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DEDICATIONThis thesis is dedicated to:I. God Almighty, the giver ofall good and perfect gifts2. Oluwabunmi Mercy Baba and Abdul Naafiu Otoobong Baba (children) fortheir love and patience for me

CERTIFICATIONI certify that this work was carried out by Mrs. Marycelin Mandu Baba in theDepartment ofVirology, College of Medicine, University of Ibadan, Ibadan, Nigeriaand Institut of Pasteur De Dakar, Senegal.(SUPERVISOR)J. A. Adeniji, Ph.D.Senior LecturerDepartment ofVirology, College ofMedicine,University ofIbadan, Ibadan, NigeriaDate / E> (_/_?---+I_~_t>_O_.£ _ll\

ACKNOWLEDGMENTI would like to express my very sincere gratitude to my Supervisor, Dr J, AAdeniji for his wonderful interest in the work and constant encouragement towardsthe completion of the work. He a llowed me free access to himself and contributedimmensely towards the completion of the project. I remain eternally grateful to himfor everything and trust God in His infinite mercy to continually bless him and hisfamily abundantly.I would also like to a ppreciate with to appreciate with t hanks and place 0 nrecord the assistance of my Head of the Department, Prof Olaleye. He assisted me inso great measure that words could not express my indebtedness to him. May GodAlmighty bless him richly?My sincere appreciation is hereby extended to Prof. F. D Adu, who gave meboth moral and technical support. He diligently guided me towards the completionof the work. May he remain blessed in all his endeavors? I am equally grateful to Dr(Mrs.) G. Odaibo for her moral and technical assistance towards the completion ofthis work. May God greatly reward her for a work well done.This work would not have been possible without the financial support Ireceived from the Third World Organization for Women in Science (TWOWS). Iconfess tothe fact t hat it w as rare 0 pportunity well utilized a nd I hereby place 0 nrecord my heartfelt appreciation of the award. TWOWS,.I say more grease to yourelbow. May the good seed you are sewing towards the development of Science andWomen in the Third World countries continue to yield positive fruits.Dr. Christian Mathiot (former) and Dr Simon (current), Directors of! nstitutPasteur De Dakar, (IPD), Senegal, hosted and provided me with all that I neededduring my Sojourn in Senegal. My two Supervisors in !PD, Dr. Marie-Francois Saronand Dr. Ousmane Diop, I am eternally grateful to them for their support, patience andexpertise guidance towards the completion of this project. I remain greatly indebtedto you all. And to all the staff of IPD especially Mme Mbayne Niag, Mme Mondo,Modu Diagne, Mme Girault, Merry Payne, Aishatu, Ahmed, Rougy, Ousmane Faye,Ka, to mention, I say a 'big' thank you to you all for all your support andunderstanding you accorded me during my stay at Dakar. "Merci beaucoup.'I received a very warn] and wonderful moral support from the staff of theNigerian Embassy, Dakar, Senegal especially, Mrs. O. Chukwura a friend, sister andIV

Guardian, Mrs. Hauwa Yakubu, Prof F. Oyekomi and the entire member ofInternational Association of African Women (IAAW). Their warm and hand offriendship extended to me made my stay in Dakar a memorable one. May Godcontinue to bless you all for your sacrifices towards me.I also, appreciate the warm and wonderful co-operation I enjoyed from manystaff members, students, and friends, in the Department of Virology, University ofIbadan, among whom are Mr. Nwaogu, Mr. Francis, Mr. Maxwel, Forbi lC, Funke,Adewumi M. 0., Donbraye Emmanuel, Dr. A. S. Bakarey.I wish to extend my gratitude to the Head, of the general Out PatientDepartment, and all her staff especially Mr. Peter, the entire staff of the Departmentof Microbiology, especially Or. Oni, University College Hospital, Ibadan, theManagement and laboratory staff of Adeoyo Hospital Ibadan, Federal Health center,Gombe . especially Kudi Ayuba, Bayero University teaching Hospital, Kanoespecially, University of Calabar Teaching Hospital especially Or, Mrs. Ekanem andImo King, Gwagwalada Hospital Abuja, Shalom Medical Laboratories especially Mr.G Okara, and the entire Management and staff of Immunology and Haematologylaboratory of University of Maiduguri Teaching Hospital especially B.B. Ajayi andMr. Obitremendous support.for assisting me in collecting all the samples. I say thank all, for yourTo the sponsor of thisimmensely grateful to the authorities for this opportunity.programme, University of Maiduguri, I remain. .Unfortunately, space will not permit me to list here all the people that havecontributed in one way or the other to make the completion of this project a reality.However, there are names I cannot but mention a few. These include my belovedmother, M adame M ayen B assey Nkanga, who su pported me in everyway humanlypossible. I pray that God will grant her long life and more blessings to reap the fruitsof her labour. My sisters, Rita Umoren and Simon Umoren, Irene and ClementBassey a nd my brothers, Or. Ekong Nkanga, Mr. Essicn Nkanga, and I mo Nkangawere wonderful in their own small way. My friends like Mr. and Mrs. LawrenceGboko, Emmanuel Ntuen, Alice, Fati and Or. Andrew Roberts- Kwasari all assistedme in one way or the other, in the course ofmaking this project a reality.My children, 0 luwabunmi a nd Abdul N aafiu, showed lots 0 f understandingand support for which I remained grateful. The entire staff of the Polio eradicationNetwork program especially National Polio Laboratory, Maiduguri, Nigeria especiallyv

Prof. 0 Tomori, the staff of WHO national Polio laboratory, Maiduguri particularlyProf T.O. Harry, David Bukbuk, Lamide, Monilade who supported me in one way orthe other. I remain deeply grateful to you all.For everything, I give God all the Glory for great and mighty things he hasdone to me. He has been merciful, kind and faithful and most especially sparing mylife and granting my children and me good health in various circumstances. IN THEEo LORD I PLACE ALL MY TRUST.VI

ABBREVIAnONSADECDCcDNACFCFACFTCNSCSFDENDFDHFDMSODNADSSEDTAELlSAFAGDPHAHCVHIHLAIFIFNIgGIgMJE- VAX®Antibody- dependent EnhancementCenter for Disease ControlComplementary Deoxyribonucleic AcidComplement FixationComplement Fixing AntibodiesComplement Fixation TestCentral Nervous SystemCerebrospinal FluidDengue VirusDengue FeverDengue Hemorrhagic FeverDimethyl sulphur dioxideDeoxyribonucleic AcidDengue Shock SyndromeEthylenediaminetetraacetic AcidEnzyme- Linked lmmunosorbent assayFluorescentAntibodyGross Domestic ProductHaemagglutinationHepatitis C VirusHaemagglutination Inhibition. Human Leukocyte AntigenImmuIlofluorescenceInterferonImmunoglobin GImmunoglobin MFormalin Inactivated Mouse Brain Vaccineagainst closely related Japanese EncephalitisVirusesJEJEVKEDJapanese EncephalitisJapanese Encephalitis VirusKedougouVII

KFDKUNLIMAC-ELISAMHCmRNAMAFMVENANKVNSNTRODOHFORFPDRPBSPOWPRNTPSpeRRNART-PCRRPSLESLEVSPSPOTBEUSUSAVax WHOKyasanur Forest DiseaseKUI~inLouping IIIIgM Capture ELISAMajor Histocompatibility ComplexMessenger RNAMouseascitic fluidMurrayValley EncephalitisNucleic AcidNo Known VectorNon - Structural ProteinsNon - Translational RegionOptical DensityOmsic Haemorrhagic FeverOpen Reading FramePathogen derived resistancePhosphate buffered salinePowassanPlaqueReduction Neutralization TestPorcine KidneyPolymerase Chain ReactionRibonucleic AcidReverse Transcription - Polymerase ChainReactionRheumatoid factorSI. Louis encephalitisSI. Louis encephalitis VirusStructural ProteinSpondweniTick - Borne EncephalitisUnitedStatesUnitedStatesof AmericaYF - WN Chimeric VaccineWorld HealthOrganizationviii

WNFWNVYFYFVWest Nile FeverWest Nile VirusYellow FeverYellow Fever VirusIX

Title of DissertationTABLE OF CONTENTSPAGEDedication 11CertificationiiiAcknowledgementAbbreviationsTable of ContentsListofTablesListof FiguresAbstractIVVIIxXVIxviuXIX-CHAPTER ONE 11.1 Introduction I1.2 Aims and objectives 221..2.1 Specific objectives 2';':i,~ , CHAPTER TWO 32.0 Review of Literature 32.1 Historical Review ofFlavivirus Infections 32.1.1 TheOrigin of Flaviviruses 32.2 The family Flaviviridae 62.2. I Flaviviruses 62.2.2. Morphology of Flaviviruses Physical and chemical properties of Flaviviruses 72.2.4 Genome Organization ofFlaviviruses 92.2.5 Structural Proteins ofFlaviviruses The C Protein The prM and M Proteins The E protein 112,2.5.4 NonstructuraI Proteins ofFlaviviruses 122, The NS3 Protein The NS5 Protein 14X

XI2. The NS2A, NS2B, NS4A, and NS4B Proteins 142.2.6 Phylogenetic Relationships of Flaviviruses thatCorrelate With Their Epidemiology, Disease-Association and Biography 152.2.7 Infections ofFlaviviruses in arthropod vector 182.3 Dengue viruses 202.3. I Recombination ofgenetic materialsbetween dengue viruses 232.3.2 Dengue Fever virus infections in Nigeria 232.3.3 Dengue fever, Dengue hemorrhagic feverand dengue shocksyndrome 242.3.4 Antibody dependent enhancement indengue virus infections 252.3.5 Epidemiology ofdengue hemorrhagic fever 272.3.6 Molecular epidemiology ofdengue virus infections 322.3.7 The pathogenesis of dengue fever 332.3.8 Dengue virus structural differences thatcorrelate with pathogenesis 342.3.8.I Significance ofE protein differences 342.3.8.2 Signi ficance of prM differences 362.3.8.3 Significance ofNS4b differences 362.3.8.4 Signi ficance of Non transnational region differences 372.3.9 Host genetic factors that may influenceseverity ofdengue infections 392.3.I0 Clinical manifestation ofdengue fever 392.3.11 Clinical manifestation ofdengue hernorrhagicfever and dengue shock syndrome 402.3.I2 Treatment ofdengue virus infections 412.4 West Nile virus infections in Nigeria 412.4.I The pathogenesis ofWest Nile virus 412.4.2 West Nile fever, and West Nile encephalitis 422.4.2.1 The aetiological agent 422.4.3 The clinicalmanifestations of West Nile fever 452.4.4. The external habitats ofWest Nile virus 46

3.4.6 Enzyme immuno assay for the detection ofIgG antibodies to dengue, West Nile andYellow fever viruses 673.4.6.1. The principle ofthe test 673.4.6.2 The test procedure 683.5 Vectoral studies 683.5.1 Reasons for the vectoral studies 683.5.2 Methods used in catching the mosquitoes 693.5.3 When and where the mosquitoes were trapped 693.5.5 Virus isolation from field caught mosquitoes 713.5.5. I Cell preparation 713.5.5.2 Passage ofthe cells 713.5.5.3 Cell freezing for storage 713.5.5.4 Preparation ofmosquito suspension 723.5.5.5 Innoculation of mosquito suspension onto monolayerofAP61 723.5:5.6 Indirect immuno fluorescence antibody test 733. The principle ofthe test 733. Test procedure 733.6 RT-PCR in sera and mosquitoes 743.6.1 The principle ofthe test 743.6.2 Semi-nested Rt-PCR for dengue viruses 743.6.2.1 The extraction ofRNA from serum/mosquitoes suspension 753.6.2.2 Selection ofoligonucleotides primers for dengue viruses 763.6.2.3 The first round ofamplication in PCR fordengue viruses (TTTAN) 763. The principle ofthe test 763. The procedure 763. Mixture ofreactants for amplification 773.6.2.4 Dengue virus typing by second -round amplificationwith type specific primers 773. The principle ofthe test 773. The procedure ofthe second amplification 783. Polymerace chain reaction by multiplex mcthod 78XIII Mixture ofreactant 783. Second polymerace chain reaction by single method 793.6.3 Reverse- Transcription- polymerace chain inCulex mosquitoes for West Nile virus 813.6.3.1 Reverse transcription (performed separately from PCR) 813. The principle 813. Preparation of the Mix 813. The procedure 813.6.3.2 Polymerace chain reaction for the detection forWest Nile virus in Culex mosquitoes/sera 813. The principle 813. The procedure 823. Preparation ofthe Mix 823. Preparation of amplification product 823.6.3.3 RT-PCR for West Nile virus in onereaction vessel (TITAN) 833.6j.3.1 Mixture ofreactant for amplification 833.6.4 Identification ofamplification productsby gel electrophoresis 85CHAPTER 4 864.0 Results 864.1 Optimization ofantigens and hyperimmune mouse ascitic 864.2 Pattern ofdengue virus infections in Nigeria 914.2.1 IgM capture ELISA for dengue virus 914.2.2 Seasonal distribution ofdengue virus IgM antibodiesin Sahel savanna zone 954.2.3 Age distribution ofpatients with IgM to dengue viruses 994.2.4 Gender distribution ofpatients with IgM to dengue viruses 1014.2.5 The prevalence ofDEN IgG antibodies in3different ecological zones in Nigeria 1034.3 IgM capture ELISA for West Nile virus 1074.3.1 The seasonal distribution ofWNV IgM antibodies I 104.3,2 Age distribution of patients with WNV IgM antibodies 114XIV

xv4.3.3 Gender distribution of patients with WNV IgMantibodies 1164.3.4 Seasonal distribution of WNV IgG antibodies I 184.4 The prevalence ofYFV IgM and IgG antibodies 1214.5 Cross reactivity between DEN, WNV, andYFV by MAC-ELlSA 1244.6 The results ofMAC-ELlSA with problematic seraAftertreatment to remove non-specific immunoglobulins 1264.7 Dilution ofDEN positive IgM to determinethe infecting serotype 1274.8 Vectoral studies 1294,8.1 Virus isolation from mosquitoes 1294.8.2 RT-PCR on mosquitoes 1294.8.2.1 RI-PCR for DEN with Aedes mosquitoes 1324.8.2.2 RI-PCR for WNV with Culexmosquitoes 137CHAPTERS 1435.0 Discussion 1435.1 Summary and Conclusion 1586.0 References 1607.0 Apendix I: Inoculation ofmice with seed virus 1997.1 Apendix 2: Preparation ofreagents 2017.2 Apendix 3: Pooled mouse ascitic fluid fordifferent arboviruses 2047.2.1 Apendix 4: IgM ELlSA results after the treatmentof problematic sera 205

SERIALNO.LIST OFTABLESPageI Membersof Flaviviridae 52 The ecological zones in Nigeria where sera were obtained 583 Serum samples collected at different seasons of theyear from Sahel savanna zone (Maiduguri), Nigeria 604 The mosquito vector and the ecological zoneswhere they were caught 705 Oligonucleotide primers used to amplifyand type dengue viruses 806 Oligonucleotide primers used to amplifyand type WNV 847 The chessboard titration for DENI-4 against thecorresponding hyperimmune mouse ascitic l1uid 878 Titration of DEN 1-4 by IgM capture ELlSA 899 Distribution oflgM and IgG antibodies againstdengue viruses in Nigeria 9210 Dengue virus infections in different ecologicalzones in Nigeria 9311 Seasonal distribution ofdengue virus infections inSahel savannazone 9612 Monthly distribution ofdengue IgM positive sera 9813 Age distribution ofpatients with IgM to dengue viruses lOO14 Gender distribution ofpatients with IgM to DEN viruses 102IS The Prevalence ofDEN IgG antibodies according to zones 10416 Seasonal distribution ofDEN IgG antibodies 10617 The prevalenceofWNV IgM antibodies in patients with fever 10818 Seasonal pattern ofWNV IgM in patients with fever I1119 Monthly distribution ofWNV IgM antibodies 11320 Age distribution ofWNV IgM positive patients lIS21 Gender distribution of WNV IgM positive patients 11722 The prevalenceofWNV IgG according to seasons 11923 The prevalenceofYellow Fever IgGaccording to seasons 122XVI

24 A table showing the level ofcross reactivitybetween DEN, WNV, and YFV by IgM ELISA 12525 Dilution od DEN positive IgM todetermine the infecting DEN serotype 12826 The summary ofRT-PCR on Aedes species 13327 T-PCR with DEN IgM positive sera 13428 Comparison ofRT-PCR with WNV IgM positive sera 140XVII

SERIALNOUST OF FIGURESPAGEI. Titration of antigen and mouse ascitic fluid by chessboard method 882. Titration of antigen and mouse ascitic fluid by IgM captureEL/SA 903. DENIgMand the corresponding IgG antibodies 944. Seasonal distribution of/gM to DENsin Sahel savannazone 975. Zonal distribution ofDEN IgG antibodies 1056. West Nilevirus IgM and the corresponding IgG antibodies 1097. Seasonal distribution ofWNV IgM antibodies 1128. Seasonal distribution ofWNV IgGantibodies 1209. Seasonal distribution ofYFV IgGantibodies 12310. Firstamplification products for DEN viruses duringoptimization ofreagents 130Il. Semi-nested PCR with Aedes mosquitoes for the detectionofDEN-I-4 (Multiple) 13112. Semi-nested PCR with Aedes mosquitoes for thedetectionofDENI-4 13513. Semi-nested PCR with Aedes mosquitoes for thedetection of DENI-4 continued 13614. RT-PCR on culex mosquitoes for WNV 13815. RT-PCR on culex mosquitoes for WNV continued 14916. RT-PCR on WNV positive IgM sera 14117. Comparison ofTITAN and RT-PCR 142xviii

ABSTRACTDengue viruses (DENs) and West Nile virus (WNV) are etiologic agents offebrile illness, which often lead to hemorrhagic fever/shock syndrome in DENs ormeningoencephalitis with WNV. The principal vector of DENs is Aedes aegypti whileCulex and Mansonia species transmit WNV. There are four distinct serotypes ofDENs (1-4) and infection by one serotype does not protect against subsequentinfection by another. Since the initial symptoms of these diseases mimic other febrileillnesses, diagnosis of these viruses could be confusing. Consequently, misdiagnosedcases could result in high morbidity, complications and mortality. This study wastherefore designed to determine the significanceofthese viruses in febrile patients.A total of 1948 serum samples were collected from June 2001 to July 2002from six ecological zones [Grass savanna (Abuja), Rain forest (Ibadan), Woodedsavanna (Gornbe), mangrove forest (Calabar), and Sahel savanna (Maiduguri)] inNigeria. In addition, 973 sera were obtained at different seasons [Hot (January toApril), rainy (May to August) and cold (September to December)] of the year fromSahel savanna zone. Crespuscular/scoop net catches of 3395 mosquitoes from Rainforest and Sahel savanna zones were made, identified and pooledby species as Aedes(295) Culex (3050) and Mansonia (50) species. MAC- ELISAwas used to test all thesera for IgM and IgG. Reverse Transcriptase- Polymerase Chain Reaction (RT-peR)was also used to analyze the IgM positive sera for specific viral RNA. Each mosquitopool was subjected to RT-PCR and inoculated into AP61 'cell line for virus isolation.Detection of the virus in AP61 was by Indirect Immunofluorescence Antibodytechnique (IFAT) using a panel of polyvalent mouse ascitic hyper immune sera forspecific arboviruses.Thirteen (0.67%) ofthel948 sera were positive for DEN land 2 IgM from 4(Rain forest, Mangrove forest, Grass and Sahel savanna) zones. Twelve (1.2%) and5(0.5%) of973 sera had IgM for WNV and DENs respectively. Six (33.3%) of 18and4(33.3%) of 12 of the IgM positive sera had detectable RNA to DENs and WNVrespectively. Two (0.2%)ofthe 973 patients had mixed infections ofDEN and WNV.DEN IgM was significantly higher during the rainy season (1.3%) than cold (0.3%).However, WNV IgM was more during the cold (2.9%) than rainy (0.1%) season.Neither DEN nor WNV IgM was detected during the hot season. A high proportion(>59%) of the study population in four ecological zones had IgG to DENs but slightlyXIX

lower in Sudan (32.6%) and Grass savanna (38.1 %). Also, DEN, YFV and WNV IgGwere detected in 86.2%, 86.9% and 80.6% of the 973 patients respectively. No viruswas isolated from mosquitoes after two passages in AP6J. However, DENs and WNVRNAs were detected in Aedes species from the rain forest and Culex species fromSahel savanna zones respectively.Infections by theses viruses are often unrecognized in different parts of thecountry. Therefore there is need to include DENs and WNV routinely in thedifferential diagnosis of febrile illness in Nigeria.KEYWORDS: Dengue, West Nile viruses, IgM, Nigeriaxx

1.1 INTRODUCTIONCHAPTER ONEArboviruses cause some of the most devastating diseases known to humanand veterinary medicine. There are over 500 identified arboviruses, most of whichare members of the family Togaviridae, Flaviviridae, Bunyaviridae, Rhabdoviridae,and Reoviridae (Blair et al., 2000). Dengue viruses (DENs) and West Nile viruses(WNVs) belong to the family Flaviviridae and are maintained in natural cycles inwhich they multiply in a hematophagous mosquito. They are transmitted in saliva toa vertebrate host when the arthropod takes a blood meal.Generally the information on the current status of arboviral infections andtheir vectors in Nigeria is poor. In other countries, several arboviruses are frequentlyconsidered in the etiology of acute febrile illness. Surprisingly, most healthinstitutions in Nigeria lack appropriate diagnostic facilities for this group of virusesand seem to attach less importance to viral fever. Yet many cases in Africa thatpresent with fever are documented as pyrexia of unknown origin (PUO), especiallyif they fail to respond to anti-malaria or anti- microbial drugs. Moreover, the earlysymptoms of arbovirus infections mimic malaria, typhoid, measles and influenza,thereby rendering the elinical diagnosis of these cases very confusing.In addition, few atypical clinical features had been reported to cause problemin diagnosis. For example, atypical clinical features in patients with hepaticencephalopathy and dengue encephalopathy presentations were initially thought tohave hepatitis or encephalopathy but later tested pos!tive for dengue viruses, Similarsituations may be occurring in Nigeria where many cases of DHF are mistaken fortyphoid fever. Reports so far had demonstrated the existence of dengue (Moore etal.,1975) and West Nile virus (Kemp et al.,I973) in Nigeria. Nearly 3 million casesof DHF have been officially reported worldwide with more cases occurring in Asia(Monath and Heinz 1996). Surprisingly, no epidemic of DF or DHFfDSS has beenreported in Nigeria despite the fact that factors (mosquito vectors, susceptible hosts,and heterotypic dengue infection) responsible for the global re-emergence of denguevirus infections are common in the country. It may be recalled that both viralinfections have the non-specific prodromal and specific phases involvinghemorrhagic/neurological symptoms. The former phase could also be easilyconfused with other hyperendemic bacteria (typhoid) and protozoal infections(malaria) in the environment. As observed in India (Voluntary health Association ofIndia- Unpublished data), inadequate knowledge and the experience of general

2'-I.v I! I\'; , cl. aninfluence the reports, diagnosis and management of these cases.West Nile virusinfection could be so non-specific that it often escapes medical attention. In suchsituations, arbovirus infections are quite often misdiagnosed and so, inappropriatelytreated. Consequently these cases are often missed out, resulting in high rate ofmorbidity, complications and mortality. There is need to include these viruses in thedifferential diagnosis of febrile illnesses. This speculation is supported by the reportthat virological and vector surveillance serve as early warning system for anyoutbreak of arboviral diseases. Such situations underline the need for continuingmedical education for both doctors and paramedical workers besides activesurveillance. Therefore in this study, febrile cases suspected of malaria/typhoid feverwere used to survey for dengue, Yellow fever and West Nile virus infections inNigeria.1,2 AIMS AND OBJECTIVESThis study was designed to survey the epidemiology of arboviral infections, withparticular reference to Dengue viruses (DENs) and West Nile viruses (WNV) inNigeria1.2.1 Specific objectives were to:1. To determine the current status of arboval infections with particularreference to DENs and WNV in Nigeria.2. Determine the circulating serotypes of Dengue viruses in different ecologicalzones in Nigeria To assess the effect of seasonal changes on the prevalenceof these viral infections in Nigeria4. To assess the role of the mosquito vectors in the epidemiology of arbovirusinfections in Nigeria5. To assess the role of the mosquito vector in the epidemiology of arbovirusinfections in Nigeria.6. To determine the endemicity of these Flaviviruses in Nigeria bydemonstrating the presence of IgG antibodies7. To compare the prevalence of known common Flavivirus infections e.g.Yellow fever, with the less common Dengue and West Nile

