns/do-the-mozzie-wipeout-campaignhttps://www.nea.gov.sg/programmes-grants/campaigns/do-the-mozzie-wipeout-campaign[15] Spiegel JS, Bennett HL, Hayden MH, Kittayapong P, et al. Barriers and bridges to pre-vention and control of dengue virus illness: the need for a socialeecological approach.EcoHealth 2012;2:273e90.[16] McNaughton D, Duong TT. Designing a community engagement framework for a newdengue virus illness control method: a case study from Central Vietnam. PloS Negl Dis2014;8:e2794.[17] Runge-Ranzinger S, McCall PJ, Kroeger A, Horstick O. Dengue virus illness disease sur-veillance: an updated systematic literature review. Trop Med Int Health 2014;19:1116e60.[18] Global strategy for dengue virus illness prevention and control. 2012e2020. World HealthOrganization (WHO), http://apps.who.int/iris/bitstream/handle/10665/75303/9789241504034_eng.pdf;jsessionid¼D1B8C9859E520412654E9E8D0EC415CC?sequence¼1.Psychological and social aspects of Dengue Chapter | 8 149http://apps.who.int/iris/bitstream/handle/10665/75303/9789241504034_eng.pdf;jsessionid=D1B8C9859E520412654E9E8D0EC415CC?sequence=1http://apps.who.int/iris/bitstream/handle/10665/75303/9789241504034_eng.pdf;jsessionid=D1B8C9859E520412654E9E8D0EC415CC?sequence=1http://apps.who.int/iris/bitstream/handle/10665/75303/9789241504034_eng.pdf;jsessionid=D1B8C9859E520412654E9E8D0EC415CC?sequence=1http://apps.who.int/iris/bitstream/handle/10665/75303/9789241504034_eng.pdf;jsessionid=D1B8C9859E520412654E9E8D0EC415CC?sequence=1Chapter 9Economic and political aspectsof Dengue virus diseaseMushtaq H. QureshiTexas Tech University Health Sciences Center El Paso, Neurology, Texas Tech University, El Paso,TX, United States; Zeenat Qureshi Stroke Institute, St Cloud, MN, United StatesDisease burdenThe disease and economic burdens of Dengue virus infection are considerable.It is essential to quantify these burdens by the policy-makers in order toallocate resources, to select prevention and control strategies, to set priorities,and to most importantly to evaluate the cost effectiveness of interventions [1].Disease burden can be defined as the number of infections, years of life lost topremature mortality, years that are lived with disability, and/or life years thatare disability-adjusted. The cost of disease burden can be conceptualized basedon the disease effect on the society, which is associated with diagnosis,treatment, outcome, and disease prevention. The disease burden of Denguevirus infection has been estimated using different data, analytical methods, andgeographical coverage. According to World Health Organization, there are50e100 million annual cases of Dengue virus infection [2], whereas accordingto a research in 2013 by University of Oxford and Wellcome trust, it is esti-mated to be 390 million Dengue virus infections per year (Fig. 9.1). Theestimated burden of Dengue virus infection based on geostatistical models and2010 population data is to be 390 million (95% credible interval [CrI]284e528) infections per year, of which 96 million (95% CrI 67e136) arethose which are causing clinical symptoms [3]. Approximately, 70% of thedisease burdenis bourne by Asia in its large, highly populated areas(approximately 67 million infections annually). When compared with theofficial reported figures by the countries, these numbers are much larger. TheGlobal Burden of Disease Study 2013 estimates a temporal trend in the burdenof Dengue virus infection, with the number of apparent cases more thandoubling each decade between 1990 and 2013 [4]. If we look at the children inparticular, it is not only a higher burden of symptomatic infections, but there isa significant numbers of these infections which resulted in hospital admissionDengue Virus Disease. https://doi.org/10.1016/B978-0-12-818270-3.00009-6Copyright © 2020 Elsevier Inc. All rights reserved. 151https://doi.org/10.1016/B978-0-12-818270-3.00009-6(ranging from 4.9% to 45.5% in 10 Asian and Latin American countries) [5].Looking at the deaths, however, the estimates seem low and do not go with theincreasing trend, which is observed in the apparent cases [4,6]. The GlobalBurden of Disease Study 2013 estimates that Dengue virus infection isresponsible for 1.14 million disability-adjusted life-years in 2013, which is aclear 61% increase from 1990 [4,7]. Many countries, where Dengue virusinfection is endemic, have insufficient data about deaths [4]. Data analyzedfrom a surveillance system from Puerto Rico recorded the highest Denguevirus infection mortality rate ever detected (1.05 per 100 000 people), and therates were higher in adults with comorbidities. (1.66 per 100,000 people aged65 years or older) [8]. These numbers, however, are still an underestimate. Sofar, only two studies have provided the global estimations of the diseaseburden [9,10]. Combining the disease burden estimates provided by Bhatt andcolleagues(3) and the cost associated with Dengue virus infection treatmentand loss of productivity (as provided by World Health Organization), theglobal estimated cost was estimated to be approximately US$39.3 billion(about $414 per symptomatic case) for 2011 [9]. Another study performed byShepard and Colleagues (9) reviewed and combined data from differentsources (including the Global Burden of Disease Study 2013, household data,expert panel surveys, and empirical cost data) in a modeling exercise that wasable to produce the first worldwide estimation regarding the economic burdenof Dengue virus infection, which was comparable across regions and countries[9]. This global health burden was estimated to be 58.4 million symptomaticcases, resulting in an estimated global cost of $8.9 billion (95% uncertaintyinterval 3.7e19.7) [9] which is higher than various other infectious diseases(Fig. 9.2).FIGURE 9.1 Distribution of global Dengue virus infection risk.152 Dengue Virus DiseaseDefining Dengue in monetary terms means the disease can be compared withother economic problems. Public health systems can then leverage that infor-mation to secure resources from their Ministry of Financedand possibly thedonor communitydto control the disease.Professor Donald Sheperd, PhD. (lead author and economist).Challenges in measuring the burden of diseaseThe challenges of measuring the burden of Dengue viral illness often result inuncertain estimates and hamper cross-country comparisons. Common chal-lenges can be grouped into the following categories: finding out true number ofcases, estimating the heterogeneous costs, quantifying the cyclical variations,and assessment of the burden. Usually dengue virus infection cases and deathnumbers that are reported by the endemic countries is an underestimate. Thereason being, private health practices only provide limited information [11];inability to detect Dengue virus infection in symptomatic patients seeking caredue to surveillance gaps; when patients does not seek health care; financialrestraints leading to restricted access to primary health care and no access tosensitive and specific diagnostic tests; in the health-care world when there isfailure make a diagnosis of dengue virus infection [11]; lack of informationtechnology and reliance on paper records; inadequate reporting of Dengue virusinfection to the national authorities. Effect of Dengue virus infection on in-dividual’s productivity is also worth considering. For e.g., number of days lost atschool or work is routinely not collected. Even more challenging is estimatingthe cost of reduced performance at work due to fatigue and other short-term andlonger-term consequences of Dengue virus infection [12]. Quantifying costFIGURE 9.2 Cost associated with Dengue virus infection compared with major infectiousdiseases.Economic and political aspects of Dengue virus disease Chapter | 9 153during the outbreak can be very challenging for various reasons: during emer-gency periods when supplemental financial resources are disbursed, withoutdetailed allocation to specific activities, it will make it difficult to categorize thedifferent cost component and also the additional cost during out breaks is noteasily quantifiable through routinely acquired data. According to a review [13],50% or less studies estimating the economic burden of Dengue virus disease,based their reports on the basis of the costs of outbreaks.Impact on low-income and middle-income tropicalcountriesThe economic burden of individual country or region often vary in severalaspects: number of years of data used, types of costs, decisions on how toextrapolate the data, choice of cost analysis perspective, monetary reference,and geographical coverage. Estimates of the economic