CHAPTER TWO2.0 LITERATURE REVIEW2.1 HISTORICAL REVIEW OF FLAVIVIRUS INFECTIONS2.1.1 THE ORIGIN OF FLAVIVIRUSESYellow Fever Virus (YFV) is the prototype of the family Flaviviridae, (hencethe name derived from Latin word for 'yellow'). In the 20 th century, about 350 yearsafter the first clinical description of the disease, YFV was the first filterable agentshown to cause a human disease and the first virus proved to be transmissible by anarthropod vector (Zanotto et al., 1996). YF was the first Flavivirus to be isolated(in1927) and cultivated in -vitro (in 1932). Moreover, during the first decade of thiscentury, dengue viruses (DENs) were shown to be filterable agents transmitted byarthropod but were not isolated until 1943. These authors further reported thatduring 19 th and 20 th centuries, diseases characterized by meningoencephalitis wererecognized as nosologic entities. Later, there were evidences that these diseases werecaused by Flaviviruses. Such diseases include Louping ill (a disease of sheep,recognized in Scotland since 1807), Japanese encephalitis (in Japan, 1873), andAustralian X disease [now known as Murray Valley encephalitis (MVE), (Australia,1917). Thereafter, between 1931 and 1937, the viruses responsible for Louping ill,St Louis encephalitis (SLE), Japanese encephalitis (JE), and tick-borne encephalitis(TBE) were isolated. (Monath and Heinz, 1996). In that report, although initiallythey were believed to be unrelated agents, it was lat~r discovered that neurotropismand arthropod transmission were common among these viruses (Monath and Heinz,1996). Furthermore, in late 1930s and early 1940s, these authors demonstratedrelationships between St, Luis encephalitis (SLE) viruses, Japanese encephalitisvirus (JEV), and West Nile virus (WNV) by neutralization and complement fixationtests. However, with the advent of hemagglutination -inhibition test, which definesthe broadest spectrum of antigenic relatedness, Cassals and Brown, (1954) separatedthe Flaviviruses (Group B arboviruses) from the Alphaviruses (Group A viruses)and defined the cross-reactions among a set of 10 F1aviviruses. As the Flavivirusfamily grew with continued isolations of new agents from wild vertebrates andarthropods, so did the complexity of their serologic classification (Cassals, 1957).During the 1970s thirteen new F1aviviruses were isolated for the first time, and onenew virus recovered in 1980 (Monath and Heinz 1996).3

Despite the lack of serologic relationship, the group A and B arboviruses wereoriginally linked on the basis of their mode of transmission and physico - chemicalcharacteristics into a single family, Togaviridae (Monath and Heinz, 1996). Thisfamily encompasses small RNA viruses with lipid envelopes and cubid nucleocapsidsymmetry. Nevertheless, as the knowledge of the morphogenesis, biochemistry andreplication strategy of the Flaviviruses expanded; it was clear by 1984 that theirdifferences when compared to other Togaviruses were sufficiently great to placethem in a separate family (Westaway et al., 1985). Presently, Togaviridae is now aseparate family and arbovirus is not classified taxonomically. Great strides havebeen made in the molecular characterization of Flaviviruses in the last decade. Theseinclude the elucidation of the genome organization and function, replicationstrategy, and crystallographic structure (Monath and Heinz, 1996).4

TABLE 1: MEMBERS OF THE FLAVIVIRIDAE (Monath and Heinz 1996)SGROUPTYPE MEMBERFlaviviruses Tick - Borne encephalitis Central European encephalitis virusvirues (IZ',T b )(TBE-W)Far Eastern encephalitis virus (TBE-FE)Rio Bravo' (6,T)Rio Bravo virusJapanese encephalitis viruses Japanese encep~alitis (JEV)(IO,M)Kunjin virus(KUNV)Murray Valley encephalitisvirus (MVEV)St. Louis encephalitis virus(SLEV)West Nile virus(WNV)Tyuleniy (3,T) viruses Tyuleniy virusNtaya" (S,M) virusesNtaya virusUganda S (4,M) viruses Uganda S virusDengue (4,M) virusesDengue Type 1 virus (DEN-I)Dengue Type Z virus(DEN-Z)Dengue Type 3 virus(DEN-3)Dengue Type 4 virus (DEN-4)Modoc (S,U) virusesModoc virusUngrouped C (l7,M) viruses Yellow Fever virus (YFV)Bovine viral diarrhea virusesPestiviruses Classical swine fevervirusesBovine viral diarrhea virus (BVDV)Hog cholera or classical swine fever vim" II,Border disease viruses(CSFV e )Border disease virus (BDV)?,V",-',. viruses dCHepatitis CvirusesHepatitis C virus (HCV)a- Number of recognized members in eachantigenic group(Monath and Heinz 1996)b- Arthropod vectors: T. tick, M, mosquito; U unidentified or no vectorc- Arthropod vectors for some members of these groups have not been identified. The ungrouped Flavivirusesinclude mosquito- and tick- transmitted viruses as well as some with no known vectord- The Hepatitis C viruses include a large number of isolates that can be divided into several groups orgenotypes on the basis of genetic divergence (Sirnmonds et al., 1994). An official name for this genus and astandardized nomenclature for different genotypes have not yet been agreed uPOI}.e- In the Pestivirus literature, HeV has been a common abbreviation for hog cholera virus. Most recentpublication and this chapter use CSFV to avoid confusion with the human Hepatitis C virus.

2.2 THE FAMILY FLAVIVIRIDAE:The Flaviviridae was recently established as a separate family (Marshall1988). distinct from the Togaviridae and currently includes three genera, theFlaviviruses, the Pestiviruses (from Latin pestis, or plague), and the Hepatitis Cviruses (from Greek hepar, hepatos or liver) (Chiewslip et al., 1981). These threegenera have diverse biological properties, show no serological cross-reactivity andappear to be similar in terms of virion morphology, genome organization andpresumed RNA replication strategy (Monath and Heinz, 1996).Flaviviridae comprises 69 viruses, 67 of which are arthropod-borne or closerelatives of these arboviruses. The family also includes Simian lIemorrhagic Fevervirus and Hepatitis C virus. Of the 67 arboviruses, 34 (50%) are mosquito-borne, 19(28%) are tick-borne, 12 (18%) are zoonotic agents transmitted between rodents orbats without known arthropod vectors and two have unidentified transmission cycles(Monath and Heinz, 1996). That report also revealed that, 38 viruses (55%) havebeen associated with human disease. These include the most important arthropodborneviral afflictions of humankind-Dengue fever, Yellow Fever, JapaneseEncephalitis. Eight Flaviviruses are pathogenic for domestic or wild animals ofeconomic importance.2.2.1 FLAVIVIRUSESThe Flavivirus genus includes more than (i~ members separated into groupson the basis of serological relatedness (Blok et al., 1984). This genus contains manyviruses associated with emerging and re-emerging human diseases. Entities of.globalconcern include Dengue Fever with its associated DHF/DSS (Halstead, 1988;Monath and Heinz, 1996), lE (Monath, 1988), and YF (Monath and Heinz, 1996),Tick-borne encephalitis (TBE), Kyasanur Forest hemorrhagic disease, St Louisencephalitis (SLE), WN encephalitis (Lanciotti et al., 1999) and Murray Valleyencephalitis (MVEl are other important agents of regional endemic and epidemicdiseases (Monath, 1986). Flaviviruses are a useful model for studying the evolutionof vector-borne diseases since they comprise mosquito-borne, tick-borne, and noknown-vector(NKV) viruses (Porterfield, 1980). The genus contains about 70recognized Flaviviruses that are antigenically related and have a widespreadgeographical dispersion. They are positive -strandcd RNA viruses with a genome of6

approximately 1O.5kb. Virions contain three structural proteins, capsid, membraneand envelope (E) and infected cells have been known to contain seven non-structural(NS) proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (Rice, 1996).The evolution, dispersal patterns and epidemiological characteristics ofFlaviviruses are believed to have been determined through a combination ofconstrains imposed by the arthropod vector, the vertebrate hosts, the associatedecology and the influence of human commercial activity (Zanotto et aI., 1995). Theemergence and the expansion of DHF in the tropics have followed an increase inhuman and mosquito population densities brought about by urbanization andindustrialization (Zanotto et al., 1996). Previously, the trans-Atlantic dispersal ofYFV and possibly many other Flaviviruses was thought to have coincided with thetransportation of people and mosquitoes from Africa to the Americas on slave ships(Strode, 1951; Gould et al., 1997).2.2.2 MORPHOLOGY OF FLA VIVlRUSESFlavivirus virions are spherical in shape with a diameter of 40 to 60nm(Murphy, 1980). An electron-dense spherical nucleocapsid of 30nm in diameter issurrounded by a lipid bilayer. Particles typically have a rather smooth appearanceand regular surface projections are usually not apparent (Murphy, 1980), although7nm ring-shaped structures have been observed on the surface of DEN virusparticles (Smith et al., 1970). Mature virions sediment between 170 and 21OS, havea buoyant density of 1.19 to 1.23 glml, and are composed of 6% RNA, 66% protein,9% carbohydrate, and 17% lipid (Russel et al., 1980). The envelope proteins- E andM (membrane) are type 1 membrane proteins embedded by the lipid bilayer by c­terminal hydrophobic anchors. Released virions also contain variable amount of theun processed Membrane precursor membrane (prM) (Russel et. al., 1980). PHYSICAL AND CHEMICAL PROPERTIES OF FLAVIVlRUSESFlavivirus virions consist of a spherical ribonucleoprotein core surroundedby a lipoprotein envelope with surface projections. The projections seen in electronmicrographs are clarified by X-ray crystallography and represent molecules ofenvelope glycoproteins, which form rod -like structures anchored to the viralmembrane at their basal ends. Envelope lipids constitute approximately 17% of the7

virion dry weight (Trent and Qureshi, 1971) and are derived from the host cellIipids. Lipases and lipid solvents disrupt Flaviviruses particles. Inactivation bychloroform and sodium deoxycholate provides a useful preliminary step inidentifying Flaviviruses (and other envelope arboviruses). Acetone, which is oftenused to extract Flavivirus antigens from infected mouse brain tissue, also destroysinfectivity, whereas addition of sucrose partially preserves it. Treatment with betapropiolactoneis an effective inactivating procedure that retains Flavivirus anti genicreactivity to a greater extent than does formalin or phenol treatment.Mature Flavivirus virions contain three structural proteins: a nucleocapsid, orcore protein, (C; 12kd), a non-glycosylated membrane protein (M; 8kd), and anenvelope protein E; 53kd) which is usually glycosylated (Trent and Qureshi, 1971).The M and E protein are both associated with the lipid envelope by means ofhydrophobic membrane anchors. The E protein is the major component of the virionsurface and contains the major antigenic determinants subserving hemagglutinationinhibitionand Neutralization. Thus, it induces immunological responses intheinfected host. Structural elements of the E protein determinants are assumed to beinvolved in the binding of virions to the cell receptors and intraendosornal fusion atlow PH. These protein constituents are sensitive to enzymatic digestion with trypsin,chymotrypsin, and papain, which render the virus noninfectious but preserve certainantigenic reactivities, depending upon the degree of proteolysis.Detergents and proteases have been used to characterize the structure ofFlaviviruses and to isolate immunologically reactive' subunits. (Stolar, 1969; Trentand Qureshi, 1971). Nonionic detergents such as Triton-X solubilize the entireenvelope, releasing E and M proteins; where as sodium deoxycholate appears toremove only E, leaving M associated with nucleocapsid. Protease treatment showedthat a portion of the E glycoproteinis located within the lipid bilayer (Heinz andKunz, 1979). Analysis of the primary structure of the Flavivirus glycoprotein hasconfirmed the presence of a hydrophobic membrane anchor region at the carboxylterminus of the E protein molecule (Nowak and Wengler, 1987). The Flaviviralenvelope protects the genome from cellular nucleases, and naked nucleocapsidsreleased by detergent treatment are degraded by ribonuclease. Flavivirus infectivityand hemmagglutinin are optimally stable at pH 8.4 to 8.8 (Karabastos, 1980).Sensitivity to acid pH (also to bile and proteolytic and lipolytic enzymes) generally8

precludes infection by the oral route. Tick-borne encephalitis, may however, beacquired by ingestion of infected milk (Pogodina, 1958)Flaviviruses are rapidly inactivated by high temperature. At 50°C, 50% ofinfectivity is lost in 10 minutes. Practically total inactivation of the virus suspendedin blood or other protein solutions occurs within 30 minutes at 56°C. Nevertheless,low temperatures preserve infectivity, with stability being greatest at -60°C orbelow. Aerosols present a hazard of laboratory infection (Schercr et al., 1980). StLouis encephalitis virus (SLEV) is stable for 6 hours in aerosol suspension at roomtemperature and 23% to 80% humidity (Karabatsos, 1980). For unknown reasonsaerosol laboratory infections with tick-borne Flaviviruses are more frequent thanwith mosquito- borne viruses.In addition, flaviviruses are inactivated by ultraviolet light, gammairradiation,and disinfectants, including 3% to 8% formaldehyde, 2% glutaraldehyde,2% to 3% hydrogen peroxide, 500-. 5,000 ppm available chlorine, alcohol, 1%iodine, and phenol iodophores. The tick- borne viruses appears to be relatively moreresistant to these measures than mosquito- borne agents. (Monath and Heinz 1996)2.2.4 GENOME ORGANIZAnON OF FLA VIVIRUSES .The genome of Flaviviruses is a single-stranded RNA of approximately llkb(Chambers et. al., 1990). Genome-length RNAs appear to be the only virus-specificmessenger RNA (mRNA) molecules in Flavivirus-infected cells. The genomic RNAhas a type 1 cap at its 5' end (m'Gppparnl') followed l;y the conserved dinucleotidesequence AG. Genomic RNAs of mosquito-borne and tick-borne Flaviviruscs lack a3' terminal poly (A) tract and terminate with the conserved dinucleotide CV.The major portion of the genome RNA consists of a long open reading frame(ORF) of more than 10,000 bases (Chambers et al., 1990). Flanking this ORF are the5' (95 to 132 bases in length) and 3' (114 to 624 bases in length) nontranslatedregions (NTR) containing conserved RNA elements, which may play as yetundefined roles in RNA replication. Previous studies predicted potential secondarystructures near 5'termini of several Flavivirus genomic RNAs with thecorresponding structures possible at 3' end of minus strand RNA (Mandl et. al.,1993). Secondary structures in all Flaviviruses are conserved in conformation andstability but not in primary sequence, and can be predicted for the 3' terminal 909

10bases of the genome RNAs (Mandl et. al., 1993). Conserved RNA sequence are alsofound near the 5' and 3' ends of the genome RNAs, but these elements are distinctfor both mosquito and tick-borne Flaviviruses. For the mosquito-borne Flaviviruses,two short conserved RNA sequence (called CS I and CS2) are located 5'to theputative 3' terminal secondary structure (Halm et al., 1987) CSI is about 26nucleotide in length and is located immediately a~jacent to the terminal secondarystructures. Part of CS I is complementary to a conserved sequence near the 5' end ofthe genome in the region encoding the capsid protein (5'CS).Base-pairing of these or other terminal sequences could lead to a cyclizationof the viral genome, which may be important for regulating translation, replication,or packaging (Mandl et. al., 1993). CS2 is about 24 nucleotides in length and islocated 12 to 22 bases 5' to CSI. This sequence is duplicated in members of the JEand DEN subgroups. Blocks of conserved RNA sequence and potential cyclizationsequences are also found in the genome RNAs of tick- borne Flaviviruses (Mandl, 1993). Interestingly, some TBE isolates have an internal poly (A) tract in the 3'NTR (Monath and Heinz, 1996), which was earlier thought to represent the 3'terminus (Mandl et al., 1991). Besides these conserved RNA sequences andstructures, short subgroup-specific repeated sequences of unknown function are alsoobserved (Mandl et. al., 1993).2.2.5 STRUCTURAL PROTEINS OF FLAVIVIRUSES2.2.5.1 The C Protein:The virion C protein is a small [predicted molecular mass (M) l l kd], highlybasic protein that forms a structural component of the nucleocapsid. Regions ofhydrophobic and hydrophilic amino acids are conserved (Mandl et al., 1988). Cprotein determinants that participate in RNA and protein interactions important fornucleocapsid assembly have not yet been identified. The prM and M protein:The prM protein is the glycosylated precursor (M, 26kd). PrM undergoes adelayed cleavage to form M and the N- terminal pr segment, which is secreted intothe extracellular medium (Murray et al., 1993). This cleavage occurs shortly orcoincident with virion release because prM and M are found on intracellular and

extracellular virions respectively. The N-terminal pr segment is predominantlyhydrophilic and contains one to three N-linked glycosylation sites (Chambers er. aI.,1990) and six conserved systemic residues, all of which participate in intramoleculardisulfide bridges (Nowak and Wengler, 1987). The structural protein M located inthe C-tenninal portion of prM is present in mature virions and contains a shortenedectodomain (41 amino acids) followed by two potential membrane-spanningdomains. Antibodies to prM can mediate protective immunity (Kaufman et aI.,1989) perhaps by neutralization of released virions that contain some uncleavedprM. The E protei":This is the major envelope protein of the virion. It is believed to play keyroles in a number of important processes including virion assembly, receptor bindingand membrane fusion. It is also the principal target for neutralization antibodies(Heinz, 1986). Not surprisingly, mutations in this protein can result in dramaticeffects on viral pathogenesis. Comparison of deduced E protein sequences showareas of striking homology as well as divergence amongst the Flaviviruses(Chambers et. al., 1990; Heinz et. aI., 1990). All twelve Cys residues in the Eectodomain are strictly conserved and involved in intramolecular disulfide bonds(Nowak and Wengler, 1987). The E protein is glycosylated for some, but not all,Flaviviruses and the role of N-linked glycosylation on E function is unclear(Chambers et. al., 1990)The detergent- solubilized TBE E protein (Hcinz and Kunz, 1980) as well asthe soluble tryptic fragment used for crystallography is a dimer. The X-ray structuredemonstrates that the dimer is a head-to-tail oligomer in the shape of a 170 a rod andpredicts that the dimer is anchored in the bilayer at both dista1 ends, The curvature ofthe dimmer fits with its location on the surface of the SOOA virion. This fittingsuggests that, these oligomers do not form long projections or spikes. Two of thethree-distinct structural entities present in the monomeric unit correspond topreviously defined anti genic domains (Mandl et. aI., 1989). Potential neutralizationsites defined by amino acids substitutions present in monocIonal antibody escapemutants are mostly distributed on the surface and can be present on any of thestructural domains. The role of the highly conserved sequence from the residues 1911

through 111 is not apparent from the dimmer structure. However, the structure of theactive fusogenic unit is likely to be an E protein trimer (Johnson and Roehrig, 1999),which forms by the organization of the virion surface upon exposure to acid pH.The E glycoprotein, which is exposed on the surface of the dengue virion,represents the dominant virus antigen, conferring protective immune responses byeliciting neutralizing, hemagglutination inhibiting, antifusion and virus enhancingantibodies. It is also for virus attachment, virus specific membrane fusion in acid pHendosomes and virus assembly (Chen et al., 1996; Roehrig et. al., 1998). Ananalogous situation has been described for other Flaviviruses: single amino acidsubstitution in the envelope protein at positions 52 (Hasegawa et al., 1992) 138(Sumiyoshi et al., 1995),271 and 336 in JE (Cecillia and Gould, 1991), at position390 in Murray Valley virus (Lobigs, et al., 1990), at positions 104 and 107 in a tickborneencephalitis-DEN-4 chimera (Pletnev, et a11993) and at position 171 and 384for TBE virus (Mandl et al., 1988) were shown to have dramatic effect on virulencein vitro and in vivo. NON-STRUCTURAL PROTEINS OF FLAVIVIRUSESThe NSl protein: NSl includes 12 strictly conserved cysteine residues, 110 3N-linked glycosy1ation sites, and region of high sequence conservation (Chamberset. al., 1990). NS1 is secreted slowly from mammalian cells and not from mosquitocells (Mason, 1989; Post et al., 1991). A detergent stable dimer, which becomesapparent 20 to 40 minutes after synthesis, is the predominant form of both cellassociatedand secreted NSl (Lee et al., 1989; Mason, 1989). NSl expressed by itcan dimerize and mutagenesis results suggest that the C- terminal portion of NS1 isimportant for dimmer stability and secretion (Parrish et al., 1991; Pryor and Wright,1993)The function of NS1 in virus replication has not been elucidated. Anotherstudy suggests the role of NSl protein in the early part of replication because RNAaccumulation is blocked at the non- permissive temperature (Monath and Heinz,1996). Mutations in NSl can also affect virulence (Pletnev et al., 1993). NaturalFlavivirus infections elicit antibodies to NS1 with complement -fixing activity, andthe secreted form of this protein has been called the soluble complement -fixing ­SCF) antigen (Russell et al., 1980; Smith and Wright, 1985). Type- specific,.12

complex- specific and group- reactive epitopes have been defined for NSI and someappear to play a role in protective immunity. Protection may occur throughantibody-dependent complement-mediated lysis of virus -inf.ected cells expressingNSl on thecell surface (Schlesinger et al., 1990). The NS3 protein:NS3 is the largest viral protein (predicted M, 68 to 70 kd) and is highlyconserved among Flaviviruses (Mandl et al., 1989). It is believed to be an enzyrnaticcomponent of the RNA replication machinery. Although NS3 does not contain longstretches of hydrophobic amino acids, the protein is membrane associated (Wengleret al., 1990) perhaps through its interaction with the hydrophobicNS2B protein(Arias et al., 1993; Chambers et, al., 1993). Studies suggest that NS3 is probably atleast trifunctional, containg protease,helicase, and RNA triphosphatase activities.The N-terminal one third of NS3 contain the catalytic domain of the NS2B-3protease (Bazan and Fletterick, 1990; Chambers et al.,1990). The sequence ,surrounding the serine protease nucleophilic serine is conserved among Flaviviruses(GXS-GXP; YFNS3 residues 136 to 141) (Gorbalenya et al., 1989). The conservedAsp-13'1 residue and the conserved sequence GLYYYGNG (residues 151 to 156 forYFNS3) have been hypothesized to form part of the substrate- binding pocket.Besides cleavage at the virC/anchC, 2N2B, 2B/3, 3/4A, 4N2k 4B/5 sites, theNS2B-3 protease also appears to cleave additional sites in the NS2A (Monatli andHeinz, 1996) and NS3 regions (Arias et. al., 1993). Although polyprotein contextand conformation are important determinants of cleavage site specificity (Lin et al.,1993), cleavage sites usually consists of two basic residues followed by an aminoacid with a short side chain [(ArglLys/Gin)-(ArglLys) (Gly/Serl AlafThr)]. (Chambers et. al., 1990; Rice and Stauss, 1990)NS3 also contains significant regions of homology with the DEAD family ofRNA helicases (Gorblenya et. al., 1989). This motif is .also present in thehomologous proteins of other positive stranded RNA viruses and in proteinsinvolved in NTP binding. The sequence alignment of these various proteins generateseven regions of amino acid conservation (located between NS3 residues 191 to508) including conserved motifs GAGKT, DEAD, and GRXGR, which are.13

postulated to be involved in nucleotype cofactor binding and hydrolysis (Gorbalenyaet. al., 1989). RNA- stimulated Nl'Pase activity has recently been demonstrated forpurified NS3 (Warrener et. al., 1993) and for a 50-kd C-terminal fragment derivedby proteolysis. Interestingly, this fragment also contains an RNA triphosphataseactivity that is likely to be involved in the formation of the cap structure at the 5' endof Flavivirus genome RNAs (Wengler and Wengler. 1993). Although there is noevidence of cleavage between the protease and helicase domains, autocatalyticprocessing possibly at a dibasic site near the end of the NS3 helicase region andpreceding the proposed RNA triphosphotase domain (Wengler and Wengler, 1993),has been observed in mammalian but not in mosquito cell line (Arias et al., 1993;Pugachev. et al., 1992). The NS5 protein:The last protein encoded in the long ORF is NS5, the largest (predicted M,103 to 104 kd) and most highly conserved Flavivirus protein (Mandl et. al., 1989).NS5 is a basic protein, lacking any long hydrophobic stretches. It is believed to be"Flavivirus RNA -dependent RNA polymerase. This assumption, although notverified directly, is based on the presence of a highly conserved region, including thesequence motif GDD (YFNS5 residues 666 to 668), which is characteristic of knownor putative RNA-dependent RNA polymerase of positive strand RNA viruses (Riceet. al., 1986). The N-terminaI domain of NS5 (between residues 60 and 145) ishomologous to a region of mcthyltransferases implicated in S- adenosylmethioninebinding (Koonin, 1993). That report suggested that this domain might be involved inthe methylation of the 5' cap structure. Although the enzymological characterizationof the protein is lacking, it seems likely that the NS5 protein is at least bifunctional,possessing both methyltransferase and RNA polymerase acdvities (Monath andHeinz, 1996). The NS2A, NS2B, NS4A and NS4B proteins:These are the less conserved regions of the polyprotein, which are processedto at least four additional nonstructural proteins. They are found between the highlyconserved NSl, NS3, and NS5 proteins (Arias et al., 1993) little is known abouttheir functions in the Flavivirus life cycle. C-terminal truncations can inhibit14

cleavage at the NS1/2A site, but the precise role of NS2A in processing is unclear(Falgout et al., 1989). In the case of YF, at least one additional cleavage site for theNS2B-3protease occurs in the NS2A region, and a mutation that block at this site islethal for virus replication.The NS2B protein (predicted M 27kd) contains a highly charged andconserved central region flanked by hydrophobic segments (Falgout et al., 1993).This protein together with NS3 serineprotease domain, have been shown to beessential for processing at all of the known structural and nonstructural dibasic sites(Amberg et al., 1994). Evidence of a stable complex between NS2B and NS3 hasbeen obtained (Arias et al., 1993; Chambers et al., 1993). Mutagenesis data suggestthat the charged central region participate in the formation of this complex and theactive protease (Chambers et al., 1993; Falgout et al., 1993). Besides its function inproteolysis, it has been suggested that the interaction between the hydrophobicNS2B protein and NS3 may be partially responsible for the localization of the RNAreplication machinery to cellular membranes.No data is available concerning the function of the hydrophobic NA4A and" NS4B proteins.2.2.6 PHYLOGENETIC RELATIONSHIPS OF FLAVIVIRUSESTHAT CORRELATE WITH THEIR EPIDEMIOLOGY,DISEASE ASSOCIATION AND BIOGEOGRAPHYMolecular sequenciug and phylogenetic re~onstructions have prov~dedimportant insights into the taxonomy (Heinz et al., 2000) and dispersal ofFlaviviruses (Gould et al., 1997). It was demonstrated previously (Marin et al.,1995; Kuno et al., 1998) that the Flavivirus genus was monophyletic and threedistinct groups of viruses, namely tick-borne, mosquito-borne and NKV viruses,diverged at the deepest nodes. Recently, it has been demonstrated that the mosquitoborneviruses are subdivided into culex and aedes clades (Gaunt et al., 2001).Moreover, the evolution of the culex clade appears to have occurred after theseparation of the mosquito-borne from the tick-borne and NKV viruses. Theseobservations were supported by the congruence between NS5 and E genephylogenies as well as by Monte Carlo simulation and quartet puzzling support.15