burden demonstratethat low-income and middle-income tropical countries are the ones, which arethe most affected by Dengue virus infection. A major fraction (50%e60%) ofthe estimated economic costs of Dengue virus infection is related to loss ofproductivity [14,15], as well as vector control (40%e72% of the estimatedcost) [1,16,17]. Vector control represents a significant portion of Dengue virusinfectionerelated cost. The costs associated with Dengue virus infection insouth Asian countries are comparatively higher when compared to otherconditions such as, Japanese encephalitis, hepatitis B infections, upper respi-ratory infections, etc. [14] (Fig. 9.2) Also, when looking at the daily cost as aresult of Dengue virus infection in India, the cost is twice the cost per day thanof tuberculosis case [18]. When looking at the Americas, the economic burdenof Dengue virus infection exceeded that of other viral diseases, e.g., humanpapillomavirus [15]. Assessment of the economic cost of Dengue virusinfection in Puerto Rico (14) showed that households incurred 48% and em-ployers 7% of the total Dengue virus infection cost, whereas the governmentand insurers bore 24% and 22% of the cost, respectively. Households incurred90% of the cost that was associated with fatal cases, 21% for hospitaladmission for a child and 37% of the costs for hospital admission for an adult,and 51% and 63% of the costs for ambulatory child and adult cases, respec-tively. Also, when estimates were drawn for the direct medical costs in India[18], a country bearing one third of global disease burden of Dengue virusinfection [3], suggest that private sources, mostly households, bore 80% of thecost. However, the burden could vary by country, depending on the financestructure of the health-care system. Uncertainties, associated with the esti-mation of the burden of Dengue virus infection reflect problems with avail-ability and quality of data, and also raise important points: 1. Undoubtedly, theburden is large and growing; 2. Uncertainties associated with estimation ofdisease burden (i.e., infections and deaths) carry over into the estimation of theeconomic burden of disease; and 3. The greater the uncertainty in estimates,154 Dengue Virus Diseasethe more serious are the challenges that policy-makers face in prioritiessetting, resources allocation, and interventions planning.A comparison with Ebolavirus diseaseAccording to the World Bank, budget deficits of the affected countriescontinue to increase by amount equal to 1.8% of gross domestic product incountries including Sierra Leone and Guinea and 4.7% in Liberia (Fig. 9.3).With the continuous virus surge in the three worst affected countries with spanto neighboring countries, the 2-year regional financial impact by 2015 reachedto be $32.6 billion, causing a potentially catastrophic blow to already fragilestates. On the other hand, when comparing the Dengue virus infection diseaseburden estimates provided by Bhatt and colleagues(3) and the cost associatedwith its treatment and loss of productivity (as provided by World Health Or-ganization), the global estimated cost was estimated to be approximatelyUS$39.3 billion (about $414 per symptomatic case) for 2011.A comparison with Zika virus diseaseZika virus disease which is primarily spread by Aedes aegypti mosquito wasdeclared a public health emergency of international concern in February of2016 [19]. Although no longer considered a public health emergency of in-ternational concern, Zika virus disease is still considered as a health issuewhich has the potential to hit the most vulnerable communities the hardest.Several factors had played a role in the economic burden posed by this disease.According to the statement from World Bank Group, “Initial estimates of theshort-term economic impact of the Zika virus epidemic for 2016 in the LatinAmerican and the Caribbean region (LCR) are a total of US$3.5 billion, or0.06% of GDP” (Table 9.1). What they used were a few factors to calculateFIGURE 9.3 Estimated short-term impact on the overall fiscal balance (2014), in $US millionsof ebola virus disease.Economic and political aspects of Dengue virus disease Chapter | 9 155these numbers. These factors included behaviors to avoid transmission, itseffect on economic staples like tourism, loss of worker productivity, publicperceptions of risk from Zika virus disease which included media attentionsand the urgent need to take the action against the virus’s spread.Dengue virus infection vaccine, a promising toolA tool that has shown promise against Dengue virus infection is the use of avaccine. Even if a vaccine is partially effective, it would still affect the burdenof the disease, will decrease the number of new infections and thereforedecreasing the costs associated with illness from the perspective of healthproviders, individuals or households, and society. A vaccine with effectivecontrol would be an excellent addition to the limited number of tools which arecurrently available to reverse the growing burden of Dengue virus infection[20]. Several countries (e.g., Brazil, Mexico, the Philippines, Indonesia, CostaRica, Paraguay, and El Salvador) have approved the use of a live recombinanttetravalent Dengue virus infection vaccine that is administered in three dosesin individuals between 9 and 45 years of age. Several phase 3 trials in Asia andLatin America have shown reductions in numbers of severe Dengue virusinfection cases as well as the need for hospitalization among certain groups[21e23], but if we consider the protective efficacy, it is varied by serotype andpresence of antibodies from previous infection at the time of vaccination.These trials have also shown possible disease enhancement in young children[23].However research continues with other vaccine candidates [24e27].TABLE 9.1 World Health Organization estimates of the short-termeconomic impact of the Zika virus epidemic for 2016 in the Latin Americanand the Caribbean region.Mexico $744 millionCuba $664 millionDominican Republic $318 millionBrazil $310 millionArgentina $229 millionJamaica $112 millionBelize $21 millionOther $1.08 billionTotal $3.48 billionSource: World Bank.156 Dengue Virus DiseaseReferences[1] Carrasco LR, Lee LK, Lee VJ, Ooi EE, Shepard DS, Thein TL, et al. Economic impact ofdengue illness and the cost-effectiveness of future vaccination programs in Singapore. PLoSNeglected Trop Dis 2011;5(12):e1426.[2] Dengue: guidelines for diagnosis, treatment, prevention and control. New Edition. Geneva:WHO Guidelines Approved by the Guidelines Review Committee; 2009.[3] Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL, et al. The globaldistribution and burden of dengue. Nature 2013;496(7446):504e7.[4] Stanaway JD, Shepard DS, Undurraga EA, Halasa YA, Coffeng LE, Brady OJ, et al. Theglobal burden of dengue: an analysis from the Global Burden of Disease Study 2013. LancetInfect Dis 2016;16(6):712e23.[5] L’Azou M, Moureau A, Sarti E, Nealon J, Zambrano B, Wartel TA, et al. Symptomaticdengue in children in 10 Asian and Latin American countries. N Engl J Med2016;374(12):1155e66.[6] Wilder-Smith A, Byass P. The elusive global burden of dengue. Lancet Infect Dis2016;16(6):629e31.[7] Hotez PJ, Alvarado M, Basanez MG, Bolliger I, Bourne R, Boussinesq M, et al. The globalburden of disease study 2010: interpretation and implications for the neglected tropicaldiseases. PLoS Neglected Trop Dis 2014;8(7):e2865.[8] Tomashek KM, Rivera A, Torres-Velasquez B, Hunsperger EA, Munoz-Jordan JL,Sharp TM, et al. Enhanced surveillance for fatal dengue-like Acute Febrile illness in PuertoRico, 2010e2012. PLoS Neglected Trop Dis 2016;10(10):e0005025.[9] Russo M, Bevilacqua P, Netti PA, Torino E. A Microfluidic platform to design crosslinkedhyaluronic Acid nanoparticles (cHANPs) for enhanced MRI. Sci Rep 2016;6:37906.[10] Selck FW, Adalja AA, Boddie CR. An estimate of the global health care and lost pro-ductivity costs of dengue. Vector Borne Zoonotic Dis 2014;14(11):824e6.[11] Shepard DS, Undurraga EA, Betancourt-Cravioto M, Guzman MG, Halstead SB, Harris E,et al. Approaches to refining estimates of global burden and economics of dengue. PLoSNeglected Trop Dis 2014;8(11):e3306.[12] Barnighausen T, Bloom DE, Cafiero ET, O’Brien JC. Valuing the broader benefits of denguevaccination, with a preliminary application to Brazil. Semin Immunol 2013;25(2):104e13.[13] Constenla D, Garcia C, Lefcourt N. Assessing the economics of dengue: results from asystematic review of the literature and expert survey. Pharmacoeconomics. 2015;33(11):1107e35.[14] Shepard DS, Undurraga EA, Halasa YA. Economic and disease burden of dengue inSoutheast Asia. PLoS Neglected Trop Dis 2013;7(2):e2055.