The dominance of Aedes and culex species in F1aviviruses transmission isexplained by the species prevalence of each of the genera, which contain 975 and769species respectively, and comprise more species than all other mosquito generacombined (1522) (Gaunt et al., 2001). Aedes and culex mosquitoes are also amongthe small number of genera that are globally dispersed. Blood-meal data obtained forAedes species suggest that mammals are the primary host of most species, whichcould explain the Aedes c1ades-primate/mammal association (Christensen et al.,1996; Clements, 1999). The feeding pattern of only relatively few species of culexmosquitoes is known, although a small number of birds -or mammal-specificspecies have been identified. Many culex species feed indiscriminately on bothmammals and birds and they include the principal vector for several Flaviviruses inculex clade, such as C amulirostris (MVE virus), C tritaeniorhynchus (Jevirus), Ctarsalis (SLE virus) and C univittatus (WNV) (Christenson et al., 1996; elements,1999). The difference in feeding behavior between Aedes and culex mosquitoesprovides a clear explanation for the association between Aedes-borne Flavivirusesand mammals and between culex-borne Flaviviruses and birds. Moreover, itexplains why the association between Aedes clades and mammals appear to beunequivocal, while the association between the culex elade and birds contain anumber of notable exceptions (Gaunt et. al., 2001).Another correlation was between the type of disease produced and themosquito c1ade in which each virus appeared. Generally, several infections causedby some Aedes species viruses result in hernorrhagic disease, whereas many culexspecies viruses cause encephalitic disease (Gaunt et. aI., 2001). However, there hadbeen reported cases of DEN (Aedes species-associated) encephalitis, but this seemsto be very rare (Lum et al., 1996; Solomon et al., 2000). Until the precise basis ofFlaviviruses pathogenicity has been defined at the molecular level, it is not possibleto understand why these different disease associations can be seen in thephylogenetic tree. In contrast with the mosquito-borne Flaviviruses, different virusesin the tick borne virus groups produce encephalitic disease, but OHF and KFDviruses may also produce hemorrhagic disease in humans and this does not appear tocorrelate either with their phylogenetic or geographical characteristics.Phylogenetic divisions between Old and New World Flaviviruses were seenthroughoutthe NS5 and E gene phylogenies (Gaunt et. al., 2001). In some instances,16

dispersal of Flaviviruses could be readily linked with the vertebrate host, providingevidence of the importance of the host in Flavivirus evolution. For viruses thatestablish infections in bats, it is easy to imagine dispersal to remote sites, as the OldWorld bats from which Flaviviruses have been isolated are known to migratehundreds of kilometers (Shilton et al., 1999). On the other hand, individual rodentassociatedNKV viruses might be expected to show a more restricted distributionand this is demonstrated by theirdetection almost exclusively in the New World andby their localized or niche-like distribution.Virtually all of the tick-borne Flaviviruses are exclusively Old World, withthe exception of POW virus (Gaunt et al.. 200 I). The seabird-associated tick-borneviruses were dispersed to geographical areas where they established niches inseabird colonies in both the Northern and the Southern hemispheres (TYU, SRE,MEA viruses) (Chastel et al., 1985). At the early period of their evolution the TBEcomplex viruses appear to have been dispersed either by seabirds or rodents andtheir associated ticks. As they reached the forest of Asia, they became establishedpredominantly in Ixodes species, where they continued their clinal evolution into.' Europe (Zanotto et. al., 1995;Gould et. al., 1997).The earliest evolutionary lineages in the mosquito-borne virus cladesappeared to have radiated to geographically distant pats of the Old World and to awide variety of species, Le. bats, Aedes species, sandflies and large animals,including simians and humans (Gaunt et. al., 2001). Only YF and four DEN.serotypes, which cause human epidemics, are found in the New World. There is astrong evidence to support the notion thatYF virus was introduced to the Americasfrom the Old World during the past few centuries when slaves were transportedacross the Atlantic ocean (Strode, et al 1951; Gould et. al., 1997). There are reasonsto believe that DEN viruses have been an African ancestry (Gaunt et. al.• 2001). Theother members of the Aedes c1ade containing the DEN, ZIKa, Sl'O, and KEDviruses were all isolated from Africa and formed two paraphyletic lineages to theDEN viruses. In addition the E gene phylogenies of endemic/epidemic and sylvaticDEN viruses show a basal position for Old World sylvatic lineages of DEN-I, DEN­2 (Africa and Malaysia) and DEN-4 (Wang et al., 2000). The vector of DEN viruses,Aedes aegypti, is also believed to have originated in Africa (Tabachnick, 1991).There is no reason to believe that DEN virus could not have been shipped to the17

Americas from the Old World in the same way as YF virus (Gaunt et. al., 2001).Therefore these authors reported that most of the other Aedes species-associatedviruses are found solely in Africa and that the culex species- associated virusesappear to be descendents of the Aedes species-associated viruses. They postulatedthat the mosquito-borne Flaviviruses appear to have evolved out of Africa.Furthermore, these authors conclude that the Flaviviruses that are recognized today,represent a diverse group of viruses that could have emerged and dispersed duringthe past 10,000 years Le. since the most recent ice age (Zanotto et. al., 1996). Thecharacteristic epidemiological groupings of the viruses that are apparent in thephylogenetic trees illustrate the significant influence of the invertebrate vectors, thevertebrate hosts, and the particular ecological niches into which these species haveevolved (Gaunt et. al., 2001).2.2.7 INFECTIONS OF FLAVIVIRUSES IN ARTHROPOD VECTORThe fact that certain arthropods depend upon blood feeding for energyrequirements and egg development provides a mechanism for salivary transmissionof Flaviviruses and many other infectious agents (Monath and Heinz, 1996). Theseauthors revealed that mechanical transmission involving host to host by simpletransfer of virus on the mouthparts of mosquitoes during the process of interruptedfeeding do occur occasionally. Nevertheless, biological transmission is the rule anddepends upon the following: -a. Ingestion of blood meal containing virus; infection of the epithelial cellslining the mesenteron (midgut)b. Escape of virus from the midgut epithelium into the hemocoelec. Infection of the salivary glandd. Secretion of virus in saliva during refeeding on a susceptible vertebratehost (Monath and Heinz, 1996)Many F1aviviruses exhibit a high degree of specificity in their ability toinfect and be transmitted by individual insect or tick species (or even strains ofindividual species). Other reports showed that vector competence is under geneticcontrol, with the susceptibility of the midgut epithelium being the primarydeterminant (Hardy, 1988; Turell, 1988). In a susceptible arthropod, a sufficientconcentration of the virus must be ingested to exceed the mesenteronal infection18

threshold. The time interval between the ingestion of an infectious blood meal andthe salivary secretion of the virus (extrinsic incubation period) must not exceed thelife span of the arthropod (Monath and Heinz, 1996). In that report increasedtemperature shortens the extrinsic incubation period, and may significantly increasethe rate of virus transmission in nature. Other extrinsic factors affecting Flavivirustransmission by arthropod vectors include:I. Mosquito larval crowding and nutritional deprivation which couldincrease transmission by adult vectors2. Rearing temperature (Kay et al., 1989)3. Co - infection with other infectious agents such as microfilaria, .whichmay include lesions in the midgut epithelial barrier and therebyfacilitate viral dissemination to the hemocoele (Turell, 1988).Transfer of virus in mosquito saliva occurs during the process of probinghost tissues with the piercing mouthparts in an attempt to canulate a small vessel(Monath and Heinz, 1996). The probing process often results in micro hematomaformation, which facilitates location and canulation of the vessel (Edman andspeilman, 1988). Salivary enzymes play important role in feeding, particularlyapyrase, which prevents ADP-dependent platelet aggregation and coagulation(Mamotti et al., 1990). Salivary virus is deposited principally in the extravasculartissues of the host during probing and the mosquito rapidly reingests saliva that isinjected intravascularly during blood feeding (Turell and Spielman, 1992; Turell andTammarielo, 1993). This results in virus replication at the site of inoculation andrelatively slows the spread ofinitial infection through lymphatic channels to regionallymph nodes, rather than a rapid viremic dissemination of the inoculums to theblood stream. The delay engendered in this initial phase of virus infection may beimportant in the initiation of the immune response and eventually the abrogation ofinfection. Conversely, if the virus does enter the vascular space during mosquitofeeding and is disseminated early in infection, the incubation period may beabbreviated and infection accelerated (Monath and Heinz, 1996). In that report, thedifferences in these early events in infection could partially explain variations in thecourse and outcome of infections in individual hosts.The vector must find and successfully feed on a vertebrate host for virustransmission to occur (Monath and Heinz, 1996). Factors that influence the search19

for vertebrate host include vector and host behaviour and density, chemical signalsemitted by the host, and environmental variables (Edman and Speilman, 1988). Ifthe vertebrate host behavior is altered, virus infection may influence the rate oftransmission, since infected animals may be lethargic and exhibit reduced defenseagainst mosquito and tick bite. Interestingly, the reduced defense against mosquitoattack by immature nestling birds appear to be offset by their intrinsically lowerattractiveness to blood -seeking mosquitoes (Scott et aI., 1990).2.3 DENGUE VIRUSESDengue Virus (DEN) is a single-stranded positive-polarity enveloped RNA(of approximately l Ikb in length), a Flavivirus that causes DF, DHF/DSS (Diamondet al., 2000). They comprise four distinct serotypes (DEN-l to DEN-4), which noweo-circulate in many localities. The four serotypes constitute distinct anti geniccomplex. DENl and 3 forms a sub complex defined by monoclonal and polyclonalantibodies. Monoclonal antibodies have also shown unexpected relationships at thesub complex level [e.g. between DENl and 3, 2 and 4, and between DEN andviruses of the JE, and TBE complexes (Monath and Heinz 1996). Within the DENs,amino acid sequence positional homology of 63% to 68% was found, compared to44% to 51% between DEN, YF, and WNV. Genetic variants of DEN-2 showedmore than 90% similarity (Monath and Heinz, 1996). DEN genome is ofapproximately 10,700 bases in length, surrounded by a icosahedral nucleocapsid andcovered by a lipid envelope containing the envelope and the membrane proteins(Leitmeyer et al., 1999). The genome contains a single open reading frame, whichencodes a precursor polyprotein and is flanked by two nontranslated regions (5' and3'NTR). COe and posttranslational proteolytic cleavage of the precursor results in theformation of three structural proteins, capsid (C), membrane (M), and envelope (E),and seven nonstructural proteins arranged in the order 5' C-prm-E-NSI- NS2a­NS2b-NS3- NS4a- NS4b- andNS5 3' (Chambers et al., 1990; Rice, 1996). The viralgenome is the only mRNA found in the infected cells and is translated as a singlepolyprotein (350kDa) which is cleaved eo- and post - translationally by both hostcell and virus-encoded protease to generate individual proteins (Chambers et al.,1990), These authors observed that the glycosylated prM protein (22 to 24 kDa) isreleased by host cell signalase cleavage of the polyprotein. They further reported20

that, in susceptible vertebrate cells, prM protein is further cleaved by host cellproteases late in infection to produce the nonglycosylated M protein found in themature virion. However, in mosquito cells, this cleavage is much less efficient ordoes not occur during virus assembly (Murray et. al., 1993). For dengue, severalstudies of cloned or cell culture-passaged wild and attenuated viruses showed thatdifferent sites of nucleotide and amino acid changes are associated with virulence(Mangada and Igarashi, 1997; 1998). Infection with one dengue virus serotype doesnot provide protective immunity against others, and sequential (heterotypic)infection has been shown to increase virus replication and thus the probability ofdeveloping DHF by a process known as antibody-dependent enhancement (ADE)(Halstead 1970; Kliks et al., 1989). Host immune factors such as cytokines,interferon and activated complement have been suggested to increase the capillarypermeability, the hallmark of DHF (Kurane et al., 1994). Other subsequent eventssuggest the importance of viral factors in the establishment of DHF (Leitmeyer etal., 1999). For example, despite the eo - circulation of several dengue serotypes inthe Americas, it was not until the Cuban epidemics of 1981 that the first DHF casesoccurred. This incidence coincided with the introduction of a new genetic type ofdengue virus DEN-2 (Rico-Hesse, 1990). Armstrong and Rico-Hesse, (2001) alsosuggested that variants within serotype or genotypes, differ in their potential to causeDHF. Another report revealed that within each of the four-dengue virus serotypes,phylogenetic studies have identified genetic subtypes that differ in nucleotidesequence by up to 12% in the envelope gene, which determines most antigeniccharacteristics of the virus (Lewis et al., 1993). DEN-l virus comprises five knownsubtypes [I-V, (Rico-Hesse, 1990)], and DEN-2 virus comprises six, although DEN­2 virus subtype 111 has been further di vided into sublineages 111a and 111b (Lewiset al., 1993). DEN-3 and DEN-4 viruses are classed into four and two differentsubtypes, respectively (Lanciotti et. al., 1997). Phylogenetic studies demonstrateddifferent genetic types of DEN-2; the "native" American genotype has beenassociated thus far with the mild disease (DF), while the introduced Southeast Asiangenotype coincided with the appearance of DHF in four different countries. (Rico- ,Hesse, et al.,1997). Another supporting example came from epidemiological studiesin Peru, where over a period of four years (1993 to 1997), active surveillance for DFcases revealed that, inspite of secondary infection rates up to 75%, no DHF cases21

have been detected. This implied that Peru has not then imported the SoutheastAsian genotype of DEN-2 (Watts- unpublished data). The genetic variation withineach serotype can be further partitioned into different viral genotypes; some haverestricted geographical distributions, while others are more cosmopolitan, reflectingtheir dispersal across the tropical world (Rico-Hesse, 1990).Dengue viruses grow in a variety of primary and continuous cell cultures;high yields but the demonstration of CPE is difficult to obtain in many systemswithout adaptation and passage. Cells of human (BSC-I and HL-CZ, a promonocytecell line), monkey (LLC-MK2, vero, primary monkey kidney), hamster (BHK-21),and mosquito origin are most susceptible. Yields up to 7 to 8 dexlml, CPE andplaque formation are obtained under appropriate conditions (Monath and Heinz,1996).DENs replicate in the brains of suckling mice and hamster inoculatedintracerebrally (Brandt et al., 1970). However, unadapted virus strains usuallyproduce subclinical infections or only scattered illnesses with paralysis and death.Neurovirulence for mice and monkeys increases with sequential passage in mousebrain. Guinea pigs, rabbits, cotton rats, adult hamsters, chickens, and lizards are notsusceptible to infection (Karabatsos, 1985; Schlesinger, 1977). Embryonated eggsreplicate some strains only after repeated passage. Adult mice inoculated with highlyadapted DEN-I and 2 become infected with or without overt encephalitis(Boonpucknavig et al., 1981). Old world and New world monkeys and apes developsubclinical infection and viraemia (Simrnons, 1931;'Whitehead et al., 1970). DENsreplicate to high titer in Aedes spp. and Toxorhynchites spp. Mosquitoes could beinoculated intrathoracically or intracerebrally (Rosen and Gubler, 1974).The virus is transmitted between human and monkey hosts by mosquitoes ofthe genus Aedes and principally, Aedes aegypti, which often breeds in areas ofhuman habitation (Holmes et ol., 1999). The rising prevalence of dengue virus andthe greater mixing of host and vector populations have led to suggestions thatgenetic exchange between strains is an increasing possibility (Kuno, 1997).22

2.3.1RECOMBINATION OF GENETIC MATERIALS BETWEENDENGUE VIRUSESOne undoubted contributing factor to the genetic diversity of dengue virus isits relatively high mutation rate, a characteristic of many RNA viruses that lack theproof-reading enzymes used by DNA-based organisms to enhance the fidelity ofgenome replication (Worobey et al., 1999). These authors further reported thatrecombination is another largely ignored but potentially important means by whichgenetic diversity could be generated in dengue virus. For recombination to takeplace different viruses need to eo-infect the same individual human or mosquito hostand then replicate in the same cell. Simultaneous infection of human hosts withdifferentserotypes of dengue virus has, in fact, been carefully documented andsimultaneous presence of more than one species of Flavivirus is known to occur inthe vector Aedes aegypti (Gubler, 1985). Although, the possibility of geneticexchange between divergent dengue virus strains- and the serious consequences thishas for the generation of new types of dengue viruses- have been consideredpreviously (Monath, 1994; Kuno, 1997), nearly all of these previous studies so far" on biology, evolution, and control of dengue virus has rested on the implicitassumption that recombination plays little or no role in producing genetic differencebetween strains. However, Worobey et. al., (1999) demonstrated sevenrecombinants among three of the four-dengue virus serotypes, with break points inthe premembrane/membrane gene, the envelope gene, and at the junction of theenvelope and firstnonstructural genes. Many of'the individual recombinantscontained certain sequence representing separate genetic sub types. They concludedthat recombination might play a very significant role in shaping genetic diversity inDEN viruses and consequently, have important implications for its biology and itscontrol. Also, Holmes et al., (1999) revealed two recombinants in DEN-l withbreakpoints at similar positions, within the fusion peptide of the envelope protein.This demonstrated that a single recombination event occurred prior to the divergenceofthese two strains.2.3.2 DENGUE VIRUS INFECTIONS IN NIGERIASeveral reports have consistently shown that dengue virus infections areendemic in Nigeria (Adekolu-John and Fagbarni, 1983; Fagbami et al., 1988). Other23

eports also revealed that the principal vector for dengue virus infections is highlyprevalent in Nigeria (Wagbatsoma and Ogbeide, 1995; Rodhan, 1996). Thedemonstration of dengue neutralizing antibodies in monkeys, and galagos suggestthe occurrence of a forest cycle of dengue in Nigeria (Fagbami et al., 1977;).Fagbami et. al. (1988) demonstrated that 17 f1avivirus neutralizing antibody-positivesera contained infection-enhancing antibodies to dengue 2. In that reportheterologous infection-enhancing antibody titers were lower than the homologousones. In a study in Oyo state, Nigeria, involving mild febrile illness, 81% of thepatients examined had antibody to dengue type 1. All the studies showed that dengue1 and 2 are prevalent in this environment. In some of these cases, virus isolationattempts yielded dengue viruses (Moore et. al., 1975; Fagbami et. al., 1977).Several serological studies have consistently indicated high activities of denguevirus serotypes in Nigeria (Fagbami, 1979; Adekolu-John and Fagbami, 1983). Inboth the serological and virological studies of dengue virus in Nigeria, Dengue 3 and4 have not yet been reported. In a study involving four ecological zones in Nigeria,the highest percentage (63%) of dengue-neutralizing antibody was observed in the.' derived savannah zone. This was followed by the rain forest zone (42%) while thesouthern guinea savannah and plateau zones had lowest (Fagbami et. al., 1977). In arelated study, it was found that the monthly arbovirus activity was highest during therainy season of the months of June, July, and August and lowest in the dry monthsof January and February (Moore et. al., 1975). In addition, a high prevalence ofantibody to DEN-2 has been reported in Kainji Lake area of Nigeria (Adekolu-JohnandFagbami,1983).2.3.3 DENGUE FEVER (DF), DENGUE HEMORRHAGIC FEVER (DHF)AND DENGUE SHOCK SYNDROME (DSS)All four dengue virus serotypes cause DF, DHF and DSS (Monath andHeinz, 1996). Dengue is an acute viral disease, a world -wide public health problem,.with recurring epidemics involving thousands of persons in tropical and subtropicalregions of Asia, Africa, Australia and the Americas where Aedes mosquitoes arepresent (Usuku et al., 2001; Uzcategui et al., 2001). Dengue virus infections can besubclinical or cause illness ranging from DF, which is a mild, flu-like syndrome. with rash to DHF, a severe and sometimes fatal disease. The latter is characterized24