[15] Shepard DS, Coudeville L, Halasa YA, Zambrano B, Dayan GH. Economic impact ofdengue illness in the Americas. Am J Trop Med Hyg 2011;84(2):200e7.[16] Undurraga EA, Betancourt-Cravioto M, Ramos-Castaneda J, Martinez-Vega R, Mendez-Galvan J, Gubler DJ, et al. Economic and disease burden of dengue in Mexico. PLoSNeglected Trop Dis 2015;9(3):e0003547.[17] Castaneda-Orjuela C, Diaz H, Alvis-Guzman N, Olarte A, Rodriguez H, Camargo G, et al.Burden of disease and economic impact of dengue and severe dengue in Colombia, 2011.Value Health Reg Issues 2012;1(2):123e8.[18] Shepard DS, Halasa YA, Tyagi BK, Adhish SV, Nandan D, Karthiga KS, et al. Economicand disease burden of dengue illness in India. Am J Trop Med Hyg 2014;91(6):1235e42.Economic and political aspects of Dengue virus disease Chapter | 9 157[19] Organization WH. Fifth meeting of the Emergency Committee under the InternationalHealth Regulations (2005) regarding microcephaly, other neurological disorders and Zikavirus. 2005.[20] Rodriguez-Barraquer I, Mier-y-Teran-Romero L, Schwartz IB, Burke DS, Cummings DA.Potential opportunities and perils of imperfect dengue vaccines. Vaccine 2014;32(4):514e20.[21] Hadinegoro SR, Arredondo-Garcia JL, Capeding MR, Deseda C, Chotpitayasunondh T,Dietze R, et al. Efficacy and long-term safety of a dengue vaccine in regions of endemicdisease. N Engl J Med 2015;373(13):1195e206.[22] Villar L, Dayan GH, Arredondo-Garcia JL, Rivera DM, Cunha R, Deseda C, et al. Efficacyof a tetravalent dengue vaccine inchildren in Latin America. N Engl J Med2015;372(2):113e23.[23] Capeding MR, Tran NH, Hadinegoro SR, Ismail HI, Chotpitayasunondh T, Chua MN, et al.Clinical efficacy and safety of a novel tetravalent dengue vaccine in healthy children inAsia: a phase 3, randomised, observer-masked, placebo-controlled trial. Lancet 2014;384(9951):1358e65.[24] Durbin AP, Kirkpatrick BD, Pierce KK, Elwood D, Larsson CJ, Lindow JC, et al. A singledose of any of four different live attenuated tetravalent dengue vaccines is safe andimmunogenic in flavivirus-naive adults: a randomized, double-blind clinical trial. J InfectDis 2013;207(6):957e65.[25] Osorio JE, Huang CY, Kinney RM, Stinchcomb DT. Development of DENVax: a chimericdengue-2 PDK-53-based tetravalent vaccine for protection against dengue fever. Vaccine2011;29(42):7251e60.[26] Coller BA, Clements DE, Bett AJ, Sagar SL, Ter Meulen JH. The development of re-combinant subunit envelope-based vaccines to protect against dengue virus induced disease.Vaccine 2011;29(42):7267e75.[27] Beckett CG, Tjaden J, Burgess T, Danko JR, Tamminga C, Simmons M, et al. Evaluation ofa prototype dengue-1 DNA vaccine in a Phase 1 clinical trial. Vaccine 2011;29(5):960e8.158 Dengue Virus DiseaseChapter 10Treatment and therapeuticagents and vaccinesSargun Singh Walia1,2, Ngan Nguyen3, Mohammad F. Ishfaq4,51Clinical Research Fellow, Zeenat Qureshi Stroke Institute, St. Cloud, MN, United States;2Department of Neurology, University of Missouri, Columbia, MO, United States; 3Department ofInternal Medicine, University of Tennessee Health Science Center, Memphis, Tennessee, UnitedStates; 4Resident physician, University of Tennessee Health Science center, Memphis, Tennessee,United States; 5Zeenat Qureshi Stroke Institute, St. Cloud, MN, United StatesManagement of Dengue viral illnessThe mainstay of management for Dengue viral illness is supportive care. Thereis not antiviral treatment directed toward Dengue viral illness. As the mainpathogenesis of Dengue viral illness is fever, plasma leakage and shockmainstay therapy is directed toward managing these.Overall assessmentAs mentioned in any treatment guidelines published by World Health Orga-nization (WHO; 2009) and World Health Organization South East AsiaRegional Office, the first step in management involves taking a history fromthe patient about the symptoms along with past medical and family history.A good history should include the onset of fever, mucosal bleeding or anyinternal organ bleeding, any urinary symptoms, change in mental status,seizure, dizziness and recent travel history including travel to or living inDengue viral illness-endemic areas. Assessment of warning signs, coexistingconditions and poor social circumstances is important in determining the levelof care (Table 10.1).Second part of the initial assessment is physical examination. A thoroughphysical examination includes assessment of volume status, hemodynamicstatus, mental state, examination for rash and bleeding manifestation, checkingfor abdominal tenderness, hepatomegaly or ascites, any signs of respiratorydistress including tachypnea, kussmaul breathing sign or pleural effusion onimaging. A mental assessment should also be performed along with physicalexamination (Table 10.2).Dengue Virus Disease. https://doi.org/10.1016/B978-0-12-818270-3.00010-2Copyright © 2020 Elsevier Inc. All rights reserved. 159https://doi.org/10.1016/B978-0-12-818270-3.00010-2Laboratory testA complete blood count and comprehensive metabolic panel should beperformed at the first visit to assess for any signs of leukopenia andthrombocytopenia as these make the diagnosis of Dengue viral illness highlylikely. A rapid decline in platelet count with an increase in hematocrit abovebaseline indicates plasma leakage and progress to critical phase of the dis-ease. In addition, patient with Dengue viral illness may develop electrolyteabnormalities, mildly elevated transaminitis and elevated serum creatininelevel.After a thorough examination and laboratory workup, clinicians shoulddetermine whether Dengue viral illness is the most likely disease, and phase ofthe disease (febrile, critical and recovery phase).TABLE 10.1 List of Warning Signs, Coexisting conditions and Poor SocialCircumstances.Warning signsCoexistingconditionsPoor socialcircumstancesl Abdominal painl Persistent vomitingl Clinical fluid accumulationl Mucosal bleedl Lethargy, Restlessnessl Liver enlargement >2 cml Laboratory showing elevated hematocritwith rapid declined platelet count.l Infancyl Pregnancyl Obesityl Diabetesmellitusl Hypertension.l Living alonel Living far awayfrom hospital.TABLE 10.2 Important items of history and physical examination of Dengueviral illness patients.History Physical examinationl Fever onsetl Oral fluid intakel Diarrheal Urine outputl Mental state change/seizure/dizzinessl Family historyl Travel historyl Sexual historyl Drug usel Mental state assessmentl Hydration state assessmentl Hemodynamic status assessmentl Look for rash and bleeding manifestations.l Tourniquet testl Respiratory: Look for tachypnoea/pleuraleffusionl Gastrointestinal: Abdominal tenderness/hepatomegaly/ascites160 Dengue Virus DiseaseThe World Health Organization handbook for clinical management ofDengue 2012 has divided patients into three groups to help aid in the diseasemanagement.The groups are:1. Group A (outpatient management)dDengue viral illness without warningsigns2. Group B (inpatient management)dDengue viral illness with warning signs3. Group C (emergency management)dsevere Dengue viral illnessGroup A (outpatient management)Patients who do not present with any warning signs or coexisting conditionsand able to tolerate adequate volume of oral fluid and pass urine every 6 h canbe managed in the outpatient setting [1]. Serial cell count using completeblood count should be followed. An increase in hematocrit and a decline inplatelet count indicate presence of plasma leakage and increased risk ofbleeding complication. Patients should be monitored closely for any warningsigns as they may decline rapidly in the critical phase. Patients who presentwith symptoms of Dengue viral illness for more than 3 days should bemonitored on a daily basis for disease progression. Evaluations are done basedon reduction in white blood cells and platelet counts along with an increase inhematocrit levels. Patients with a stable hematocrit level can be managed asoutpatient.Adequate oral fluid intake is the mainstay of management in these patientsand can decrease the rate of hospitalization. Oral fluids in the form of coconutwater, rice water, barley water, oral rehydration solutions (ORS), fruit juices,and soup are advised. Carbonated drinks should be avoided as they are rich insugar content and can exacerbate physiological stress hyperglycemia presentin Dengue viral illness patients. Oral fluids should be taken until the urinaryfrequency increase to 4 to 6 times per day. A record of fluid intake and urineoutput is helpful. Bed rest is advised to minimize any trauma or bleedingcomplications.High fever