., by capillary leakage, thrombocytopenia and sometimes, hypovolemic shock(Leitmeyer et al., 1999). Other authors described DF asan acute, debilitating. althoughrarely fatal illness (Gaines et al., 1996).For more than 20 years DF and DHF have emerged as the most importantarthropod-borne viral diseases of humans (Gubler and Trent, 1994; Monath, 1994).Over 2 billion persons inhabiting tropical areas are at the risk of dengue infection,and up to 100 million cases of DF and 250,000 cases of DHF occur annually on aworldwide basis (Monath and Heinz 1996). Cases of hemorrhages and death hadbeen described during outbreaks of classical DF in Australia (1897), Greece (1928),and Formosa (1931) (Johnson et al., 1967). In 1954, a febrile disease withhemorrhagicsigns (Philippine hemorrhagic fever), occurred in epidemic form inManila and was shown to be caused by DEN-3 and 4 (Monath and Heinz, 1996).Dengue hemorrhagic fever was subsequently described in many other areas ofSoutheast Asia (Hammon, 1969). The disease was first reported in China in 1985.The incidence of DHF has risen steeply in Asia for more than 20 years now and over450,000 cases are now reported annually, making it a leading cause of morbidity.Case- fatality rate currently ranges from 1% to 10%, depending on the availabilityand sophistication of hospital care. Sporadic cases of DHF were recognized in theAmericas during the 1970s and epidemic of DHF appeared in the 1980s. Thepathogenesis of DHF remains incompletely understood. However, it has beenproposed that the severe form of the disease had an immunopathologic basis and,occurred in an individual previously sensitized by infection with heterologousdengue serotype (Halstead, 1988; Halstead et al., 1970).2.3,4 ANTIBODY DEPENDENT ENHANCEMENT IN DENGUE VIRUSINFECTIONSIt has been shown by epidemiologic and clinical associations thatenvironmental (Diamond et. al., 2000) immunologic and viral factors determine the.severity of the disease (Halstead, 1988; Rosen, 1977). Previous studies gave three. possible explanations for the occurrence of DHFIDSS to include.- increased viral:' load caused by enhanced infection of monocytes and macrophages in the presence of,.,I :.pre-existing dengue antibody at subneutralizing levels (Gubler, 1988; Halstead,j' 1981), hyperviremic variant strains (Lanciotti et al., 1994), and exacerbated>,.;if',',..~ ."I"~25

immunopathological response (Kurane, et al.,1994). Zanotto et. aI., (1996)suggested that eo circulation of heterologous serotypes of dengue might becomemore common, resulting in rapid increase in viral diversity which could lead to theemergence of viral lineages with increased transmission potential and pathogenicity.Infection with one DEN serotype does not provide immunity against the others, andsubsequent (heterotypic) infection has been shown to increase virus replication andthus the probability of developing DHF by a process known as antibody -dependentenhancement (ADE) (Kliks et. al., 1989, Rothman et al., 1996). In addition, viralserotypes may differ in their potential to cause severe disease (Diamond et. al.,2000). In Thailand, for example, secondary infection with serotype 2 contributeddisproportionately to DHF cases versus other serotypes of DEN (Vaughn et al.,1997; 2000). Althongh secondary infection contributes to the pathogenesis of DHF,the sequence of infection by particular serotypes may also affect disease outcome(Armstrong and Ricco-Hesse 2001). These authors further demonstrated that denguevariants within serotypes or genotypes differ in their potential to cause DHF. Forexample, during 1960s and 1970s, DEN outbreaks in Puerto-Rico, Colombia, the.' Dominican Republic and Jamaica involved DEN-2 and at least one other serotype,yet none was associated with epidemic DHF (Halstead, 1980). This virtual absenceof DHF ended in America, however, abruptly ended with Cuban outbreak of DEN-2in 1981. Subsequent DHF outbreaks became evident in Brazil, Venezuela,Colombia, and French Guiana and in each of these instances; DEN-2 was thepredominant serotype (Gubler, 1997). Phylogenetic analysis indicated that twodistinct viral lineages of DEN-2 are maintained in the Western Hemisphere (Rico­Hesse, et al.,1997; Leitmeyer et. al., 1999). The American genotype has beendetected in the region since the 1950s and thus far, associated solely with DF. Thenthe Southeast Asian genotype in contrast was introduced into the region morerecently and is associated with DHF outbreaks (Gubler and Meltzer, 1999,Uzcategui et. al., 2001).Dengue viruses were shown to replicate to higher titer in humanmononuclear cells in the presence of cross-reactive neutralizing dengue antibodies,;.than in its absence. Infectious complexes of virions and IgG antibody gained accessto monocytic cells through their Fe receptor-bearing (FeR) cells, This led to theconcept that the presence of enhancing antibodies increased the number of dengue-26

v »infected cells. Also, lysis or immune clearance of these cells led to the release ofvasoactive mediators and procoagulants, which results in increased capillarypermeability. Consequently the blood cells cripple, and bleeding develops in thegum, the skin, and the intestinal tract. Eventually the blood vessels do not workquite right and the blood pressure drops precipitously; as a result the blood fails tomeet the metabolic demands of the cells in the body -which is shock (Halstead andDeen, 2002). Host immune factors, such as cytokines, interferon and acti vatedcomplement have been suggested to increase the capillary permeability, the hallmarkofDHF (Kurane et al., 1994, Annstrong and Ricco-Hesse, 2001). Loke et al., (2001)observed that there seems to be a genetic component that places certain individualsat high risk of severe disease during secondary dengue infection. For two decadesADE has served as an explanatory hypothesis (Halstead and Deen, 2002). It hasbeen demonstrated that severity is correlated with peak virus titers early in theillness with children with secondary dengue infections (Vaughn et al., 1999).Halstead and Deen, (2002) therefore, suggested that with ADE as an explanation fordisease severity, proposed vaccines need to be capable of producing protectiveneutralizing antibodies to all the four-dengue viruses.2.3.5 Epidemiology ofDHFNearly 3 million cases of DHF have been officially reported worldwide withmost cases occurring in Asia. In Thailand alone, 874,207 cases of DHF werereported between 1958 and 1990, with a case-fatality rate of 1.57% (Monath andHeinz, 1996) and the disease ranks fifth as a cause of morbidity, third as a cause ofdeath, and fifth as a cause of years of productive life lost (Grats, 1993). Factorsresponsible for the emergence of DHF as an epidemic disease in Asia in the 1950sand 1960s and for the dramatic rise in incidence in the last decade include:1. Demographic changes (human population growth and urbanization)favoring contact with the domestic mosquito vector, Aedes aegypti.; ..2. Ecologic changes linked to urbanization (poor sanitation, inadequatepiped water necessitating domestic water storage) favoring Aedesaegypti breeding;3. The rapid rise in air travel, providing the means for movement ofviremic human beings and dissemination of multiple dengue serotypes.27

4. Establishment of hyperendemic dengue infection and increasingfrequency of sequential infections of children (Gubler and Trent, 1994;Monath, 1994).The factors responsible for the emergence of DHF in Asia have come to bearon the American region in the last 20 years (Monath and Heinz, 1996).distribution of Aedes aegypti changed dramatically, due to the collapse of mosquitocontrol efforts. The pattern of dengue infections changed from outbreaks at intervalsof multiple years by a single serotype to annual outbreaks and eo -circulation ofthree of the four-dengue serotypes (Monath and Heinz,The1996). In that report,sporadic cases ofDHF were recognized in the 1970s, and major epidemics occurredin Cuba, Venezuela, and Brazil in the reported the occurrence of DHF.Eleven countries in the region haveDengue hemorrhagic fever in Asia is a disease of childhood. Two peaks havebeen noted in age-specific incidence rates and these include children less than 1 yearof age and children 3 to 5 years of age (Monath and Heinz, 1996). Furthermore, thatreport stated that the disease in infants is associated with primary infection in thepresence of maternal antibody, whereas the vast majority of cases of older childrenare the result of secondary infections. Studies in Thailand have estimated thefrequency of DHF and DSS in children 1 to 14 years of age to be 31 and 11 casesper 1,000 secondary dengue infections, respectively, whereas the incidence of DHFand DSS in primary infections is 1.9 and 0.07 per \.000, respectively (Durke et al..1988). An age-dependent excess in cases of severe DHF with shock syndrome ingirls, compared with boys, has been noted and appears to be related to host factors,since serosurveys have shown no difference in sex-specific antibody prevalence. Inthe 1980s, DHF began a second expansion into Asia when Sri. Lanka, India and theMaldive islands had their first major DHF epidemics, while Pakistan first reportedan epidemic of DF in 1994 (eDC, 2001). According to these authors, the recentepidemics in Sri Lanka and India were associated with multiple dengue serotypesbut DEN-3 was predominant and was genetically distinct from DEN-3 virusespreviously isolated from infected persons in those countries. After an absence of 35.years, epidemics of DF occurred in Taiwan and the People's Republic of China in, i,the 1980s. The latter had a series of epidemics caused by all the four serotypes and28

its first major epidemic of dengue fever, caused by DEN-2, was reported on HainanIslandin 1985. Singapore also had a resurgence of DFfDHF from 1990 to 1994 aftera successful control program had prevented significant transmission over 20 years.In other countries of Asia 'where DHF is endemic, the epidemics have becomeprogressively larger in the last 15 years. In the Pacific, DENs were reintroduced inthe early 1970s after an absence of more than 25 years. Epidemic activities causedby all the four serotypes have intensified in recent years with major epidemics ofDHF on several islands (CDC, 2001).Despite poor surveillance for dengue in Africa, epidemic of DF caused by allthe four serotypes has increased dramatically since 1980. Most activity has occurredin East Africa and major epidemics were reported for the first time in the Seychelles(1977), Kenya (1982), DEN-2, Mozambique (1985, DEN-3), Djibouti (1991-92,DEN-2) Somalia (1982, 1993, DEN-2 and Saudi Arabia (1994, DEN-2). rcoc,2001) These authors reported that epidemic of DHF has been reported in neitherAfrica nor the Middle East, but sporadic cases clinically compatible with DHF havebeen reported from Mozambique, Djibouti and Saudi Arabia. The emergence ofDFfDHFas a major public health problem has been most dramatic in the Americanregion. In an effort to prevent an urban YF, the Pan American Health Organizationorganized a campaign that eradicated Aedes aegypti from most central and SouthAmerican countries in the 1950s and1960s. Consequently, epidemics of DFoccurred only sporadically in some Caribbean islands during this period. The Aedesaegypti eradication program, which was officially discontinued in the United Statesin 1970 gradually, eroded elsewhere, and this species began to re - infect countriesfrom which it had been eradicated. In 1997, the geographic distribution of Aedes(Ae) aegypti was wider than its distribution before the eradication program.In 1970, only DEN-2 virus was present in the Americas, although DEN-3may have had a focal distribution in Colombia and Puerto Rico. In 1977, DEN-Iwas introduced and it caused major epidemics throughout the region over a 16-yearperiod. DEN-4 was introduced in 1981, and it caused similar widespread epidemics.Also in 1981, a new strain of DEN-2 from Southeast Asia caused the first majorUHF in the Americas (Cuba) in which there were 10312 cases and ISO deaths due toDHFIDSS, followed by 4 years after DEN-I virus epidemic (Uzcategui et al., 2001).This strain has spread rapidly throughout the region and has caused outbreaks of29

DHFin Venezuela, Colombia, Brazil, French Guiana, Surinarae and Puerto Rico. By1997, 18 countries in the American region had reported confirmed DHF cases andthis diseaseis now endemic in many of these countries.DEN-3 virus recently reappeared in the Americas after an absence of 16years.This serotype was first detected in association with a 1994 DFmHF epidemicin Nicaragua (Miagostovich et al., 1997). In that report, almost simultaneously,DEN-3 was confirmed in Panama and in early 1995, in Costa Rica. In Nicaragua,considerable numbers of DHF cases were associated with the epidemic, which wasapparently caused by DEN-3. In Panama and Costa Rica, the epidemic was classicdengue fever. Viral envelope sequence data from the DEN-3 strains isolated fromPanama and Nicaragua had later shown that the virus was introduced from Asiasince it was genetically distinct from the DEN-3 strain found previously inAmericas. However, it was identical with the DEN-3 virus that caused the majorDHF epidemics in Sri Lanka and India in 1980s (CDC, 2001). With the finding ofthe new DEN-3 and the susceptibility of the population in the American tropics to it,the virus spread rapidly throughout the region and caused the major epidemics of" DF/DHF in Central America in 1995. (CDC, 2001). According to that report theglobal distribution of dengue is comparable to that of malaria, Attack rates inoutbreaks in the Americas have ranged from 20% to (Ehrenkranz et al., 1971;Figueiredo et al.• 1991) and that the case fatality rate of DHF. in most countries isabout 5%; with most fatal cases among children and young adults ..There is small but significant risk of dengue outbreaks in the continentalUnited States. This is because two competent mosquito vectors, Ae aegypti and Aealbopictus are present and under certain circumstances, each could transmit DENs.About three (1980,1986, and 1995) of this type of transmission has been detected inthe last 16 years in South Texas (CDC, 2001; 2002) and has been associated withthe Dengue epidemics in northern Mexico. Travellers returning from tropical areaswhere dengue viruses are endemic do introduce numerous viruses annually (CDC,2002).Many more cases probably went unreported each year because surveillance intheUnited States is passive and relies on physicians to recognize the disease, inquireabout the patient's travel history, obtain proper diagnostic samples, and report thecase. This data therefore suggest that. southern Texas and the southeastern UnitedStates, where Ae aegypti is found. are at the risk of dengue transmission and30

sporadic outbreaks (CDC, 2001). In addition, the largest epidemics occurred in thesouthern United States (1922) affecting over one million people in Australia (1925and 1942), Greece (1927), and Japan (1942-45). Vector density and factorsdetermining exposure to infected female mosquito vectors are importantdeterminants of the rate of dengue virus transmission (Monath and Heinz, 1996).The domestic habits of the principal vector, Ae aegypti, assure that infection occursin and around human habitations. Inhabitants of screened houses are at significantrisk of dengue epidemic (Ko Y-C, 1992). Where dengue is transmitted it is commonto find 10-20 female Ae aegypti per room, of which 5% to 110% may be infected(IIkal et al., 1991). Ae aegypti commonly exhibit interrupting feeding behavior, andmost female feed on the blood multiple times between egg laying; these factorscontribute to the rapid transmission of dengue virus and the explosive nature ofdengue epidemics (Monath and Heinz, 1996).CDC (2001) reported that the reasons for this global emergence of DF/DHFas a major health problem are complex and not well understood. That reporthowever, identified several of such important factors: First, effective mosquitocontrol is virtually nonexistent inmost dengue -endemic countries. Considerableemphasis for the past 20 years has been placed on ultra-low-volume insecticidespace sprays for adult mosquito control, a relatively ineffective approach forcontrolling Ae aegypti. Secondly, there are major global demographic changes, themost important of which has been uncontrolled urbanization and concurrentpopulation growth. These demographic changes 'have resulted in substandardhousing and inadequate water, sewer, and waste management systems, all of whichincrease Ae aegypti population densities and facilitate the transmission of Aeaegypti-bome diseases. Thirdly, increased travel by airplane provides the idealmechanism for transporting dengue viruses between population centers of the tropicsresulting in a constant exchange of dengue viruses and other pathogens. Lastly, inmost countries the public health infrastructure has deteriorated. Limited financialand human resources and competing priorities have resulted in a 'crisis mentality'with emphasis on implementing so-called emergency control methods in response toepidemics rather than on developing programs to prevent epidemic transmission.This approach has been particularly detrimental to dengue control because, in mostcountries, surveillance is Gust as in the U.S) very inadequate; the system to detect31

increased transmission normally relies on reports by local physicians who often donot recognize dengue in their differential diagnosis. Consequently, the epidemic hasoften reachedor passed transmission before it is detected.: 2.3.6 MOLECULAR EPIDEMIOLOGY OF DENGUE VIRUS INFECTIONSt., ' Molecular analysis of dengue viruses is a useful adjunct to epidemiologic-;:~;' investigation of their distribution over distance and time (Kanesa-thasan et al.,;.JI~·:1998).Nucleotide sequencing of the entire E gene is the modem tool of determining,ti. the origin and spread of dengue epidemics (Deubel et aI., 1993; Gubler and Trent,~F;,;1994). Phylogenetic and nucleotide sequence signature patterns of the E gene permit'.,. classification of each dengue serotype into a number of E- genotypes, which:, generally correlate with geographic origin. However, the current classification will.. ,:'h:expand as more strains of DEN serotypes are examined. Within a single, ii":~)'geographical area, genetic changes in. the virus population may be found over time~; ."i.(Walker et. al., 1988), with the appearance of new variants by mutation or byi( lrecombination and selection as well as by introduction from afar. Inconsistencies in?,";~;'>';' .;'~~~e geographical classification e.g., the finding of sequence homology between a~~Virusgenotype of one region and a strain from a widely separated region imply that:~a' new strain has been introduced by viremic humans or possibly by virus-infectedIf,n10SqUi~OeS or mosquito ova (Monath and Heinz, 1996). The differences in the~,\nucleotide sequences ofDEN-land 2 were reported to be closely associated with the~h -',!igeographic location as well as with different time ~f'l~'_;,,~j~ation'infection within the same(Kanesa-thasan et. al. 1998). Thus, genotype classification has been usefuli' .~~ a tool to determine the origin and spread of epidemics. Two distinct genotypes of,~ 'I{f .;DEN-I (E-genotype I and 11) coexist in Thailand. Genotype 11 has been recovered~. '~"Africa. In 1977, it was introduced into American region, where it caused a~~ -~~t_.:[: f~demic. The genotype has persisted in the Americas and its distribution hasf'li'expanded to include most Latin American countries except Argentina and Chile~" .'(Monath and Heinz, 1996). An example of a stabiy transmitted virus is the E-"genotype 1 of DEN-2. This genotype, represented by the New Guinea C strain, theprototype DEN-2, isolated in 1944, has persisted to the present day in Asia and wasintroduced into the Americas in 1981, causing the first epidemic of DHF in Cuba(Guzman et al., 1995). This genotype now coexists in multiple areas with E-32

genotype 111, which had been present In the Caribbean since at least 1969.Similarity between the Caribbean E- genotyelll strains and DEN-2 strains isolatedin the South Pacific between 1971 and 1976 indicated a potential route of spread.More than two decades ago, DEN-3 which was prevalent in the Caribbean, belongedto subtype IV but during 1996-1998 epidemic in Guatemala, DEN-3 isolatesbelonged to sub type IIIwhich was reported to be imported from Asia or Africa orfrom a Pacific island where this subtype was recovered in 1994. (Usuku et al.,2001). The DEN-4 circulating in endemic/epidemic pattems in the Americas since1981 is closely related to the strains from Niue Island and the Gilbert Islands, againindicating virus transfer between the Americas and the Oceania (Monath and Heinz,1996). Comparisons of DEN-2 strains isolated in the 1980sin Africa demonstratedthat enzootic strains associated with sylvatic vectors and nonhuman primates inWest Africa were genetically distinct from strains causing human epidemics. TheWest African epidemic strains were genetically related to isolates from Indonesia,Sri Lanka, the Seychelles Islands and Somalia, suggesting a route of spread acrossthe Indian Ocean to East Africa and thence to West Africa. The epidemics thus aroseby introduction of viremic travelers rather than from a local jungle cycle (Gubler andTrent, 1994; Rico- Hesse, 1990).2.3.7 THE PATHOGENESIS OF DENGUE FEVERNeuroadapted DEN produces typical encephalitic lesions, predominantly inthe rhinencephalus of infant, weanling, and adult mice (Monath and Heinz, 1996).Viral antigen is detectable by immunofluorescence in reticuloendothelial cells ofliver, lymph nodes and spleens of intraperineally-infected mice (Boonpucknavig, 1981). DEN antigens are detectable by Western blotting in suckling mousebrain or liver examined 7 days after intracerebral inoculation (Chardboonchart et al.,1990). A thymic nude mice peripherally infected with adapted DEN developes fatalencephalitis and viral antigens in neurons, skeletal muscles, and myocardi urn andKupffer cells In experimentally infected non - human primates, the role ofmononuclear phagocytes as principal sites of DEN replication has been establishedby tissue titration and IF staining of cells in the skin. spleen, lymph nodes, liver,lung, and thymus (Monath and Heinz, 1996) Classical DF produces self-limitedinfection in humans. Biopsies of skin lesions have shown swelling of endothelial.,.,33

cells of small vessels, perivascular edema and infiltration of mononuclear cells(Monath and Heinz, 1996)Dengue viruses multiply in the midgut epithelium, brain, fat body, andsalivary glands of mosquitoes (Rosen and Gubler, 1974). No detectable pathologicchanges result from infection and mosquitoes remain infectious for life (Monath andHeinz, 1996) DEN replicates in the female mosquito genital tract and may enter theovum at the time of fertilization, thereby infecting a portion of her progeny (Rosen,1987a). Sexual transmission also occurs from male Aedes with inherited infectionsto susceptible females, which may subsequently pass the virus to their progeny(Rosen, 1987b)2.3.8 DENGUE VIRUS STRUCTURAL DIFFERENCES THATCORRELATE WITH PATHOGENESISThe genetic differences between two DEN-2 genotypes that have beenassociated with distinct clinical presentations: the Southeast Asian genotype with DFand DHF and the American genotype with DF only showed the structural. differences that correlated with pathogenesis. Prior studies of wild and attenuatedDENs had suggested that genetic differences among strains of the four serotypescould be associated with attenuation, virulence, and for epidemic potential(Leitmeyer et. al., 1999). Despite the fact that various numbers of nucleotide andamino acid differences were found in coding and non-coding regions of the genome,no specific site(s) could be correlated with attenuation or severe disease in humans.From several studies, it has been concluded that all viruses belonging to SoutheastAsian genotype have the potential to cause severe disease (Gubler and Meltzer,1999; Uzcategui et. al., 2001). Therefore Leitmeyer et. al., (1999) demonstrated thata comparison of viruses form genetic groups with distinct clinical andepidemiological associations can better identify structural differences that correlatewith pathogenesis potential2.3.8.1 Significance of one E glycoprotein difference:- Amino acid diffcrence wasobserved between Southeast Asian genotype and American genotype virusesat position E-390.The E glycoprotein which is exposed on the surface of thedengue virion, represents the dominant virus antigen, 'conferring protcctive.....34

immune responses by eliciting neutralizing, hemagglutination-inhibit~ng,antifusion and virus enhancing antibodies. It is also for virus attachment,virus specific membrane fusion in acid pH cndosomes and virus assembly(Chen et. al., 1996; Roehrig et al., 1998). An analogous situation has beendescribed for other Flaviviruses: single amino acid substitution in theenvelope protein at positions 52 (Hasegawa et al., 1992) 138 (Sumiyoshi etal., 1995),271 and 336 in JE (Cecillia and Gould, 1991), at position 390 inMurray Valley virus (Lobigs et al., 1990), at positions 104 and 107 in a tickborneencephalitis-DEN-4 chimera (Pletnev, et al.,1993) and at position 171and 384 for TBE virus (Mandl et. aI., 1988) were shown to have dramaticeffecton virulence in vitro and in vivo. It is generally accepted that mostantigenic determinants reactive with antibodies are exposed on the surface ofthe protein (Berzofsky, 1985; Sutcliffe et al., 1983) and hence arehydrophilic. Based on this notion, algorithms, which calculate hydrophilicity,have been used to predict antigenicity. (Hopp and Woods, 1981; Kyte andDolittle, 1982). Most of the neutralization-resistant variants of Flavivirusesobtained so far have amino acid substitution, which cause a change in charge(Hasegawa et al., 1992; Jiang et al., 1993;). It is known that charged residueare important in the interaction of antigenic sites with antibodies. An analysisof the envelope glycoprotein shows that residueE-390 is located in a highlyhydrophilic region, Asn and Asp have the same hydrophilicity value, but theformer is neutral in charge while the latter is acidic. E- glycoproteins foldinto three distinct functional domains named 1,11 and III The observeddifference in residue E-390 is localized in the C-terminal domain 1I 1 (aminoacid 303 to 395) on the G sheet (amino acid 388 to 394) which formstogether with the C and F sheet, the outer lateral surface of the dimer. This isimportant because the lateral surface of thc domain 1I I is suggested tocontain residues implicated as determinants for host range, tropism, andvirulence in different Flaviviruses (Rey et al., 1995). The observed aminoacid change at position E-390 is also localized within one of the putativeglycosaminoglycan binding motifs at amino acid 284 to 310 and 386 to 411(Chen et al., 1996). These motifs have been shown to specifically bindDEN-2 viruses (Monath and Heinz 1996) to the host cell surface. Since35

glycosaminoglycan-binding motifs in position are predicted by definingmultiple regions enriched for basic amino acids (Fromm et al., 1995), it mustbe determined whether a change from acidic Asp (AM genotype) to neutralAsn (AS genotype) enhances DEN-2 virus attachment. Significance of prM differences: - While monoclonal antibodies specificfor the prM protein of DEN-3 and 4 have been shown to passively protectmice against challenge both homologous and heterologous DENs (Kaufmanet aI., 1989), prM monoclonal did not display neutralizing activity in vitro(Bray and Lai, 1991). The comparison of DEN-2 strain to its vaccinederivative, revealed an amino acid change from Asp to Val at position prM­29 (Kinney et al., 1997). Also 2 amino acid at prM -28 and prM-31distinguished the SE from the AM genotype of DEN-2. However, since theantigenic structure of prM is not known and the locations of protectiveepitopes have not been identified, the role of these amino acid changesbecomes difficult to assess (Leitmeyer et. al., 1999)., Significance of NS4b and NS5: Although some structural properties ofdengue virus NS4b and NS5 have been elucidated, little is known about theirantigenic structure. For NS4a and NS4b proteins, it has been shown that,despite marked amino acid heterogeneity, their hydrophilicity profiles areremarkably conserved among DENs and Flaviviruses in general. In terms offunction, both NS4 proteins may be involved in membrane localization ofNS3 and NS5 replication complexes through protein-protein interaction(Chambers et. al 1990). The observed residue change at NS4b-17 did notaffect the hydrophobicity profiles, and its importance in protein function isunknown. Because the NS5 gene presumable encodes a protein withimportant enzymatic functions, the RNA polymerase (Koonin, 1993), therewas conservation on its structure, especially in the presumed active sites. Of5 amino acids substitutions observed here, 3 at NS5-645 (Asparagine-->Aspartic acid), NS5-676 (Ser-->ArgininefLysine),and NS5-800 (Lysine -­>Serine), conferred a charge change, but were not .predicted to change thestructure while the side chain changes at NS5-271 [(Isoleusine-->Threonine)and NS5-819 (Gln--> Leu)] resulted in significant differences In36

hydrophilicity and antigenicity. However, since the three-dimensionalstructure or antigenic function of the NSS protein has not been determined,the relative contribution of these changes to infectivity and immunogenicityisunknown (Leitmeyeret. al., 1999). Significance of NTR difference: The NTRs of several positive-strand RNAviruses are predicted to fold into stem-loop structures that interact with viralor cellular proteins (Day et al., 1992; Andino et al., 1993) binds to the 3'terminus of the minos strand to initiate transcription of the positive strands,thus regulating replication. Mutations that modify these structures, andthereby alter the RNA-protein interactions, have been shown to affectvirulence or cause attenuation (lizuka, 1989;Kinney et al., 1993). Engineeredmutations and deletions in the S'NTR of a full-length DEN-4 cDNA clonewere shown to restrict DEN-4 growth in cell culture and inoculatedmosquitoes (Cahour et al., 1995). Most mutations within thc long stemstructure were lethal, but RNA transcripts containing deletions in the loop orshort-term regions were usually infectious. However, a mutant bearingdeletions in the 3'-terminal loop was least efficient in translation. It has beenshown that an Avto-U mutation that distinguishes South East dengue -2 fromAmerican genotype viruses at position 69 was predicted to change thesecondary structure of the viral RNA: a 4-nt long bulge was formed at the S'terminus of the American genotype, which reduced the length of the stem,but increase the length of the 3' -tenminalloop. Whether the presence of thissmall bulge could reduce translation efficiency remains unclear. Bulges assmall as 1nt have been shown to reduce RNA -protein interactions in otherviral systems (Weber and Konisberg, 1975; \Vu and Uhlenbeck, 1987)The 3' NTR contains sequences essential for virus replication andgrowth, serving as signals for the initiation of minus -strand synthesis, andpossiblypackaging in YF and DEN-4 (Hahn et al., 1987; Men et al., 1996).A stable secondary structure motif, formed by the 3' terminal 100nt, wasdescribed for all, mosquito-borne Flaviviruses studied to date (Rauscher etal., 1997; Shi et al., 1996). Within this region and farther upstream, threehighly conserved sequences termed CS1, CS2, and RCS2, are thought to be37

functionally important elements of the 3' NTR (Hahn, et al.,1987). Whileconservation of the 3' -terrninal region is assumed to be essential forFlaviviruses, there is evidence that regions farther upstream determine virusreplication efficiency. Deletions introduced into full- length DEN-4 cDNAclones that did not extend beyond the 3' terminal 113 nt were viable whentransfected into cells in culture, but they exhibited a range of growthrestrictions (Men et al., 1996). Leitmeyer et. al., (1999) observed aconserved region of 110 nt at 3' terminus, which 'folded in a form seen inother Flaviviruses (Rauscher et al., 1997). However, a striking size andsequence heterogeneity was observed in the 300-nt upstream regiondistinguished the genotypes, based on nucleotide alignments (Leitmeyer, et.aI., 1999). All the AM genotype samples revealed deletions in the regionimmediately downstream of the stop codon as well as 5 intersperseddifferences when compared with the SE genotype. Secondary structurepredictions yielded drastically different conformations that werecharacteristics for each genotype. The importance of these structures indefining DEN pathogenicity is unknown, but 3' NTR structure has also beenshown to correlate with virulence in YF (I'routski et. al., 1997) and TBE(Rauscher et al., 1997) viruses Leitmeyer et al., (1999) concluded that virusvirulence characteristics have been shown to be due to synergistic effect ofvarious genomic loci. It is possible that DEN pathogenesis is a result ofcohesive activity of the sites pinpointed above, in addition to the hostimmunologic factors. The amino acid substitutions within the coding region,which may affect the antigenicity or cell attachment and nucleotide changeswithin the NTRs, affecting secondary structure and thereby replication, maybe the viral determinants of severe dengue in humans. Genome differences innonstructural genes or in the NTRs potentially altering replication efficiencycould possibly be measured most effectively in terms of human viraemia38