should be controlled with the use of paracetamol. Maximumdose in children is 10 mg/kg/dose, with an upper limit of 3e4 times in a 24 hperiod. Maximum dose of paracetamol in adults is 3 grams/day in a 24 hperiod. If the fever is still not controlled then sponging can be done with tepidwater. A randomized, double-blind, placebo-controlled trial was conducted totest the use of paracetamol in children suffering from fever (>38�C perrectum) less than 4 days. It was found that children taking paracetamoldemonstrated improvement in activity and comfort [2]. Aspirin and nonste-roidal anti-inflammatory agents are not advised as they can lead to gastritis andbleeding complications.Treatment and therapeutic agents and vaccines Chapter | 10 161Specialcare should be taken to look for development of any signs ofvolume depletion, dry mouth, cold extremities or severe abdominal pain,persistent vomiting, mucosal or internal organ bleeding such as difficultbreathing, decreased urination frequency, black stools, or coffee groundvomiting warrant prompt clinical evaluation. Patient should be instructed toreturn to hospital immediately if these warning signs develop (Table 10.3).Group B (inpatient management)These are the patients that present with warning signs or have preexistingconditions or social circumstances that can make management of Dengue viralillness a complicated as an outpatient. The most important key to managementin these patients is fluid resuscitation to prevent progression to a state of he-modynamic shock.Appropriate use of intravenous fluids can be vital in the management ofthese patients. The initial step is to get a baseline hematocrit level to use asreference to manage the intravenous fluid therapy. Intravenous fluid therapy isstarted in the form of 0.9% normal saline or lactated Ringer. Therapy is startedat an initial rate of 5e7 mL/kg/h for 1e2 h, then decreased rate of 3e5 mL/kg/h for 2e4 h, with further decrease to 2e3 mL/kg/h or less based on clinicalresponse of the patient.TABLE 10.3 Dengue viral illness Group A management algorithm.ClassificationDengue viral illness without warning signsGroup A (outpatient care)Criteria Patients without warning signs orl Patients able to take oral fluidsl Patients able to urinate at least once every 6 hLab tests l Full blood countl HematocritTreatment l Bed restl Fluid intakel Paracetamoll Stable hematocrit patients can be sent homeMonitoring l Daily disease progressionl Reduction of white blood cellsl Defervescencel Warning signsl Immediate return to the hospital if warning signs developAdapted from World Health Organization: Handbook for Clinical Management of Dengue (2012).162 Dengue Virus DiseaseAfter fluid replacement, the hematocrit should be repeated. If there is no orminimal increase then the fluids can be maintained at a reduced rate of2e3 mL/kg/h for another 2e4 h. But if the vitals are not stable or the he-matocrit is increasing then the rate of fluid should be increased to 5e10 mL/kg/hour. The clinical status should be reassessed and hematocrit levelsreviewed again to change the fluid rate accordingly.The minimum intravenous fluids required to maintain perfusion and urineoutput of 0.5 mL/kg/hour should be given to all patients. Intravenous fluids areusually not needed after 24e48 h. As the urine output and oral fluid intakeimproves the fluids should be reduced at a gradual rate.Health care providers should monitor these patients suffering with Dengueviral illness with warning signs. Fluid balance charts should be maintained indetail. Peripheral perfusion and vital signs should be monitored every 1e4 huntil the patient is stable and out of the critical phase. Urine output should bemonitored every 4e6 h. Hematocrit levels should be recorded before startingfluid replacement therapy and also after administering fluids. Then it should berepeated every 6e12 h. Renal profile, liver profile, coagulation studies, andblood glucose should be recorded as indicated. There is no clinical advantageof colloid over crystalloid [3].In patients of Dengue viral illness with coexisting conditions, in theabsence of warning symptoms, the treatment plan is different. The patients areadvised to take oral fluids but if not tolerated then intravenous fluids are startedin the form of 0.9% saline or Ringer lactate with or