392.3.9 HOST GENETIC FACTORS THAT MAY INFLUENCE SEVERITYOFDENGUE INFECTIONSAn Association between HLA haplotype and disease expression was foundin Cuban patients with DHF. HLA-B "blank", HLA-Al, and HLA-Cwl were morefrequent and HLA-A29 was less frequent than in normal control subjects (Paradoa etal., 1987). In Thailand, HLA-B "blank" and HLA-2 were associated with DHF(Chiewslip et al., 1981). In the Cuban epidemic, whites had higher incidence ofDHF than persons of the black race, a finding that could not be explained on thebasis of a racial difference in the background of immunity (Bravo et al., 1987).Among acquired factors that influence disease expression, nutrition and underlyingdiseases may play a role. The prevalence of malnutrition is lower in children withDIlF patients than in general population (Thisyakom and Nimmannitya, 1993),possibly reflecting the requirement for robust immunologic responsiveness in thegenesis of DHF. Chronic diseases such as sickle cell disease, diabetes mellitus, andbronchial asthma appear to increase the risk of developing severe disease (Bravo etal., 1987)2.3.10 CLINICAL MANIFESTATION OF DENGUE FEVERThe clinical manifestions of dengue fever described below were reported bySiler et al., (1926), Simmons et al., (1931), and Sabin, (1952):-ln the typical case,the disease begins abruptly, a 2- to 7- incubation period, with high fever, headache,retrobulbar pain, lumbosacral aching pain, conjunctival congestion, and facialflushing. Fever may be sustained for up to 6 to 7 days or may have a biphasic(saddle back) course. Initial symptoms are followed by generalized myalgia or honepain that increases in severity, anorexia, nausea, vomiting, weakness and prostration.The pulse rate may be slow in relation to the fever. Respiratory symptoms (cough,sore throat, and rhinitis) are not uncommon, especially in children. A transientgeneralized macular or mottled rash may appear on the first or second day.Coincident with defervescence (day 3 to 5) or shortly after, a secondary rash,maculopapular or morbilliform in nature and nonirritating, appear first on the trunkand then spreads centripetally to the face and the limbs but spares the soles andpalms. The rash may desquamate.Fever may rise again, creating the second phaseof the saddleback course. Generalized lymphadenopathy, cutaneous hyperesthesia,

and altered (metallic) taste sensation may accompany this step of the disease. Theperipheral white blood cell countis depressed with an absolute granulocytopenia andthe plateletcount may fall to lOO,OOO/mm 3 (Monath and Heinz 1996)2.3.11 CLINICAL MANIFESTATIONS OF DENGUE HEMORRIIAGICFEVER AND DENGUE SHOCK SYNDROMEOHF is distinguished from OF by the presence of fever, thrombocytopenia(platelet count, 100,000mm J ) , and hernoconcentration (hematocrit increased by20%). DSSis a more severe form of the disease, characterized by hypotension (pulsepressure

2.3.12 TREATMENT OF DENGUE VIRUS INFECTIONSThe World Health Organization has formulated specific guidelines for themanagementof cases (WHO, 1982). Principles of treatment are dictated by the needto closely monitor the patient's vital signs and hematocrit and to replace plasmavolume by judicious fluid replacement. Oxygen should be administered; moreover,if disseminated intravascular clotting is documented, consideration may be given toheparin therapy. Blood transfusion is indicated only in the case of severehemorrhage. Salicylates and hepatoxic drugs should be avoided. Corticostreroidsare widely used; evidence for their usefulness is conflicting, but some studiesindicate that they are of no value (Sumarmo et al., 1982).Specific, antiviral' therapy has not been extensively evaluated. Anuncontrolled trial of interferon was conducted during the 19~ 1 epidemic in Cuba,with some indications that deaths may have been averted. Although ribavirin showsill vitro activity against dengue virus, concentrations required for ill vivo efficacy arenot achievable without toxicity. A study of ribavirin in dengue-infected monkeysfailed to show an antiviral effect (Malinosky et al., 1990).2.4 WEST NILE VIRUS INFECTIONS IN NIGERIAIt has been reported that WNV and other viral types including otherarboviruses were isolated from livestock in the northern part of Nigeria between1966-1970 (Kemp et al., 1973). Since then, the most recent study on the status ofWNV infections in Nigeria was in 1990 where haemagglutinating Inhibitionantibodies (HI) were demonstrated in both animals and humans (Olaleye et al.,1990). That study covered the Rain Forest (Ibadan) and Sahel savanna (Maiduguri)ecological zones. Another report also revealed the presence of complement fixingantibodies (CFA) in humans and dornestiic animals (Omilabu et al., 1990).Therefore the endemicity of the virus in Nigeria cannot be over emphasized2.4.1 THE PATHOGENESIS OF WNVThe pathogenesis of WNV is similar to that of other Flaviviruses (Kundin etal., 1963; Nathanson, 1980) described above. Avian pathogenesis is distinctive inthat visceral organs are affected, with deaths from myocarditis as well asencephalitis (Stee1e et al., 2000) Virus is shed in cloacal fluids and bird -to bird41

transmission may occur by the oral-fecal route. By pecking, or ingestion duringpredation (Monath et al., 200l). That report also revealed laboratory infections ofhuman presumably through aerosols. Autopsies performed. on patients who diedwithin four weeks after inoculation of WNV resulted in WNV isolation from spleen,lymph nodes, liver, and lungs (Southam and Moore, 1951; 1954).2.4.2 WEST NILE FEVER AND WEST NILE ENCEPHALITIS2.4.2.1 The aetiologicalagent:-West Nile virus is a single stranded RNA VIruS with positive sense polarity.It has a molecular weight estimated as 3.8x106 to 4.2x 106; 3,000 kD (llkbp). It isspherical in shape, with icosahedral nucleocapsid of mean diameter of 43nm; 35­55nm. The virus has a diameter of 45nm and is enveloped. The envelope contains athin layer «5nm) of surface projections which under electron microscope areclarified byX-ray crystallography and represent molecules of envelope glycoproteinwhich form rod like structures anchored to the viral membrane at their basal ends(McLean et al., 2002). West Nile virus causes West Nile fever, West Nileencephalitis (Ludwig et al., 2002; Monath and Heinz, 1996) andmeningoencephalitis (Samuelsson, 2003). This virus is a member of the antigeniccomplex of Flaviviruses, which includes MVE, SLE, and JE viruses (Monath andHeinz 1996, Nasci et al., 2002). Kunjin virus (another agent in the virus complex) ismore closely related to WNV than to other members of the complex (Coia et al.,1988; Westaway, 1966). A high degree of cross-protection was found in hamstersimmunized with lE or SLE viruses and challenged peripherally with WNV(Hammon and Sather, 1956). Also, monkeys immunized with WNV were partiallyprotected from lethal intranasal lE virus challenge. whereas. lE-immune animalswere fully protected againstWNV challenge (Goverdhan et al., 1992).There are significant strain differences between isolates from different partsof the world and even between virus isolates within a given geographical region(Mcl.ean et al., 2002). Two distinct antigenic groups are recognized within WNVand these include: the African Middle East group (including isolates from Congo,Egypt, France, Israel, Pakistan, Uganda, the former Soviet Union and some fromSouth Africa) and a separate set from India and the Far East (Gaidamovich andSokhey, 1973; McLean et. al.. 2002). Other studies have shown considerable42

heterogeneity among strains isolated within a single region (Besselaar andBlackbum, 1988). In Madagascar for example. rnonoclonal antibody analysisidentified five antigcnic variants, of which four represented the African- Europeanantigenic group and one resembled the India- Far East group (Morvan et al., 1990).A Madagascan strain was found to be distinct from other WNVs at a nucleotidesequence level (Porter et a!.. 1993). The primary mechanism for the maintenance ofthe virus in 'endemic areas is the transmission cycles involving a variety of wildbirds and culex species mosquitoes (Ludwig et, 01.. 2002). Wild birds living in thevicinity or' water are the main host for virus propagation, presumably because ofadaptation between virus and the host over a long period of time (Samuelsson,2003). These birds do not become ill, despite the fact that they develop protracted,severe viraemia. Migratory birds living in tropical and subtropical areas cantherefore spread the virus to new areas, and probably temperate areas of the world.Mosquitoes that have the capacity to transfer the virus from migratory to local birdsarc required for the virus'to become established in the fauna. For this to occur, thevirus must be able to propagate in the mosquito's salivary glands. This requires anaverage ..24-hour temperature of more than 22°C for more than 12 days. This mayoccur in summer in temperate regions with a continental climate. The prerequisitesdescribed for the establishment of WNV in fauna can hardly be fulfilled in areaswhere a coastal climate prevails, such as Denmark. Therefore outbreaks are notlikely to occur in this country, even though migratory birds probably frequentlyintroduce the virus, and suitable mosquitoes are present. Thus in local bird speciessuch as crows, pigeons, or zoo birds, no fatal infections have appeared. Humans andmost other non-avian are incidental hosts, failing to produce viremias of sufficientmagnitude to infect susceptible mosquitoes (Monath, 1990). The degree of virulenceof this virus among vertebrate hosts is variable, ranging from inapparent to lethal,depending on species, age and other factors (Hayes, 1989; Komar 2000). WNVinfections are often very mild in infants and young children, with typical signs offever, lymph node enlargement and rashes in older children and young adults, whileneurological signs have been seen most frequently in the elderly. (McLean et al.,2002) The results of antigenic and genomic analyses may be interpreted to indicateintercontinental exchange of WNV strains by migrating birds, but with' localsegregation of distinct genotypic variants in some areas (Monath and Heinz 1996).43

44In Central African Republic for example, where human diseases have beencharacterized by hepatitis, virus strains recovered from humans, mosquitoes andticks differed from the Egyptian prototype and from local avian isolates bymonoclonal antibody analysis and restriction digest profiles (Mathiot et al., 1990). Amosquito isolate from Central African Republic was placed (together with an EastAfrican strain) in a distinct group based on nucleotide sequence (Porteret al., 1993).This suggests that the distinctive human disease pattern in that part of Africa may becaused by a local WNV variant with a non-avian transmission cycle (Monath andHeinz, 1996).WNV grC1ws and produces CPE or plagues in a wide variety of cell cultures(including primary chick andduck embryo), as well as in continuous lines of human,primate, swine, rodent, and amphibian origin (Monath and Heinz, 1996). Itmultiplies in Aedes aegypti and Drosophila cells and produces CPE in Aedesalbopictus cells. Mice and hamsters of all ages are susceptible to lethal infection bythe intracerebral route. Resistance to peripheral routes of inoculation develops withage whereby some virus strains are pathogenic for adult mice. Lethal oral infectionof the adult mice has been reported (Monath and Heinz, 1996). Hamsters transmitvirus to their young through the milk but rabbits, guinea pigs and cottonrats developantibodies without overt illness by all inoculation routes' while rats succumb tointracerebral infection. Rhesus and bonnet macaques develop fatal' encephalitis afterintracerebral or intranasal inoculation (Goverdhan et al., 1992; Nathanson 1980).Almost all birds tested developed viremia including wild species, chickens andpigeons; encephalitis and death may occur but are rare (McIntosh et al., 1961;Tayloret al., 1956). The developingchick embryo is highly susceptible to the virus.Wild African rodents except Arvicanthus obyssinicus do not develop viremia(Monath and Heinz 1996).Sporadic cases of naturally acquired WN encephalitis have been reported inhorses in Egypt and France (Guillon et al., 1968). However, low-level virernia,antibody production and absence of clinical illness are the rule. Bovine species donot develop viremia after experimental inoculation but antibody in cattle areprevalent (Monath and Heinz 1996).

45Dogs are susceptible to infection and some develop mild illness, but lowviremia level appears to preclude a major role in transmission cycles (Blackburn etal., 1989):Strain variations exist in the pathogenicity of WNV for cell cultures, mice,pigeons, and lemurs (Umrigar, 1977). Experimental infections in various arthropodshave been reported (Hayes, 1988). Culex IlIlivittalls, the principal vector in Africa, isa highly efficient vector. The WNV infect soft and hard ticks under natural andexperimental conditions (Monath and Heinz, 1996).2.4.3 CLINICAL MANIFESTATIONS OF WEST NILE FEVERThe incubation period is 1 to 6 days (New York Health, 2004). Most peoplewho are infected with WNV either have no symptoms or experience mild illnesscharacterized by fever, headache, backache, generalized myalgia, and anorexiabefore fully recovering (New York Health, 2004; Kightlineger, 2003). The course offever may be biphasic. In that report, some persons may also develop mild rash orswollen lymph nodes. The onset of the rash is either during the febrile case or at theend of it. The rash is roseolar or maculopapular, is nonirritating and principallyinvolves the chest, back and upper extremities. Rash may persist for up to a weekand resolves without desquamation. Generalized lymphadenopathy is a commonfinding, pharyngitis and gastrointestinal symptoms (nausea, vomiting, diarrhea,abdominal pain) may occur. The disease runs its course in 3 to 6 days, followed byrapid recovery (Marberg et al., 1956). Children normally experience milder illnessthan adults. The infection may also result in aseptic meningitis ormeningoencephalitis in a small proportion of patients, especially in the elderly(Monath and Heinz, 1996). Other neurologic presentations include anterior myelitisresembling poliomyelitis and encephalopolyradiculitis (Buletsa et al., 1989; Gradothet al., 1979). In an outbreak of encephalitis in persons aged 12 to 49 who acquiredthe infection in Israel, four patients died. Flatau et al., (1981) described three casesof encephalitis in the young, one of whom had papillitis. Eleven percents of cancerinoculated with the prototype (Egypt 101) strain showed clinical signs ofencephalitis (Southam and Moore, 1954), an incidence much higher than expectedfrom experience in naturally acquired infection probably due to the alteredsusceptibility and poor immune responsiveness of the patient population. Non-

neurologic, rare complications include myocarditis, and pancreatitis (Pletnev et al.,1992). In the Central African Republic, WNV has been responsible for cases ofhepatitis including fatal disease resembling YF (Georges et al., 1988). Clinicallaboratory findings include leukopenia' and in cases with CNS signs, CSFpleocytosis and elevated protein. According to New York Health, (2004), at its mostserious, WNV can cause permanent neurological damage and death (Monath andHeinz, 1996).Inapparent and very mild infections are common. In the series of cancerpatients intentionally inoculated, (Southam and Moore, 1954) 89% of 78 infectedpatients had no clinical signs or symptoms other than fever, in 72%, fever did notexceed 1°F.2.4.4 TIlE EXTERNAL HAUITATS OF WEST NILE VIRUSThe ecological habitats in which virus transmission occurs include coastalplains and river delta areas, forests, semi-arid areas andhighland plateaus. The rangeof WNV is determined by the habitat needs of the host and vector species and the.viral range has the potential to expand wherever suitable vectors and definitive hostsco-exist and where an infected vector or host can reach (McLean et al., 2002). Theseauthors further stated that the habitat Biornes where the virus appears to be able toreplicate and transfer between species sufficiently well to become permanentlyestablished in Biome(become Endemic) include the following:a) Permanent poolb) Transient waterc) Flood waterd) Artificial containers andTree holes2.4.5 TREATMENT OF WEST NILE VIRUS INFECTIONTreatment is supportive, often involving hospitalization, intravenous fluids,respiratory support and prevention of secondary infections for patients with severedisease (Kightlinger, 2003). Ribavirin in high doses and interferon alpha-2b werefound to have some activity against WNV and other flaviviruses in vitro (, 2000),' but there are no preclinical data to support its use in the treatment ofhuman disease, 'and attempts to treat Flavivirus infections in animals (including YF46

48where 22% of children and 61% of young adults were immune (Monath and Heinz,1996). A survey. conducted 10-15 years later demonstrated that the infectionprevalence in adults had decreased by 50% (Darwish and Ibrahim, 1975). In anotherstudy conducted in 1968, 14.6% of febrile children attending the fever hospital inAlexandria were diagnosed as having WNV infections (Mohammed et al., 1970).The h~perendemic virus circulation has precluded "harp epidemics but piaces a highburden of infection on chi\dhood populations, which experienced largelyunrecognized and clinically undifferentiated febrile disease. The risk of epidemicsmay increase as endemic transmission declines and a larger segment of the adultpopulation becomes susceptible.Summer time epidemics of West Nile fever (WNF) were recognized as earlyas 1950 in Israel and recurred there at frequent intervals during the 1950s (Marberget al., 1956).These epidemics involved hundreds of unrecognized cases but the trueincidence was undoubtedly much higher, and attack rate of over 60% were reportedin Rome localities, The epidemics were the result of amplified virus transmission andspillover to human population with low immunity. In South Africa with relati velylow background of immunity to WNV (13% to 20%), outbreak in 1974 resulted ininfection of 55% of the population (Monath and Heinz, 1996). Hundreds tothousands of clinical cases occurred but they were mild without any recognizedcases of encephalitis (Mclntosh et al., 1970). A small outbreak of WNV infectionoccurred between 1962 and 1964 in Camargue region of France, in which 13 casesweredocumented, some with encephalitic complications (Panthier, 1968).The incidence of Central Nervous System (eNS) infection has not beenclearly defined but this complication appears to be rare. However, cases have beendescribed in Israel, India, France and Egypt (Monath and Heinz, 1996). In Egypt, 4of 133 patients with aseptic patients or encephalitis admitted to one hospital between1966 and 1968 were shown to have WN infection (Abdel, 1970)WNV has been isolated from mosquito species; culex univittatus and culexpipiens molestus appears to be the most important vectors in Africa and the MiddleEast (Hayes, 1988). In addition the virus has been isolated from the extended list ofculex, Aedes, Anopheles, Mimomyia and Mansonia mosquitoes in Africa. (Monathand Heinz, 1996). In that report, Culex tritaeniorhynchus is an important vector intropical Asia; WNV has been isolated from ticks especially in bird rookeries in the

former Soviet Union. There had been numerous isolation from wild birds in manyareas; with birds sustaining high viraemia after experimental infection and are theonly known amplifying host (McIntosh et aI., 1970). Also, WNV has been isolatedfrom a camel and a grass mouse in Nigeria and frugivorous bat in India (Hayes,1988; Karabatsos, 1985). Bats have also been implicated as possible hosts inMadagascar, while humans and horses susceptible to clinical infections areincidentalhosts and are not thought to play a role in the mosquito transmission cycle(Monath and Heinz, 1996). Transtadial transmission hy ticks may play a significantrole in virus maintenance (Nuttall et aI., 1994).2.4.7 EPIDEMIOLOGY OF WEST NILE ENCEPHALITISWest Nile encephalitis in humans has been reported from Israel, India,France, Egypt, Belarus, Ukraine, Romania, the Czech Republic, and the US(Hubalek and Halouzka, 1999). The most dramatic upsurge in West Nile virusactivity has occurred in the last 5 years. In 1996, a large epidemic affected Bucharestand surrounding areas in Romania, with over 800 cases of CNS infection, an attackrate of 12/100,000 and a 15% case-fatality rate in patients with encephalitis (Tsai etal., 1988). In 1998 and 2000, equine epizootics occurred in Italy (Cantile et al.,2000), and France, and a human outbreak occurred in the democratic Republic ofCongo (Nur et. al., 1999). In 1999, an extensive epidemic of encephalitis occurred inSouthern Russia (Volgograd and Astrakhan), with >800 human cases, (Lvov et al.,2000). In 2000, Israel suffered the largest outbreak on record, with over 200 cases.In 1999, the virus was identified, the virus was identified for the first time in the US,causing 62 cases (7 deaths) in Queens, New York, and surrounding areas (Komar,2000). The disease re-emerged in New York City in 2000, with 18 cases (2 deaths).Virus activity in birds and mosquitoes during 2000 showed an expansion of thegeographic range within the Northeast and Mid-Atlantic States compared to 1999(Enserink, 2000).Outbreaks of equine encephalitis in horses have occurred in North Africa,West Africa, Italy, France and the US, and sporadic cases of West Nile encephalitishave been reported in cats and dogs (Monath et. al., 2001). The disease in horses hascaused considerable alarm and disruption of the equine industry in the US.49