without glucose. The rateof fluid is decided based on the ideal body weight (Table 10.4).For adults with ideal body weight (IBW) > 50 kg, 1.5e2 mL/kg can beused as quick calculation for maintenance of fluid per hour.For adults with ideal body weight (IBW) < 50 kg, 2e3 mL/kg can be usedas quick calculation for maintenance of fluid per hour.Healthcare providers should monitor these patients for temperature pattern,fluid intake and output volumes, volume and frequency of urine output, whiteblood cell count, platelet count, and hematocrit. Renal profile, liver profile,coagulation studies, and blood glucose should be recorded as indicated(Table 10.5).Group C (emergency care)Patients with severe Dengue viral illness can be categorized based on thefollowing:l Severe Dengue viral illness leading to shock. This is characterized bycirculatory collapse due to an increased systemic vascular permeability andsevere plasma leakage.l Respiratory distress due to fluid accumulation caused by severe plasmaleakage.Treatment and therapeutic agents and vaccines Chapter | 10 163l Severe hemorrhagic symptomsl Severe organ impairmentPatients presenting with these symptoms should be immediately admittedin a medical facility capable of blood transfusion. The mainstay in man-agement is the use of fluid replacement. The preferred intravenous fluidtherapy is crystalloid solution. Crystalloid solution used should be isotonic.Plasma leakage should be replaced immediately and rapidly with enoughvolume of crystalloid solution so that effective circulation is maintained.Colloid solution is the preferred fluid replacement therapy in cases withhypotensive shock. Hematocrit levels are assessed before and after the fluidresuscitation.Fluid replacement therapy is continued for at least 24e48 h to maintaineffective circulation. Blood group and cross match should be in all patients inshock due to Dengue viral illness. In patients in whom severe bleeding isoccurring or the patients with unexplained hypotension and suspicion of severehemorrhage prompt treatment with blood transfusion is recommended.As a trial, boluses of 10e20 mL/kg fluid are administered for a shortduration of time under vigilant supervision to look for development of pul-monary edema. It is important to keep these boluses of fluid free of glucose. Inpatients suffering from severe shock due to Dengue viral illness, the input offluid is typically greater than output seen, so the fluid input/output ratio cannotbe used to guide the fluid resuscitation therapy.The targets for fluid resuscitation in these patients are:l Improving peripheral and central circulation. This is seen by reducingtachycardia, improved pulse volume, blood pressure, and warmextremities, <2 s capillary refill time.TABLE 10.4 Guide for maintenance intravenous fluid infusion.Calculator for maintenance of intravenous fluid infusionNormal maintenance fluid per hour based on the Holliday Segar formulal 4 mL/kg/h for first 10 kg body weightl þ 2 mL/kg/h for next 10 kg body weightl þ 1 mL/kg/h for subsequent kg body weightIn overweight/obese patients maintenance fluid is based on ideal body weight (IBW),using the following formula:Female 45.5 kg þ 0.91(heighte152.4) cmMale: 50.0 kg þ 0.91(heighte152.4) cmAdapted from World Health Organization: Handbook for Clinical Management of Dengue (2012).164 Dengue Virus Diseasel Improvement in end-organ perfusion in the patient. This is seen byimprovement in the conscious level of the patients. These patients typicallybecome more alert and less restless as the end-organ perfusion improves.Urine output improves as well to >0.5 mL/kg/hour and the metabolicacidosis also improves.TABLE 10.5 Dengue viral illness Group B management algorithm.ClassificationDengue viral illness with warning signsGroup B (inpatient care)Criteria Patients with any of thefollowing:l Coexisting conditionslike diabetes mellitus,old age, pregnancy,and infancyl Social: Living alone,house far from hospitalPresence of warning signs:l Abdominal pain or tendernessl Persistent vomitingl Clinical fluid accumulationl Mucosal bleedingl Lethargy/restlessnessl Liver enlargement >2 cml Increase in hematocritLab tests l Full blood count including hematocritTreatment l Encourage fluid intakel If not possible then

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