Experimental inoculation of pregnant ewes causes congenital infection and stillbirth,indicating the potential for vertical infection in other species (Monath et. al., 2001).2.5 COLLECTIONS AND HANDLING OF CLINICAL SAMPLES INTIlE DIAGNOSIS OF DENGUE AND WEST NILE VIRUSINFECTIONSAlthough dengue virus replicates primarily in cells of the macrophagelineage, serum is the sample of choice for both virologic and serologic studies.Reports on the advantages of attenuating virus isolations from blood cells have beenconflicting (Gunakasem, 1982; Waterman et al., 1985). Anticoagulants such asheparin and EDTA should be avoided if possible. Although they have no effect onserologic tests, they may interfere with the PCR test (Willems et al., 1993). Ideally,the laboratory should receive at least one millimeter of serum, although more ispreferable if a variety of tests are to be performed. Hemolysis and repeated freezingand thawing should be avoided. The former interferes with the HI and CF tests,while the latter reduces the probability of virus isolation. In situations where small. children are tested, or large numbers of people are being surveyed, blood samplesdried on filter paper may be substituted. A one - tenth millimeter sample of blood onthe paper is sufficient for several ELISAs. If fatal cases are encountered, denguevirus may be isolated from liver, spleen, lung, lymph nodes and heart (WHO, 2001).Dengue virus, like other enveloped viruses, tends to be quite labile, and care shouldbe taken to store and transport specimens properly. If the serum specimen can bedelivered to the laboratory in five days or less, storage at 4°C or on wet ice isadequate to maintain virus viability. If a longer period of storage is needed, thesample should be frozen at -60°C immediately. Under these conditions the virusremains viable for many years. Alternatively, tissue samples may be fixed informalin. For convalescent samples taken for serologic analysis only, temporarystorage at 4°C is adequate if the sample is not contaminated. Permanent storageshould be at -20°C. ;A minimum set of data is needed for each patient (name, age, sex address,date of onset, travel history), on the specimen (date of collection, sample type), andon the contributing physician or hospital (name, address, phone/lax numbers) toensure the correct tests are conducted (WHO, 2001). Frequently, a summary of. 50

clinical data is useful for epidemiological studies. Ideally, a standard form,containing fill - in spaces for this information may be distributed to potentialcontributors. Samples submitted without the above information on dates of onset andcollection present problems for the laboratories in selecting and interpreting testsand are of little value to the attending physician (Vomdam and Kuno, 1997).2.5.1 DETECTION OF ANTIBODYHistorically, tests to measure antibodies against dengue virus have utilizedbiological markers such as Haemagglutination inhibition, complement fixation andneutralization tests. These are reliable tests for measuring antibodies but they fail todistinguish IgM from IgG antibodies, the former being the best indicator of a recentinfection. For this reason, these tests usually require paired acute and convalescentsamples to make a diagnosis. The recent trend in serologic techniques has beentowards tests such as Enzyme-linked Immunosorbent Assays (ELlSA), in which theantibody isotypes are measured individually, providing a picture of the developingimmune response, which is qualitative as well as quantitative. This is particularlyimportant in those cases in which the diagnostic laboratory receives only a singleserum sample (Vomdam and Kuno, 1997).2.5.2 ISOLATION, DETECTION AND IDENTIFICATION OF VIRUSIsolation of most strains of dengue virus from clinical specimens can beaccomplished in a majority of cases provided the sample is taken in the first fewdays of illness and proceeds without delay (WHO, 2001). Specimens that may besuitable for virus isolation include acute phases serum, plasma and washed buffyfrom the patient, autopsy tissues from fatal cases especially liver, spleen, lymphnodes and thymus, and mosquitoes collected in nature. For the first periods ofstorage (up to 48 hours), specimens to be used for virus isolation can be kept at +4°Cor +8°C. For longer storage, the serum should be separated and frozen at 70°C, andmaintained at such so that thawing does not occur. If isolation from leucocytes is tobe attempted heparinized blood samples should be delivered to the laboratory withina few hours. Whenever possible, original materials (viremic serum or infectedmosquito pools) as well as laboratory -passaged materials should be preserved forfuture study (WHO, 2001). Tissues and pooled mosquitoes are triturated or51

sonicated prior to inoculation. There are different methods of inoculation and themethods of confirming the presence of dengue virus (WHO, 2001). The choice ofmethods for isolation and identification of dengue virus depends on local availabilityof mosquitoes, cells culture and laboratory capability. Methods for isolation of thesearboviruses include: mouse inoculation, mosquito cell culture, and mosquitoinoculation. The isolates can then be identified using haemagglutination inhibition,ELISA, Immunofluorescence antibody test (FAT), neutralization test, CFT etcIn addition, other virological techniques for the detection of viral nucleicacid from clinical samples include polymerase chain reaction, hybridization probesand immunocytochemistry2.6 PREVENTION AND CONTROL OF DENGUE AND WEST NILEVIRUS INFECTIONSLack of vaccine and cure, leaves the option of prevention as the bestalternative. The only method of preventing dengue and WNV infections iscontrolling the mosquito infestation. Therefore to prevent contracting these nastylittle viruses, there is need to learn more about its flying harbinger of ill. The Aedesmosquito likes to bite in the morning and afternoons, often indoors or in the shade.The mosquito causing dengue primarily breeds in man-made containers like metaldrums, earthenware jars and other water storage jars, tires or other artificialcontainers (Pediatric on Call, 2001). The presence of mosquitoes in houses andflooding of basements were identified as risk factors for infection with WNV (Hanet al., 1996). So during daylight hours, in areas where mosquitoes are present, thereis need to protect one properly by using anti-mosquito measures. These includewearing repellents, such as DEET (70-30% is safe and effective) and spraying onesclothing with pennethrin (spraying mosquito nets and tents is important too).Alternatives include Neern oil from India, which can be drunk as tea or worn as alotion (Spira, 1998). That report also stated that Avon's skin- so soft is good butwears off too quickly to be practical. Furthermore, these authors observed thatmosquito coils do work, but vitamin B and garlic, do not work. The authorsuggested that wearing clothing is usually a good idea; especially long sleeves, longpants or skirts but the clothing should be loose fitting.52

Another measure is the elimination of breeding sites for these mosquitoes(Monath and Heinz, 1996). In addition to the above, proper solid waste disposal andimproved water storage practices including covering containers should beencouraged. Insecticides should be used periodically. For travellers going toaffected areas, use of mosquito repellents is advised.2.6.1 CONTROL OF DENGUE INFECTIONSThe increases in the incidence and distribution of DEN fever and DENhemorrhagic fever are due to several factors. No effective vaccines are available forDENs and WNV (Monath and Heinz, 1996). Vector control programs have beencurtailedor discontinued, and pesticides-resistant insect have emerged (Gaines et aI.,1996). Increasing urbanization, in the tropics and rapid world travel have greatlyextended the range of Aedes aegypti. Failure of conventional means to control thisimportant arthropod-borne disease suggests that novel strategies are required, suchas genetic manupulation of vector mosquitoes to render them incompetent for virustransmission (Crampton et al., 1990). The genetics of vector competence are poorly. understood. An alternative genetic mechanism to alter host susceptibility to virusinfection is the phenomenon known as pathogen -derived resistance (PDR). This isonly a proposal that is yet to be actualized.The development of a safe effective dengue vaccine is the high priority of theWHO, Health Ministries in some affected countries, the U.S. military and at leastone major pharmaceutical company but has been an elusive goal (Monath andHeinz.). Various strategies are also being explored toward the development ofgenetically engineered vaccines. Underlying assumptions of such vaccines are: theimmunity to protein E (and possibly also NS1) is required for durable protection;both humoral antibodies (especially neutralizing anti-E antibodies) and cytotoxic T­lymphocyte responses mediate protection; immunization with dengue virus isserotype specific (Green et al., 1993) and, therefore, simultaneous immunizationagainst all four serotypes is required. Dengue Vaccines in developmentMonath et. al., (2002) developed a live-attenuated Chimeric vaccine againstJEV. Starting with full-length 17D YF cDNA, these scientists replaced the genes53

encoding the prM and E proteins with the corresponding genes from JE SA 14-14-2.(Chambers et al., 1999). The Chimeric cDNA was then reverse transcribed to RNA,which was used to transfect African green monkey kidney (Vero) cells. Thenucleocapsid (C) protein, non-structural proteins, and non-translated terminiresponsible for virus replication remain those of the original YF 17D virus. TheChimeric virus replicates efficiently in vitro and in vivo. Monath et. al., (2002) gavea single subcutaneous injection of the vaccine to four groups of six volunteers, whowere either immune or not immune to YF from previous vaccination. All groupshad same mild symptoms and side effects as those elicited by YFI7D. The vaccineraised high titres of neutralizing antibodies against JE at doses of 10 5 and 10 4 plagueforming units. This vaccine IS itself a live-attenuated virus, sharing with other suchvaccines an ability to replicate in the host and to generate an array of antigens thatprovoke antibody and cell-mediated immune responses that result in exceptionaldurable, long-term immunological memory and usually lifelong protection.Applying this same, methodology, four dengue vaccines have been made byintroducing DEN-I-4 (prM and E genes into the backbone of YFV).Presently a total of six of such vaccines ore in late-stage development forDENs. Four of these vaccines are chimeras, generated by introducing prM and Egenes from DEN into the full-length cDNA of attenuated YF or DENs (panel)(Halstead and Deen, 2002). Passing each of the four DENs in non-human tissuecultures has made additional two vaccines. The first of these vaccines wasdeveloped at Mahidol University, Bangkok, and Licensed to Aventis Pasteur, Lyon,France. In this instance, DEN-I, 2, and 4 were serially passaged in primary dogkidney (PDK) cells while DEN-3 was passaged in African green monkey kidneycells (Bhamarapravati and Yoksan, 1997). When two doses of the resulting vaccineswere given to 130 chidren aged 3-14 years, seroconversion rates for tetravalentneutralizing antibody were observed in 80-90% (Bhamarapravati and Yoksan,2000).. A phase 3 trials in children is being planned (Halstead and Deen, 2002).In a separate effort at the Waiter Reed Army Institute of Research, SilverSpring, MD, USA, all four DENs were serially passaged in PDK cells and candidatevaccines produced by a final passage in fetal lung cells from the rhesus monkey(FRLL). After inoculation in SO adult volunteers, 80-90% developed neutralizingantibodies to all four viruses after two doses (Edelrnan et al., 2003). Phase 1 and 254

.'trials in children are planned for 2002-2003. This vaccine is licensed toGlaxosrnithkline, Rixensart, Belgium (Halstead and Deen, 2002).2.6.2. WEST NILE VIRUS VACCINE IN PROGRESS.There is currently no vaccine against WNV..However, a formalininactivatedmouse brain vaccine against the closely related JEV [JE-VAX(R)Aventis- Pasteur] is marketed in the US for travelers (Monath et al., 2001).Immunity to JEV may provide a degree of cross-protection in animals (Govemdhanet al., 1992), but there are insufficient data to warrant a recommendation for"Jennerian" of humans and horses (Monath et. al.. 2001). The safety of JE vaccinein the prevention of West Nile disease has not been established, and studies in micesuggest that heterologous immunity could potentiate brain inflammation followingneuroinvasion by WNV (Broom et al., 2000). Immunization of birds with JE­VAX(R) did not provide a high level of protection against WNV challenge (Monathet. al., 2001). DEVELOPMENT OF CIIIMERI Vax -WN as described byMonath et al., (2001)Genes encoding two proteins [prM and E] of YF 17D vaccine virus werereplaced with the corresponding genes of WNV. The resulting virion has theenvelope of WNV, containing structures involved in virus-cell attachment and virusintemalization, all antigenic determinants for neutralization, and epitope (s) forcytotoxic T Iymphocytes. The nucleocapsid (C) protein, nonstructural proteins andnon-translated termini responsible for virus replication remain those of the originalYF l7D virus. The chimeric virus should thus replicate efficiently in the host butimmunize against the heterologous (West Nile) virus (Monath et, al., 2001).The construction of a chimeric YF 17DI WNV was performed by thereversed Transcription of viral RNA to c DNA, which was then cloned in a twoplasmidsystem. The donor prM-E genes from wild typel999 strain of WNV wereusedfor the construction (Monath et. aI., 2001). That report also stated that, Plasmidencoding virus genomic sequences were propagated .in E coli. Appropriaterestriction sites were introduced to allow excision and in vitro ligation of plasmidfragments to produce full- length DNA template. Transcription of the linear DNA55

56template yields full-length messenger (infectious) RNA that was used for thetransfection of cell cultures using lipofectin or (for GMP production)electroporation. The progeny chimeric virus was amplified to produce a pre-Masterseed stock (passage 2). For GMP production, Master and Production seed virusstock is prepared at passages 3 and 4 respectively. The use of bacterial plasmid DNAto originate vaccine production, and the limited number of highly controlledpassages in cell culture significantly reduces the potential for contamination ofadventitious viruses. Nucleotide sequencing provides a means to monitor each stepof the production process and to determine the genetic stability (Monath et. aI.,2001).Vero cells (a continuous line of African green monkey kidney cells) are usedto manufacture chimeri Vax vaccine (and a number of other commercial vaccines)at passage level 5. Virus yield in supematant cell culture fluids after infection at lowmultiplicities are high. ePE are modest at the time of harvest, and cellular DNA andproteins are further reduced to acceptable levels by DNAse digestion andultrafiltrtion. The vaccine is stabilized using conventional excipients (Monath et. al.,2001).

CHAPTER THlUm3.0 MATERIALS AND METHODS3.1 STUDY POPULATION:A total of 1948 patients with fever ~ 38 D e, sent the laboratory (in TertiaryHealth Institutions in each of the zones (Table 2) used for the study) for malaria,Widal, HIV or hepatitis tests. Other clinical manifestations on these patients by timeof sample collection included: headache, abdominal discomfort, diarrhea,gastroenteritis, jaundice, etc3.2 STUDY AREAS:Six ecological zones in Nigeria were randomly selected for the study. Thezones include. Guinea! Grass savanna (Abuja), Rain forest (lbadan), Wooded /Guinea savanna (Gombe), Deltaic.! Swan savanna (Calabar), Sudan savanna (Kano)and Sudan / Sahel savanna (Maiduguri).A brief closed-ended questionnaire wasdesigned to collect demographic data of most of these patients such that include age,sex and the clinical history. Sera were obtained from febrile patients (Patients sentto the laboratory for malaria or Widal tests) from the six ecological zones in Nigeria.The zones include :(Table2 below)57

58TABLE 2: THE ECOLOGICAL ZONES IN NIGERIA WHERE SERA WEREOBTAINEDECOLOGICAL ZONES CITY WHERE SAMPLES NO OF SAMPLESWERE COLLECTED COLLECTEDGrass savanna Abuja 281Rain Forest Ibadan 442Wooded Savanna Gombe 341Mangrove Forest Calabar 317Sudan savanna Kano 267Sahel Savanna Maiduguti 300TOTAL: 1948

3.3 SERUM SAMPLES COLLECTION:About Srnl of blood was collected by radial venipuncture from febrilepatients. The blood was allowed to clot at room temperature and the serum wascarefully collected after centrifugation at 2,000rpm for 10 minutes and stored at ­20°C until used for analysis. A total of 1948 scrum samples were collected fromthese patients between June 2001 and July 2002 from six ecological zones inNigeria.In addition, 973 sera were collected from Sahel savanna zone during thethree seasons of the year namely, the cold I hot dry season (Jan-April), dry I rainyseason (May- August) and rainy I cold harmattan season (September-December)because the seasons are distinct in this zone. With the arrangement below, theseasons do overlap each other (Table3 below)59

3.3 SERUM SAMPLES COLLECTION:About Srnl of blood was collected by radial venipuncture from febrilepatients. The blood was allowed to clot at room temperature and the serum wascarefully collected after centrifugation at 2,000rpm for 10 minutes and stored at ­20°C until used for analysis. A total of 1948 scrum samples were collected fromthese patients between June 2001 and July 2002 from six ecological zones inNigeria.In addition, 973 sera were collected from Sahel savanna zone during thethree seasons of the year namely, the cold I hot dry season (Jan-April), dry I rainyseason (May- August) and rainy I cold harmattan season (September-December)because the seasons are distinct in this zone. With the arrangement below, theseasons do overlap each other (Table3 below)59

TABLE 3:SERUM SAMPLES COLLECTED AT DIFFERENT SEASONSFROM SAHEL SAVANNA ZONE, NIGERIAN=973SEASONS PERIOD NO. OF SAMPLESDry hot season January to April 352Rainy season May to August 306Cold season September to December 315Total: 97360

3.4 SEROLOGYThe IgM capture ELlSA (MAC- ELISA) was used to assess all the sera for IgM andIgG antibodies for DI - D4, West Nile Virus (WNV) and Yellow Fever Virus(YFV). The antigens were appropriately standardized.The procedure adopted was developed in WHO CRORA, IPD, SenegalThe following cell culture adapted strains of Dengue, West Nile and Yellow feverViruses were available at WHO collaborating center for Reference and Research onArboviruses (CRORA), Institute Pasteur De Dakar.DENI :DEN-2DEN-3DEN-4WNVYF(I) Hawaii /IgeB-O.5Jll at 10%DE -30/9/811(2)96619MAF490131721d 13/01/9/RefOlO121O5MB-0.5ml at 10%Vide-24/9/8(I) Ref.NO5MB-0.5ml at 10%Vide-24/9/8/(2) Ref. 96588Ref. 087074Ref. (FNUZU)(094564)TPIO-!These viruses were used in all control experiments and in some cases in thepreparation of virus stocks for ELlSA.61

3.4.1 PREPARATION OF STOCK ANTIGENS FROM TIlE SEEDVIRUSESDifferent species of Flaviviruses have different incubation periods in infected mice.All the seed viruses of Dengue, WN and YF used were reference strains, mouseadaptedkindly supplied by WHO CROM, Institute Pasteur De Dakar, (IPD),Senegal,About 10 litters of mice (each litter contained 10 suckling mice) were used for eachvirus seed. The mice used were 1-2 days old at the time of inoculation.The seed virus was diluted 1:5 with the growth medium. Exactly 0.02 ml of thediluted virus (01-4, WNV, YF) was inoculated into each suckling mouseintracerebrally. All the inoculated mice were properly fed till the experiment wasconcluded. The 11 1h liter of mice was used as negative control (the mice wereinoculated with L 16 medrn only, Dead mice within 24 hours post inoculation werediscarded. After inoculation, each cage was labelled with the virus used, date ofinoculation and the name of the researcher/scientist. The mice were observed dailytill regular deaths/sick (moribund) occurred among the inoculated ones in contrast tothe control ones. The dead/sick mice were harvested properly labelled and stored at-70°C until testcd.3.4.2 Preparations of Antigens from the infected mouse brainsAt IPD, Senegal where this experiment was carried out, crude mouse hrain isnormally antigen used in MAC-ELISA. Therefore, in this study crude antigenwas also used in MAC-ELlSAThe mice were thawed under the biosafcty cabinet. With the 18G needle, the brainwas carefully sucked out. The brain was ground unto smooth suspension for higheryield of the virus. Appropriate amount of borate buffer was added to the brainsuspension (1 mouse brain to 11111 of the buffer). Appropriate amount of tris bufferwas added to the suspension, (the total volume of the mouse brain suspension wasdivided by 10 to obtain the correct volume of tris buffer to be added). Add 0.01 mlof'[l-propiolactone to inactivate the virus (0.3% of the final volume). The suspensionwas incubated at 1'00111 temperature for 30 minutes for the inactivation of the virus.The suspension was centrifuged at 10,000 rpm for 20mins.Thc supemalant was62

aliquoted in lml amount in cryovials. Each cryovials was labeled with the virus,passage, lot number, source; date, 10% 5MB in borate I Tris O.lM, pH 9. The vialswere stored at _70°C til\ used.3.4.3 PREPARATION OF NORMAL MOUSE-BRAIN (NEGATIVECONTROL ANTIGEN)Suckling mice of the same age as those used for the antigens were harvested.With 18 gauge needle and syringe, the brain of the mouse was carefully sucked out.All the harvested mouse brains for a particular virus were. harvested and ground untoa smooth suspension. Appropriate amount of Borate buffer was added to the mousebrain suspension (l mouse brain to Iml of buffer). Appropriate amount of Trisbufferwas added to the suspension (For I mouse brain, O.1ml of Tris-buffer wasadded). The suspension was centrifuged at 10,000 rpm for 20 minutes. Thesupematant was aliquoted in lrnl amount into appropriately labeled cryovials. Allthe vials were stored at -70°C until tested3.4.3.1 TITMTION OF ANTIGEN AND MOUSE ASCITIC FLUIDThe technique developed by WHO CRORA, Institute Pasteur De Dakar, (IPD),Senegal, was adapted.1. Dilution of antigen.Each antigen was diluted serially starting with 1: 10 using carbonate buffer asdiluent in test tubes. Each dilution was added to the microtiter plate vertically (forexample antigen diluted W- 1 was added to wells Al to HI), covered and incubated at+4 oc overnight. The plate was washed 4 times with 0.5% PBS-Tween 20 washingsolution and tapped on filter paper to remove excess fluid. Apendix 22. Addition of mouse ascitic fluid (MAF)The mouse ascitic fluid is commercially available at WHO CRORA, InstitutePasteur De Dakar,(IPD), Senegal. The mouse ascitic fluid for each antigen used wasdiluted serially starting with 1:250 using the diluent (0.5% PBS-Tween plus 1%skimmed milk) in the test tubes. The dilution was added horizontally, for example1:250 was added to Al to AIO. The plate was covered and incubated at 37°C for 1hour.63

3. Addition ofconjugate: ,_-The plate was washed 4 times with washing solution and tapped on paper towel.Using a dispenser lOOMl of the appropriate diluted conjugate was added to all thewells. The plate was covered and incubated at 37 QC for 1 hour.4. Addition ofthe substrate;The plate was washed 6 times with wash solution before being tapped on filter paperseveral times to dry the plate. Then with the aid of a dispenser, lOO1L1 of thesubstrate was added to all the wells. The plate was then incubated at roomtemperature for 10 minutes. The reaction was stopped with the addition of lOO1l1 of4N H2S04. The plate was read using an ELISA reader at 450nm. Care was taken toensure that the wells' had no air bubbles before the plate was read.3.4.4 IgM-Capture enzyme-linked lmmunosorbent assay (MAC·ELISA).MAC-ELISA has become widely used in the past few years. It is a simple, rapid testthat requires very little sophisticated equipment (WHO, ZOO 1). Principle of the test:MAC-ELISA is based on detecting the dengue-specific IgM, or West NilespecificIgM or Yellow Fever-specific IgM antibodies in the test serum by capturingthem out of the solution using anti-human IgM that was previously bound to thesolid phase (WHO, 2001). If the IgM antibody fr?m the patient's serum is antidengue,or anti-WNV, or anti-Yf'V antibody, it will bind the dengue antigen that isadded in the next step and can be detected by subsequent addition of an enzymelabeled anti-dengue or anti-WNV or anti-YFV antibody, which may be human ormonoclonal antibody. An enzyme-substrate (O-tolidine) is added to give a colourreaction.J,rIIi3.4.4.2 MAC·ELISA ProcedureThe technique described by WHO CRORA, IPD, Senegal was adopted. All thereagents were brought to room temperature prior to addition into the plate. Maxisorpflat-bottomedmicroplates were used in all MAC-ELISA tests.One hundred microlitre of 1:500 dilutions in coating buffer of capture monoclonalantibody (MAb) for human IgM to each well of the microplate. The plate was(I ,tI164

covered and incubated overnight at 4°C. The plate was washed lour time, withwashing solution. Eaeh well was filled completely when washed. The residualliquid in the wells was tapped onto a paper towel One hundicd nucrolurc of a 1:100dilution in PBS-0.05% Twcen plus 1% skimmed milk (diluent) of each test humanserum, positive human conlrol scrum, and neg:1live human conlrol serum was addedto wells (e.g.AI to A2) in duplicate (skmuucu milk serves as a blocking agentagainst non-speei fie binding). Care was taken to avoid cross-contaminating thereagents in the dilution trays or plates during the addition of reagents. The plate wascovered and incubated for 1hour at 37"C. The plate was washed four times withwashing solution and tapped onto a paper towel About 100ld 01" an appropriatelytittered antigen in diluent was added to all the appropriate wells. Similarly, lOOrd ofthe negative (normal mouse brain) antigen at the same concentration as the positiveantigen) antigen, was added to the appropnarc wells. The plate was incubated at37')C for I hour after which it was washed four tunes with washing solution Then,JOOrL! of appropriate dilution (usually 1:5(0) of the specific mouse asciuc fl\lld wasadded to all the wells. The addition of mouse ascitic Iliud specific Ior each virusunder investigation became necessary to rule out any non-specific binding that musthave resulted from the use of crude antigen. The plate was covered and incubated forI hour at 37°C. Care was taken not to increase this incubation period beyond onehour i n 0 rder to a void increase inn on-spec i fie hinding and background 0 D. Theplate was washed four times with PBS-Twcen washimg solution and tapped onto apaper towel afterwhich IOOrL! of 1:15000 dilution of the conjugate (peroxidaseconjugatedSheep 19G Fraction to mouse) was added to all the wells. The plate wascovered and incubated at 37 "C for 1 hour. Cue was taken not to increase tlusincubation period beyond one hour as this may lead to increased non-specificbinding and background OD. The plate was washed 6 times with washing solutionand residual liquid was removed from the wells by tapping the plate onto a papertowel. About IOO~t1 01" the substrate (O-tolotlinc -scc appendix) was added to eachwell and the plate was incubated in thc dmk at room temperature for exactlyl Ominutes, Immediately, I(JOpl of the slop soluuon (4N H 2 SO,) was added to eachwell a Ilcr Ihc p late was r cad at 450nm. C arc was taken toe nsure I hal I he wellscontained 110 air bubbles before reading the plate CALCULATION OF RESULTS AND INTERPRETATIONBefore the results could be calculated for each clinical specimen, the validityof the test was determined as follows: The OD of the positive control serum withantigen was subtracted from the mean OD of the positive control serum withnegative antigen = (P). The OD of the negative control scrum with the viral antigenswas subtracted from the mean OD of the negative control serum on negative antigen" (N). The ratio of PIN must be greater than or equal to 2.0. This was considered asthe PIN of the positive control. The test validity was determined for each plate.Results of the test sera were only determined if the tests were valid. The tests thatwere considered as invalid were repeated.3.4.5 TREATMENT OF SERA THAT REACTED WITH THENORMAL ANTIGENThe rationale for the treatment: When the test sera were screened for IgMantibodies to dengue, West Nile and Yellow Fever viruses by MAC-ELISA, somesera reacted with the negative antigen (normal mouse brain) rendering such resultinconclusive. (For example serum 667, when the OD value of the well containingthis serum and the positive antigen read 1.903, the well containing the same serumwith negative antigen read 1.900). It was therefore assumed that some proteins insuch sera nonspecifically reacted with the normal mouse brain. Some attempts weremade to remove the nonspecific proteins in order to determine the status of thesepatients with regards to infections with the Flaviviruses concerned. The methodsadopted to achieve this include: ADSORPTION METHODa) The serum (diluted 1:100 in PBS-O.05% Tween 20 plus 1% skimmed milk)that reacted with the normal antigen (normal mouse brain was adsorbed withequal amount of the normal antigen (same concentration as in the test) forone hour at 37°C at stage one.b) Then an equal amount of the product of stage one and a fresh normal mousebrain was added and re-incubated at the same temperature and time.c) The process was repeated for the third time using the second stage product.The final product was used to repeat MAC ELlSA.66 DILUTION METHODOne of these problematic sera was diluted by double- dilution (l: 100­I: 12800) in MAC ELISA against the four serotypes of DEN viruses. ABSORPTION WITH REUMATOID FACTOR REAGENTA sample of three sera was treated with the Rheumatoid factor (RF)reagent as follows; each serum was diluted 1:50 andl50fll of the dilutedserum was added to 150fll of RF reagent. The mixture was incubated forISminutes at room temperature (RM). The treated sera were used in MACELISA against DEN- BY USING ANTIGEN OF CELLULAR ORIGIN:About 100fll DEN-2 (seed virus) was inoculated onto a 24-hour monolayerof AP61 in a 2Scm J flask. After being adsorbed for 1 hour at 28°C, 5rnl of thegrowth medium was added to the flask and was returned into the incubator at28°C.The inoculated cells were incubated for 10 days. The cell debris was collectedby centrifugation for 8000 r.m.p, for ID minutes. The presence of the virus in theinfected cells was detected using Immunofluoresence antibody technique (IPA)where % of the cells were infected. The supernatant was titrated and was used inELlSA test with a sample of the problematic sera. Controls included in the test wereantigens from the mouse brain, positive control human serum that did not react withthe normal mouse brain. The principle of the testA more sensitive format was described by Chungue et al (l989b) in whichantigen was captured in the plate, followed by incubation with the test serum.Antigen -specific antibody bound to the captured antigen was subsequently detectedwith a conjugated anti-human IgG (chain-specific) antibody.67 The test procedureThe plate was washed in PBS -Tween 20 (washing solution) and were tapped dryEach well was sensitized with lOO1l1 of appropriately diluted hyperimmune mouseascitic fluid specific for a particular antigen. The hyperimmune mouse ascitic fluidwas diluted in 0.1 M carbonate buffer. The plate was incubated at 4[)C overnightafter which it was washed 4 times with washing solution. 100111 of appropriatelydiluted antigen in PBS-0.05% Tween 20 plus 1% skimmed milk (diluent), wasadded to the appropriate wells. Again, lOUIlI of appropriately diluted negative(normal mouse brain) antigen was added to appropriate wells. The plate was coveredand incubated at 37°C for 1 hour after which it was washed four times with washingsolution. The residual liquid was tapped onto paper towel. About 100111 of each testserum, positive human control serum, and negative human control scrum, diluted1:100 was added to apprpriate wells. The plate was covered and incubated at 37°C.Care was taken to avoid cross-contamination of the reagents in the dilution trays orplate during addition of reagents. The plate was washed four times with PBS-Tweenwashing solution and was tapped onto paper towel. Then 100111 of the conjugate(Cappel peroxides-conjugated Goat F (AB ') 2 Fragment to Human IgG), diluted1:20000 was added to all the wells. The plate was covered and incubated at 37°C forI hour and was washed six times with washing solution and tapped onto papertowel. About IOOll1 of the substrate was added to all the wells. The plate wasincubated in the drawer (equivalent to a dark room) for 10 minutes. Adding lOOlkl of4N H2S04 to all the wells stopped the reation. The plate was read at 450nmwavelength.3.5 VECTORAL STUDIESFurthermore, crepuscular/scoop net catches of 3395 mosquitoes from the rainforest and Sahel savannah zones were made, identified and pooled by species.Themosquitoes caught represented the two genera (Aedes, and Culex) which are quitecommon in Nigeria were trapped. Vectoral study became necessary for the reasonsstated below:3.5.1 Reasons for the vectoral studyAvailable reports have consistently indicated that the presence of a competentvector results in an appreciable risk of Dengue Fever occurring in that68

environment. Aedes aegypti has been identified as the most efficient tropicalDengue Virus vector. Other reports have also described Aedes albopictus as anatural vector of the virus. Aedes aegypti and Aedes albopictus as well as Culexpipiens [atigans, Anopheles gambiae and Culex trigripes arc highly prevalent inNigeria and therefore could constitute important epidemiological factors in theestablishment of Arboviral infection in this environment. The vector studies areimportant in epidemic transmission of dengue and other Arboviruses3.5.2 Methods used in catching the mosquitoesHuman baits and scoop nets were used in catching mosquitoes from the field.The mosquitoes were caught alive. Some were stored at _20°C, which was the onlyoption available at that time and few in liquid nitrogen until transported to Dakar foranalysis.3.5.3 When and where the mosquitoes were trapped:Mosquitoes for this study had been caught between July and December 2001from two different ecological zones. The field- caught mosquitoes were identified inInstitut Pasteur, Dakar (IPD) with the technical assistance of a technician, Mr IRakotoarivony of Entomology Unit of IPD, Senegal. The identified mosquitoes werepooled as follows: 50 each for culex species and culex quincefasciatus except Aedesaegypti and Aedes species with 3 in each pool. Only culex quinquefasciatus, whichwere stored in Liquid nitrogen, were subjected. to virus isolation in an appropriatecell culture.Those stored at -20 o e was tested directly by RTPCR.69

Table 4:THE MOSQUITO VECTOR AND TIlE ECOLOGICALZONES WHERE THEY WERE CAUGHTIMOSQUITOES SEX NO. OF AREA OF PERIOD OFPOOLS COLLECTION COLLECTIONIbadan (Rain July toAedes aegyptiMales7 forest) DecemberIbadan (Rain July toAedes aegyptiFemales27 forest) DecemberAedes speciesAedes speciesMalesFemalesIbadan (Rain July to9 forest) DecemberIbadan (Rain July to12 forest) DecemberMaiduguri September toCulexMales31 (Sudan/ (Sahel Decemberquinquefaciatussavanna)Culex Maiduguri September toquinquefaciatusFemales9 (Sudan/ (Sahel DecemberSavanna)Ibadan (Rain July toCulex species Males8 Forest) DecemberICulex speciesFemalesIbadan (Rain13 Forest)July toDecemberMansoniaFemalesIbadan (R.1 Forest)July toDecember70

3.5.5 VIRUS ISOLATION FROM FIELD-CAUGHT MOSQUITOESWHO CRORA has recommended the use of a continous mosquito cell line(Aedes pseudoscutellaris-AP61) described by Varma et al., (1974), for the isolationof YFV and wild DENs (Digoutte et al, 1992). This cell line was known to be verysensitive to epidemic dengue virus (Race et al., 1979) and YFV (Varma et al.,1975). It also allowed the isolation of DEN-I, 2,and 4 from sporadic human cases inWest Africa (Digoutte et al., 1992). Generally AP61 cell line has been reported to besensitive to many but not all the arbovisuses (Digoutte et al, 1992). In this studyAP61 cell line was used for virus isolation from the mosquito vectors. Cell preparation:AP61 cell line adapted to growth at 28°C was used for virus isolation. The cellswere maintained on Leibovitzs (Gibco-BRL) (L-15)medium, with L-Glutamine,supplemented with 10% fetal calf serum, 10% Tryptose phosphate broth, lOOiLl /mlPenicillin, 100ugml Streptomycin, and 0.25 ml of fungizone. The medium wasaseptically prepared under a class II Biosafety cabinet and was filtered using Seitz filter.with a pore size of O.22nm. The sterility test was performed on prepared medium byplacing 5ml of the complete medium in a tube and incubating it at 28°C in the absenceof CO 2 for at least 3 days. Any medium showing bacterial or fungal growth wasdiscarded. Passage of the cells:Confluent monolayer cells were split 1:4 every 8-10 days. All the cell culturereagents were stored at _20°C except for bicarbonate and prepared medium, whichare stored at 4°C until used up. Cell freezing for storage:There was need for cell storage because persistence passage of cells couldresult in the loss of sensitivity to the growth of the virus. The growth medium wasremoved from exponentially growing cells. The freezing medium was prepared tocontain 10% fecal calf serum, 10% dimethylsulphoxide (DMSO), 100D/ml ofpenicillin, 100ug/ml of streptomycin and 0.25% of fungizone. The cells wereknocked off the plastic flask and aspirated several times to break up the clumps. Thecell concentration was adjusted to lOxl0 6 cells per ml with the freezing medium and71

dispensed Iml into each freezing tube. The tubes were placed into a double insulatedcontainer. All the containers were kept at -20°C for 6-8 hours, then at -170°Covernight. The cells were transferred into a canister and were exposed to the vaporof liquid nitrogen for 2-3 hours. The storage tubes were transferred into liquidnitrogen. The viability check was performed on he cells after 5 days as follows:Cells were rapidly thawed and transferred to flask with fresh growth medium. Therelative number of live and dead cells was checked after 24 hours. For virus isolation5ml of cells were seeded into a 25cm 3 flask at a concentration of 5xl0 5 per ml andincubated at 28°C overnight. Preparation of mosquito suspensionTwo genera of mosquitoes were used and these include Aedes species forDENS and culex species for WN viruses. Each genus of mosquito was pooled asdescribed above. Each pool marked for virus isolation was macerated with 2.5ml ofthe growth medium, (Aedes species was macerated with Iml while 2.5ml was usedfor culex), centrifuged at 10,000 rpm for 10 minutes. The supernatant was dispensedin duplicate (each for virus isolation and HT-PCR) into well-labeled 1.5 m!cryovials. About 500/11 of each pool of the mosquito was inoculated onto a 24hr oldmonolayer of AP61.The inoculum was adsorbed on the cell at 2SoC for I hourbefore Sml of the growth medium was added. The inoculated cells were incubated at28°C for 8-10 days. Since ePE is not visible on AP61, Indirect ImmunofluoresenceTechnique (IFAT) was carried out for virus detection. Inoculation of mosquito suspension on the monolayer of AP61Prior to inoculation, representative samples of the flasks were examinedunder the inverted microscope. Only the cells that formed at least 90-95% confluentmonolayer were used. Flasks that showed many rounded or floating cells werediscarded. About 200fll of mosquito suspension was inoculated into each flask. Itwas adsorbed for 1 hour at 280C before 5ml of the growth medium was add. Apositive control seed virus was included in each batch of the inoculated flasks. Allthe inoculated cells were returned to the incubator and were incubated for S to 10additional days at 2Soe without C02. During the period of incubation the pH of themedium was maintained at approximately 7 for maximum recovery of the virus.This was achieved by adding sodium bicarbonate (7%), drop wise until a red-orange72

color was obtained. Flask that became contaminated during the incubation periodwas repeated using mosquito suspension filtered through O.22n01 syringe filter. Thepresence of the virus was detected in each infected cell using Indirect FluorescenceAntibody technique (lFAT) because viral infection on AP61 is usuallynoncytopathic. INDIRECT FLUORESCENCE ANTIBODY TEST3. The principle of the test:This test makes use of a fluorescein- conjugated anti-human antibody todetect the binding of a patient's antibody to a solid phase (Shope and Sather 1979).The antigen substrate composed of infectcd cells mounted on a slide. The fourdengue serotypes and WNV were tested separately. With this technique, virusdetection was carried out in cell cultures using 7 pools containing 10 hyperimmunemouse ascitic fluids prepared with the reference arboviruses (Digoutte et al 1992).According to that report, the use of a panel of polyvalent hyperimmune mouseascitic fluids enhances the detection of 70 of the 80 arboviruses transmitted bymosquitoes in Africa. It also, facilitates the detection of arbovirus associations byusing either monospecific or monoclonal immune ascitic fluids (DEN-I, 2,3,4 andYFV) used in Indirect Immunofluoresence Technique. Test procedureInfected cells were scraped and the cells .collected by centrifugation at 1,000revolutions per minute for 10 min at 25°e in an IEC Centra GP8R centrifuge.Following re-suspension in 1m! of phosphate buffered saline (PES) solution,aliquots of the cell suspension were dropped onto welllabeled multi-well of Teflonmaskedslides, dried at room temperature for 3 hours and fixed in acetone for 20 minat -20 0e. Each mosquito pool was treated with the 7 pools of MAF and about 3slides were prepared with each mosquito pool. Whilst two of the fixed slides werestored at -700C for reservation in case of any accidental breakage, one was used forthe test at a particular point in time. The fixed slides were probed with a panel ofpooled (pools 1-7) arbovirus mouse ascitic fluid (MAF) diluted 1:20 with PES togive clear immunofluorescence with the corresponding reference arbovirus. (Sec thelist of arboviruses in each pool in below kindly supplied by WHO CRORA). Theslides were incubated in a humid chamber at 37°e for 30 minutes. Following73

subsequent washings in PBS and distilled water, the slides were probed with thesecondary anti-mouse monoclonal fluorescein isothiocyanate conjugate and wereincubated at 37°C for 30minutes. The conjugate was diluted 1: 80 along with 1%Evans Blue. The slides were then washed 2-3 times with PBS and were air-dried for20-30minutes. A drop of 70% glycerol in PBS was added to all the wells, coveredwith cover slips before visualization through fluorescent microscope. Appropriatepositive and negative controls were added to each slide. Positive control consisted ofcells inoculated with a particular virus and treated with its corresponding pool ofMAF while the negative control consisted of uninoculated cells treated with eachpool of the MAF used.3.6 RT - PCR IN SERA AND MOSQUITOES3.6.1 The principle of the test:In the PCR test, viral RNA from virtually any clinical source is extractedusing a combination of denaturing agents, usually the choatropic agent, guanidiumthiocyanate, phenol, chloroform. (Chomczynski and Sacchi, 1987). The viral RNA is.then transcribed into a complementary DNA (cDNA) using3.6.2 Semi-nested RT·PCR for dengue viruses:an enzyme (reverse­Transcriptase (RT)). The cDNA is then amplified a million fold or more using athermostable DNA polymerase (Mullis and Faloona, 1987) in a process calledPolymerase chain reaction (PCR). The application of this technology to dengue andWNV diagnosis has been made possible by the publication of genomic sequencesfor all of the medically important Flaviviruses. Analysis of these sequences hasshown a wide variety of conserved and variable regions from which cross-reactive~. and type-specific primers may be designed to fit individual requirement.~~;This assay uses six oligonucleotide primers within the capsid and the prMgenes of dengue viruses. A pair of cross-reacti ve primers among Flavi virusesincluding dengue viruses was used in the RT step and for the first round ofamplification. This was followed by a second amplification using a set of typespecificprimers. Each dengue serotype produced a DNA strand of different size,which was identified by gel electrophoresis (Lanciotti et al 1992).The procedure of RT-PCR for dengue viruses involved the following steps:74

a) The extraction of RNA from the clinical specimensb) The first round of amplificationc) The second round of amplificationd) The detection of amplification product by gel electrophoresis3.6.2.1 THE EXTRACTION OF RNA FROM SERUMlMOSQUITOSUSPENSIONffISSUE CULTURE EXTRACTAll the procedures adopted for the RNA extraction was according to thespecifications of the kit's manufacturer and the kit used was QI a Amp viral RNAMini Kit. The procedure involved was as follows: Four hundred microlitre of thebuffer AVL was dissolved at 80°C for 5 minutes in a 1.5 eppendoff tubeOne hundred microlitre of the serum or mosquito suspension or cell culturesupematant was added. It was shaken for 15ml seconds for proper mixing. The tubeswere centrifuged briefly to descend the products. Four hundred microlitres ofabsolute alcohol was added to the tube. The tube was shaken for 15 seconds. Fivehundred microlitres of the solution (AYL-supematant-ethanol was carefully)introduced into fresh column (from the kit). The column was centrifuged at 8000rpm for 1 minute. Part of the column that collected the liquid was replaced with newones. The remnant of the AVL-supematant-ethanol solution was added to thecolumn. The column was centrifuged for 8000 rpm for 1 minute. Five hundredmicrolitres of the wash buffer AWl was added to the column and was centrifuged at8000 rpm for 1 minute. The wash buffer was poured off and 500JlI of the bufferAW2 was added and centrifuged at 1400 rpm for 3 minutes. The wash buffer waspoured off and the column was re-centrifuged at 1400 rpm for 1minute. The columncontaining the extracted RNA was placed in a fresh 1.5 mIeppendoff tube. Sixtymicrolitre of the elution buffer (AYE) was added to the column in 1.5 tubes. Thetube was incubated at room temperature for 1 minute and was centrifuged at 8000rpm for 1 minute. The extracted RNA was stored at -70°C until tested or testedimmediately. For each batch of mosquito suspension/serum! extracted, positivecontrols (cell culture of the seed virus concerned) and uninoculated cell as negativecontrol were included.75 Selection of Oligonucleotide primers for dengue virusesDengue virus consensus primers DS 1 and DS2 and the type-specific primers(TSI, TS2, TS3, TS4), which, were ordered (Eurogentec) and used in this study3. first round of amplification in peR for dengue virusesThe principle of the test:The test combines the reverse- transcription of viral RNA and subsequent Taqpolymerase amplification in a single reaction vessel. This method consistentlyyielded an equal or a greater level of double-stranded DNA product as separatereverse-transcription reactions and PCRs (Lanciotti et al 1992). The target viralRNA was converted to a DNA copy (cDNA) prior to enzyrnatic DNA amplificationby using RT and the dengue virus downstream consensus primer (DS2), homologousto the genomic RNA of the four serotypes. Subsequently, Tag polymeraseamplification was performed on the resulting cDNA with the upstream dengue virusconsensus primer (DS I). The procedure of the first amplification for dengue viruses(TITAN):All relevant aspects of the RT-PCR (MgcI2, primers, RT. Taq polymerase, numberof cycles, and annealing temperatures) were initially optimized by using quantitatedpurified dengue virus RNA to achieve a maximum level of sensitivity.The procedure as described by IPD, Senegal was adoptcd:The peR Mix for each RNA extract was prepared as stated below:Five microlitre of DSl was added to am eppendoff tube. This was followed by 51d ofDS2. Then lOiLl of reaction buffer (5x) was added to the tube. Four microlitre ofdNTP was also added. Two microlitre of dithiothreotol (OTT) was added. Onemicrolitre of RT and a.2/-t1 of RNAse inhibitor were added to the tube...76 Mixture of reactants for amplification:Since the total volume of each tube must be 50).1I , the volume of each of the reactantthat constituted the mix was summed up and subtracted from the final volume. (Forexample: 51'1 +5).11 +101'1 +41'1 +21'1 +1).11 +0.21L! =27.2).11. Therefore, 501'1 -27.21'1=22.81'1. Another eppendoff tube was labeled appropriately. Exactly 27.2).11 of thePCR Mix was added to this tube. This was followed by 22.8fLl of RNA extract. Thecontrols included in each set of PCR were positive controls for DENJ-4 and thenegative control consisting of the Mix and the PCR grade water. The tube wasplaced on a thermocycler programmed as follows"Condition of amplification:1cycleReverse- Transcription1 cycle Denaturation35 Cycles DenaturationHybridization500C for 30 minute95°C for 2 minutes950C for 40 seconds550C for 40 secondsPolymerisation68°(: for 40.seconds1 Cycle Elongation 68°C for 7 minutes4°C for overnight3.6.2.4 DENGUE VIRUS TYPING. BY SECOND·ROUNDAMPLIFICATION WITH TYPE SPECIFIC PRIMERS (seminestedPCR) The principle of the test:In this method, type-specific primers replaced dengue virus downstreamconsensus primer, while dengue upstream primer (OSI) was retained. The Tag DNAdependent DNA polymerase amplified the products of the first amplification togenerate a DNA strand of different length, which was identified by gelelectrophoresis. Thus the second amplification differentiated dengue species intodifferent serotypes...77....._-------------_._---_. -- -.._-------._------_._--- The procedure of second amplification with type-specific primersAll the relevant aspects of this amplification were initially optimized byadopting multiplex and single step methods with the view to obtaining maximumlevel of sensitivity. Polymerase chain reaction by multiplex step:The PCR mix for each product of the first amplification was prepared asbelow:Three microlitre of DSl was added to the eppendoff tube. Three microJitre of eachTS 1, TS2, TS3, and TS4 was added to the tube. Five microlitre of the reaction buffer(lOx) was added to the tube. This was followed by 41.d of dNTP and 31;] ofmagnesium chloride (Mgcl2). Then 0.8 microliter of Taq DNA polymerase(Promega) was added. To know the volume of PCR grade water to be in the mix, thevolume of each reactant in the mix was added up. including the volume of the firstPCR product to be added. This was subtracted from the final volume- (For example:3ftl +3ftl +3ftl +3ftl +3ftl +SJll +4JlI +3Jll +0.8l1! =27.8ftl; Then 27.8Jll +SJll of thefirst PCR product =32.8Jll; therefore, the volume of the PCR water to be added tothe mix =50Jll -32.8Jll =17.2/-11). Finally 17.2ltl of PCR grade water was added tothe mix3. Mixture of reactant for amplification:Forty-five microlitre of the mix was transferred to another eppendoff tubeThis was followed by SJlI of the first PCR product diluted 1:100 with PCR water.The negative control included in each test contained 45ftl of PCR mix and SJll ofPCR grade water while the positive control tube contained 45/-11 of mix and 5/-11ofthe first PCR product of previously confirmed positive strains of DEN-l-4. Thetubes were placed on a thermocycler programmed as follows:78

Condition of amplification:I Cycle Denaturation 950C for 2 minutes20 CyclesDenaturationHybridization950C for I minute550C for I minutePolymerisationnoc for I minuteI CycleElongationnoc for 5 minutes4QC for overnight3. Second PCR: Single step: Each product of the first peR wastested against the four type-specific primers as follows:Tube I contained a mixture of 2lJI of the first PCR product (product A) diluted1:100 in PCR grade water. 31-1! of DSI, 3 fll of TSI, 51-11 reaction buffer (lOx), 4[1! ofdNTP, 3[1l ofMgCl2, 0,81-11 of Tag polymerase and 30,2[11 of PCR water. Tube 2contained 2[11 of product A, diluted I: 100 in PCR grade water, 31-11 of DS 1, 3ftl ofTS2, 5ft\ of reaction buffer (lOx), 4ft\ of dNTP, 3[l1 of MgC!2, 0.8[l1 of Taqpolymerase and 30.21-11 of PCR water. Tube 3 contained 2lJ! of product A diluted1;100 in PCR grade water, 31-11 ofDSI, 3ftl ofTS3, 5[11 of reaction buffer (IOx), 4JlIof dNTP, 3ftl of MgCI2, 0.8ftl of Taq polymerase and 30.2[l1 of peR water. Tube 4contained 2ft! of product A diluted 1:100 in PCR grade wter.Bul ofDSI, 3ftl of TS4,Sill of reaction buffer (IOx), 4ftl of dNTP, 3ft! of MgC!2, 0.8ft! of Taq polymeraseand 30.2[11 of PCR water. Negative control tube contained 2[1! of PCR water, 3[11 ofDSI, 3[11 of TSI, 5[11 of reaction buffer (lOx), 4ft! of dNTP, 3[1l of MgCI2, 0.8[11 ofTaq polymerase and 30.2ftl of PCR water. Positivc control tube contained2ft! offirst PCR product for DEN-I diluted 1:100 in PCR grade wter.Sul of DSI, 3ftl ofTSI, 5 [11 of reaction buffer (1Ox), 4ftl of dNTP, 3[11 of MgCI2, 0.8JlI of Taqpolymerase and 30.21l1 of PCR water.79


3.6.3 REVERSE-TRANSCRIPTION POLYMERASE CHAINREACTION (RT-PCR) FOR WEST NILE VIRUS IN CULEXMOSQUITOES /SERAThe extraction of RNA from culex mosquito suspension is the same as describedabove. REVERSE-TRANSCRIPTION (performed separately from PCR) The principle: Same as described above. In this method, thetranscription of RNA to cDNA by Reverse transcriptase enzyme was doneseparately from the PCR. Preparation of the MixThe mix of RT' for each extracted RNA, consisted of 4fJ.l of RT'buffer (5x), O.2fJ.1of RNAse inhibitor, 3.5fJ.1 of dNTP (8mMtotal), O.SIlI of PCR water and O.SIlI ofRT (I unit). In this case DTI was excluded.,3 The Procedure:Exactly IOfJ.1 of RNA extract from mosquito suspension/ serum/tissue culturesupematant was added to an eppendoff tube. Then IJlI of WNV downstream primerWN240 was added.The mixture was incubated at 95°C for 2minutes. The tube wasplaced immediately on ice for 5 minutes. The volume of the reaction was regularizedby brief centrifugation. Then 9/11 of the RT'mix 'was added for the final volume of20Jl!' The tube was incubated at 420C for I hour. The transcribed DNA wassubjected to PCR3.6.3.2 POLYlVlERASE- CHAIN REACTION FOR rnn DETECTION OFWEST NILE VIRUS IN CULEX MOSQUITO/ SERA3. The principleSame as described above except that a pair of primers consisting of WN downstreamconsensus primer (WN240) and WN upstream consensus primer (WN 132) wereused to transcribe and amplify the viral RNA. The amplified products were detectedusing gel electrophosis.81 The procedure of PCR for detection ofWest Nile virusA single round of amplification was carried out. Preparation of peR mixThe PCR mix for a single reaction (each cDNA) consisted of 5~llof dNTP (8mMtotal), 51ll of Taq DNA buffer (lOx), 5111 of primer WN 240 (lOOng/ugllll). 5111ofprimer WN 132 (lOOng/ulll), 3Jll of MgCI2 (25mM), 21.5JlI of PfR grade water,and 0.5111 ofTaq DNA polymerase. Preparation of amplification productsForty -five microlitre of PCR mix was transferred into another eppendoff tube. Thiswas followed by 5JlI of cDNA (the product of RT). Negative control tube contained45Jll of PCR mix and 5111 of PCR water. Positive control tube contained contained45111 of PCR mix and Sill of eDNA prepared from previously positive strain ofWNV. The tubes were placed on a thermocycler programmed as follows:Condition of amplification:1cycleDenaturation950C for 1 minute40 CyclesDenaturationHybridization950C for 30 seconds.550C for 30secondsPolymerisationnoc for 30 secondsI CycleElongationnoc for10 minutes4°C for overnight82------ -- ----------------

833.6.3.3 The procedure of RT-PCR for West Nile viruses in one reactionvessel (TITAN):The procedure as described by IPD, Senegal was adopted:The PCR Mix for each RNA extract was prepared as stated below:Exactly 5111 of WN240 was added to an eppendoff tube. This was followed by Spl ofWN132. Then lOpl of reaction buffer (5x) was added to the tube. Four microlitre ofdNTP was also added. Two microlitre of dithiothreotol (DTT) was added. Onemicrolitre of RT and 0.2pl of RNAse inhibitor werc added to the tube. Mixture of reactant for amplification:Since the total volume of each tube must be SOIL! , the volume of each of the reactantthat constituted the mix was summed up and subtracted from from the final volume.(For example: 5ftl +5ftl +lOftl +4ftl +2ftl lul +0.2ftl =27.2",1. Therefore, 50",1 ­27.2ft! =22.8ftl . Another eppendoff tube was labcled appropriately. 27.2ftl of thePCR Mix was added to this tube. This was followed by 22.8ftl of RNA extract. Thecontrols always included in each set of PCR were positive controls for DEN1-4 andthe negative control consisting of the Mix and the PCR grade water. The tube wasplaced on a thermocycler programmed as follows"Condition of amplification:I CycleI Cycle35 CyclesReverse- TranscriptionDenaturationDenaturationHybridizationSOoC for 30 minute95°C for 2 minutes950C for 40 seconds550C for 40 secondsPolymerisation68°C for 40 seconds1CycleElongation68°C for 7 minutes4°C for overnight


3.6.4 IDENTIFICATION OF AMPLIFICATION PRODUCTS BY GELELECTROPHORESIS AND ETHIDIUM BROMIDE STAININGThe method described by Lanciotti et al (J992) was used to identify theamplified products for both dengue and West Nile viruses.Preparation of the gel:One gram of agarose was dissolved in 100ml of T.A.E at a working solution of1:50. The agar was melted in the microwave at a pressure of 100 for 2 miutes andwas allowed to cool to 45°C. Five microlitre of ethiclium bromide was added to themelted agar before being poured unto the template containing the comb. The agarwas allowed to solidify for 20 to 30 minutes before the template was carefullyremoved and the agar was trnnsferrcd to the eletrophoretic tank. The tank was filledwith T.A.E (I :50).A paraffin wax was placed on the bench near the tank. A drop(2111) of Blue was spotted on the paraffin wax to correspond to the number of theamplification products. Ten rnicrolitre of each of the product was added to each spotof the blue on the paraffin paper and mixed. The mixed solution was transferred tothe corresponding wells in the tank. with the first and the last wells being alwaysreserved for bimolecular marker. The resulting DNA hand was visualized on a UVtransillurninator.. The size of the resulting DNA band should be charateristic foreither dengue or West Nile viruses·1I85'.IiI ,

CHAPTER FOUR4.0RESULTS4.1 OPTIMAZATION OFTHE ANTIGENS AND HVPERIMMUNF:MOUSE ASCITIC FLUID FOR MAC-EUSATable 7 shows the chessboard titration for DEN], 3, and 4 against thecorresponding hyper immune mouse ascitic fluid. The optimum concentration of thestock antigen was determined prior to the performance ofEllSA test. This was doneby plotting the OD values of the dilution of each antigen against its correspondinghyper immune ascitic fluid (Figure 1). According to figure 1, the OD valuesincreased Irorn lLO dilution ofthe antigen to]:40 after which there was a sharp fall.This occurred at 1:500 dilution of mouse ascitic fluid. Therefore the optimumconcentration of the antigen and mouse ascitic fluid in ELISA according to figure 1was ] :40 and ] :500 respectively. The titration of antigens against its correspondinghyperimmune mouse ascitic fluid was repeated using IgM MAC ~ELlSA method.This became necessary because in chessboard titration, the antigen was used to coatthe microtitre plate before the addition of the hyperimmune mouse ascitic fluid. Butin MAC- ELlSA, the homologous antibody was used to coat the plate. However,the Maxisorp microtitre plate used for both chessboard and MAC-ELISA adsorbsantibody better than it does to antigen. This probably accounted for the inconsistentOD values as observed in Table 7. Nevertheless in 19M EllSA; more consistentresults were obtained (Talble 8).86

"r~-TABLE 7: TITRATION or ANTIGEN AND MOUSE ASCITIC FLUID BYCHESSBOARD METHODDILUTION uie or 1:20 OF 1:40 OF 1:80 OF 1:160 OF 1:320 OF1:250 OFANTIGEN ANTIGEN ANTIGEN ANTIGEN ANTIGEN ANTIGEI\MAF* 0.918 0.747 0.458 0.257 0.149 0.0 71:500 OFMAF 1.096 0.868 0.715 0.333 0.17 0.105I:\()OO OFMAF 1.057 0.881 0.668 0.422 0.239 O.lll-1:20000FMAF 1.129 0.842 0.674 0.502 0.282 0.154MAF= mouse ascitic fluid

0.8•,..0.8> c00.4 i--1.1-0O·F-ANT IGEN'-1;--1:20 OF ANTIGENI 1:40 OF ANTIGEN:--1:80 OF ANTIGEN'--1:160 OF ANTIGENIL_-==1:~_20 OF_~r-IT~~_~N1:250 OF MAF' 1:500 OF MAF 1:1000 OF MAF 1:20000F MAFDilutions of mouse ascitic fluidFigurel:TITRATION OF A~TIGEN AND MOUSE ASCITIC FLUID BYCHESSBOARD METHOD88

TABLE 8: TITRATION OF ANTIGEN AND MOUSE ASCITIC FLUID BYIgMELISADILUTIONS 1:10 OF 1:20 OF 1:40 OF 1:80 OFANTIGEN ANTIGEN ANTIGEN ANTIGEN1:250 of MAP 0.236 0.705 0.557 0.3881:500 of MAP 0.229 0.826 0.619 0.3711:1000 of MAP 0.195 0.759 0.515 0.4161:2000 of MAP 0.233 0.663 0.525 0.37MAF= Mouseascitic fluid89

1~1:100F Antig~~i:--1:20 OF ANTIGEN~i 1:40 OF ANTIGEN--1·80 OF ANTIGEN- -_ ..._-------125001 MAF 1:500 of MAF 1:1000 01 MAF 1:200001 MAFD~Lltions of mouse ascitic fluidFIGURE 2: TITRATION OF ANTIGEN - IgM ELISA90 I

4.2 PATTER."I OF DENGUE VIRUS INFECTIONS IN NIGERIA4.2. 1 IgM CAPTURE ELISA FOR DENGlm VIRUSESThe distribution of IgM and its corresponding IgG antibodies against dengueviruses in Nigeria is presented in Table 9. Thirteen (0.6%) of the 1948 sera werepositive for DEN IgM antibodies (consisting of 11 DEN-2 and 2 DEN-I) from 4 ofthe 6 ecological zones in Nigeria studied. The zones with positive cases were Rainforest (0.9%-DEN-2), Grass savanna (0.36%: DEN-2), Deltaic savanna (0.1%_DEN2), and Sahel Savanna (1.70/0-DEN I and 2). (Table9). Figure 3 shows the IgMand its corresponding IgG antibodies to the different serotypes of DEN in the fourecological zones mentioned above. For example in Figure 3, sample 6 appeared tobe a recent infection with DEN-2 with high OD value (1.151) while sample 4seemed to be a case of anamnestic response.,.II91I

TABLE 9DISTRIBUTION OF IgM AND ITS CORRESPONDING IgG TODEN VIRUSESIgMIgGSAMPLENO DEN-! DEN-2 DEN-3 VEN-4 DEN-! DEN-2 DEN-3 DEN-'369 - + - - - + - -VF45 - + - - - + - -'VF30 - + - - - + - -30 - + - - - + - -627 - + - - - + - -I656 - + - - - + - -641 - + - - - + - -02/0681 - + - - - + - -3515 - + - - - + - -·2599 + - - - + - - -2463 + - - - + - --3778 - + - - - + - --f,:2725 - + - - - + - ---_..- _._-;,I'92


tI1 .4 - ,.,---,----- -------, r1_21 -0.8 -0.0. Values 0.6-"--0.4 .0.2-"----1 t----J BII,~lgM DEN.lgG DENiDlgM DENio 19GDEN-\.lgM DEN""gG DENI_lgM DENo '-!Lc>_D E No-02-1.1 ,.' /11 2 3 4 5 6 7 8Sample Nos9 10 11 12 13Figure 3: DEN IgM AND THE CORRESPONDING IgG ANTIBODY

4.2.2 SEASONAL DISTRIBUTION OF DENGUEVIRUS IgMANTIBODIES IN SAHEL SAVANNA ZONETable 11 shows the seasonal distribution of DEN IgM antibody. Asignificant association (X 2=5.82, df=2, P=O;054) was observed between theprevalence of DEN IgM antibodies and the seasons of the year in Nigeria. Theantibody prevalence was significantly higher during the rainy season (1.3%) than thecold harrnattan period (0.3%) and the dry season (0%). It was observed that 1I(55.6%), of the 18 DEN IgM positive sera were collected in the month of July and 2(11.1%), and 5 (27.8%) in June and August respectively (Table I I and Figure 4).95

TABLE 11: SEASONAL DISTRIBUTION OF DENGUE VIRUSINFECTIONS IN SAHEL SAVANNA ZONESEASONS PERIOD OF THE NO. TESTED NO.POSITIVEYEAR (%)Hot dry season January to April 353 0(0)Rainy season May to August 306 4(1.3)Cold season Sept to December 315 1 (0.3)TOTAL 973 5 (2.9)96


TABLE 12: MONTHLY DISTRIBUTION OF' DEN IgM POSITIVE SERAN=18DATE OF OD VALUES OD VALUES OD VALUESSINO. SAMPLE OF SAMPLE OF OFCOLLECTION POSITIVE NEGATIVECONTROL CONTROLS1 369 20-7-00 0.642 0.832 0.0142 VF45 6-6-00 0.252 0.864 0.1013 VF30 5-6-00 1.151 0.864 O.LOl4 30 5-7-01 0.976 0.864 0.1015 627 21-7-01 0.444 0.884 0.1026 656 21-7-01 Ll56 0.884 0.1007 641 21-7-01 0.398 0.860 0.0208 02/061 2-7-01 0.352 0.836 0.110.9 3515 15-11-01 0.353 0.85 0.02610 2599 31-8-01 0.30 0.927 0.015--11 2463 23-8-01 0.222 0.85 0.02612 3778 28-11-01 0.218 0.927 0.01513 2725 11-8-01 0.32 0.927 0.01514 65 5-8-01 0.353 0.85- --0.02615 216 12-7-01 0.23 0.586 0.008-,16 482 19-7-01 0.23 0.85 0.02617 407 13-8-01 0.221 0.586 0.00818 441 29-7-01 0.218 0.85 0.02698

4.23 AGE DISTRIBUTION OF PATIENTS WITH IgM TO DENGUEVIRUSESTable 13 shows the age distribution of patients with IgM to dengue viruses. Therewas no significant (X 2=3.1, Llf=3, P=0.376233) relationship between the prevalenceof DEN IgM antibodies and the age of the patients. But numerically, more patientsaged 20-29 years tested positive (1.2%) to DEN IgM than other age groups.99

TABLE 13: AGE DISTRIBUTION OF PATIENTS WITH IgM TO DENVIRUSESAGE TOTAL NO. NO. POSITIVEGROUPS TESTED (%)0-9 228 4 (1.8)10 - 19 297 2 (0.7)20-29 687 8 (1.2)30- 39 682 4 (0.6)40-49 291 0(0)50- 59 74 0(0)>60 28 0(0)Not specified 634 0(0)TOTAL 2921 18 (0.6)100

4.2.4 GENDER DISTRIBUTION OF I'ATIENTS WITH IgM TO DENGUEVIRUSESThe distribution of patients with DEN IgM by gender is presented in Table 14.Similarly, the gender of patients with DEN IgM antibodies was not significantlydifferent (X 2=3.23, df=2 P=O.l989 16).101

TABLE 14: GENDER DISTRIBUTION OF PATIENTS WITHIgM TO DEN VIRUSESGENDERTOTAL NO. NO. POSITIVETESTED (%)Male 1383 11 (0.8)Female 1134 7 (0.6)Not specified404 0(0)TOTAL 2921 18 (0.6)102

4.2.5 THE PREVALENCE OF DEN JgG ANTIBODIES IN DIFFERENTECOLOGICAL ZONES IN NIGERIAThe zonal distribution of DEN IgG antibodies is displayed on Table 15. Thepresence of DEN IgG was highested in Sahel savanna (81.7%), followed by Rainforest (69.0%) and Wooded (69.0%). However, the zones with the least prevalencerate were Sudan savanna (32.6%) and Grass savanna (38.1 %). Figure 8 equallyshows the zonal distribution of DEN IgG antibodies with the Rain forest (27%) andSahel savanna (21%) significantly different from Sudan (7%) and Grass savanna(9%). The seasonal distribution of DEN IgG is presented on Table 16. Nosignificant difference (X 2= 3.49, 1'=0.17494073) was observed. Generally significantnumber (86.2%) of patients in Sahel savanna zonc had DEN IgG antibodies.103

TABLE 15: THE PREVALENCE OF DEN IgG ANTIHODIESACCORDING TO ZONESSIN0 ECOLOGICAL CITY WIIFRE NO. NO.ZONES WHERE SAMPLES TESTED POSITIVESAMPLES WERE FOR DENWERE COLLECTED IgG (%)COLLECTE\)1 Rain forest Ibadan 442 305 (69.0)2 Sudan savanna Kano 267 87(32.6)3 Wooded savanna Gombe 341 238 (69.2)4 Grass savanna Abuja 281 107 (38.1)5 Mangrove forest Calabar 317 189 (59.6)6 Sahel savanna Mai dugUIi 300 245 (81.7)TOTAL 1948 1169 (60.0)104

90 r-----------------------~--------------- ------- ---------807080wU~ 50ZWo 40Cl:WIl.3020ECOLOGICAL ZONESFigure 5: ZONAL DISTRIBUTION OF DEN 19G ANTlBODIES105

TABLE 16: SEASONAL DISTRIBUTION OF IgG ANTIBODY TO DENVIRUSESSEASONS PERIOD OF THE NO. TESTED NO.POSITIVEYEAR (%)Hot dry season January to April 353 296 (83.9)Rainy season May to August 306 272 (88.9)Harmattan season Sept to December 315 271 (86.0)TOTAL---i'!73 839 (86.2)106

4.3 IgM CAPTURE ELlSA FOR WEST NiLE VIRUSThe prevalence of WNV IgM antibodies is presented in Table 17. Twelve(1.2%) of the 973 sera from Sahel savanna zone had WNV IgM antibodies. Possiblemixed infections of DEN-2 and WNV was observed in one sample. Figure 6 showsthe endemicity of WNV infections in the environment. It could be that samples werecollected when the WNV IgM started to drop while the IgG antibodies wereincreasing. Samples I and 6 appeared to be cases of anamnestic response for WNV.107

TABLE 17: THE PREVALENCE OF WNV IgM AND CORRESPONDINGIgG ANTIBODIESSINO SAMI'LENO. Igl\I (OD IgG (ODVALUES) VALUES) :I 1243 0.581 0.6932* 3515 0.254 07383 2172 0302 08614 3545 0.277 0.9015 3494 0.29 07576 3679 0763 0.7347 3322 0.427 0.7768** 2725 0.32 0.8469 3460 0.478 0.820.10 3255 0.49 0.72211 3481 0.25 0.10012 3500 0276 0.881* Possible crossreaction with DEN** Possible mixed infection with DEN~l08;:)...J~ 0.5ci 0.400.3l~vmv,gM1I III WI'lVlgGj0.20.11 2 3 4 5 6 7 8 9 10 11 12SAMPLE NUMBERSFigure 6: WEST NILE IgM AND THE CORRESPONDING IgGANTIBODIESWEST NILE [gM AND THE CORRESPONDING IgGANTIBODIES109

4.3.1. THE SEASONAL DISTRIBUTION OF WNV IgMANTIBODIESIn Table 18, the seasonal distribution of WN and its corresponding IgGantibodies is presented. The prevalence of these antibodies during the coldharrnattan period (IgM-2.9%) was significantly different (X 2 = 11.38, dfoo2, poo>O.003388) from the dry (IgM-O.O%), and rainy season (0.1%). Figure 7 further showsthe prevalence of this antibody during the cold period was significantly differentfrom other seasons of the year. In Figure 7, 97% of WNV IgM antibodies weredetected during the cold harmattan period in contrast to rainy (3%), and dry (0%)seasons. Table 19 shows the months during which sera of patients with WNV IgMantibodies were collected. Most (66.7%) of these samples were collected inNovember during which the environmental temperature could be as low as 18°C orbelow in Sahel savanna zone in Nigeria.110

TABLE 18:SEASONAL PATTERN OF WNV IgM IN PATIENTS WITHFEVER IN SAHEL SAVANN!!. ZOPERIOD OF THENO.IgMSEASON YEAR NO. TESTED POSITIVE (%)Dry Season January to April 352 o (0)Rainy season May 10 August 30G 3 (0.1)Cold season September to December 315 9 (2.9)TOTAL 973 12 (1.2)111


TABLE 19: MONTHLY DISTRIBUTION OF WNV IgM ANTIBODIES.SERIAL SAMPLE DATE OF OD VALUES OD VALUES OD VALUESNO. NO. SAMPLE FOR FOR FORCOLLECTION SAMPLES POSITIVE NEGATIVECONTROLS CONTROLS1 1243 19-5-01 0,581 1.584 ,0732 3515 15-11-01 0.254 1,584 0.0733 2172 6-8-0 I 0.302 1.584 0.0734 3545 30-11-01 0.277 1.584 0.0735 3494 6-11-01 0.29 1.580 0.0316 3679 19-11-01 0.763 1.580 0.0317 3322 25-10-01 0.427 1.580 0.0318 2725 11-8-01 0.32 1.580 0,0319 3460 2-11-01 0.478 1.580 0.03110 3255 22-10-01 0.49 1.574 0.055I 1'1 3481 5-11-01 0.25 1.574 0.06\5512 3500 6-11-01 0.276 1.574 0.05513 3562 9-11-01 0.150 1.574 0.05514 3222 10-12-01 0.226 1.574 0.055-,•113

4. 3. 2. AGE DISTRIBUTION OF PATIENTS WITH WNV IgMANTIBODIESTable 20 shows the age distribution of patients with WNV IgM antibodies.The prevalence of these antibodies and the ages of the patients were not significantlydifferent (X2=P>0.05). Nevertheless, numerically, more patients (2.4%) within theage group 20-29 years had WNV IgM antibodies.114

TABLE 20: AGE DISTRIBUTION OF WNV IgM POSITIVE PATIENTSAGE TOTAL NO. NO. POSITIVEGROUPS TESTED (0/0)0-9 59 1 (1.7)10-19 91 0(0)20-29 297 7 (2.4)30- 39 279 1 (0.4)40 -49 125 3 (2.4)50-59 31 0(0)>60 19 0(0)Not specified 72 o (0)TOTAL 973 12 (1.2)lIS

4.3.3 GENDER DISTRIBUTION OF PATIENTS WITH WNV IgMANTIBODIES.The gender distribution of patients with WNV IgM antibodies is presentedon Table 21. Similarly, no significant relationship (X 2:=2.00, df=2, p:= 0.368358)was observed between the prevalence of WNV IgM and the gender of patients.116

TABLE 21:GENDER DISTRIIlUTION OF WNV IgMPOSITIVE PATIENTSGENDER TOTAL NO. TESTED NO. POSITIVE (%)Male 482 8 (1.6)Female 366 4 (1.1)Not specified125 0(0)TOTAL 973 12 (1.2)117

4.3.4 SEASONAL DISTRIBUTION OF WNV IgG ANTIBODIESIn Table 22, the seasonal distribution of WNV IgG antibodies is presented.The prevalence of WNV IgG was significantly different (Xl =13.20, df= 2, p=0.001359), from the seasons of the year. Generally, a, significant number of thepatients tested (80.2%) had WNV IgG (Figure 8).118

TABLE 22: THE PREVALENCE OF WNV IgG ACCORDING TOSEASONSSEASON PERIOD OF TIlE NO.TESTED NO.POSITIVEYEAR (%)DRY SEASONRAINY SEASONCOLD SEASONJanuary to AprilMay to AugustSeptember to352 267 (75.6)I306 242 (79.0)December 315 273 (86.7)TOTAL973 780 (80.2)119


4.4. THE PREVALENCE OF YELLOW FEVER IgM AND IgGANTIBODIESSurprisingly, no YFV IgM was detected in 973 sera collected from Sahelsavanna zone. In Table 23, seasonal distribution of YFV IgG antibodies is presented.Like, DENs and WNV, significant number of patients (86.9%), tested had YFV IgGantibodies. Significant association (X"=I5.37, c1f=3, P=O.000468) was observedbetween the prevalence of YF IgG antibodies and thc seasons of the year (Figure 9)121

TABLE 23:THE PREVALENCE OF YFV IgG ACCORDING TOSEASONSPERIOD OF THE NO. NO.l'osn;iVESEASON YEAR TESTED (%)Dry season January to april 353 298 (30.6)Rain y season May to august 306 255 (26.2)September toCold season December 315 293 (30.1)TOTAL 973 846 (86.9)122


4.5 CROSS REACTIVITY BETWEEN DEN, WNV AND YF BYl\1AC-ELISAThough cross- reactions among flaviviruses render MAC-EllSA less specific, thedifference in the OD values to some extent could give a presumptive idea to theinfecting virus. Between DEN, WNV and YFV, the virus with the higher OD valuewas regarded as the infecting f1avivirus (Table 24). Samples 3515 and 2575 weresuspected cases of mixed infections of DEN and WNV while sample 2172 could bemixture of WNV and YFV124

.._-~._--TABLE 24: SHOWING THE LEVr,L OF CROSS-REACTIVITYBETWEEN DEN, WNV AND YFV BY IGM ELISAr.- ----._--._--S/NO SAMPLE DEN I WNV YFV POSITIVE!NOi FOR - II--f-.-- ----~---1 1243 0.001 0.5Xl 0.172 WNV._--- f-2 3515 ro:-352 0.254 0.192 DEN& WN,-_.._---- ------.3 2172 0.112 0302 0.215-_._-4 3545 0.131 D.277 D.1585 3494 0.121 (J.2 ') 0.114- ------_._-6 3679 0.198 0.763 D295-'-.. --7 3322 L0116 0.427 D.227WN::;~- -::~--~----WNV-i._--------------------_._.8 2725 0.23 032 0.11 DEN & WNV I------"9 3460 0.118 0.478 0.108-10 3255 0.189I0.49J= 0.1D9~-_:: .111 3481 0.119 0.25 0.08412 3500 0.121 0.276 0.08413 2599 0.23 o 104 0.08WNv-lWNVI--DEN--··'c- ..14 2463 0.221 0.094 0.093 DEN_..-15 3778 0.221 0.082 0.098 DEN--125

4.6 THE RESULTS OF MAC -ELISA WITH THEPROBLEMATIC SERA AFTER TREATMENT TO REMOVENON·SPECIFIC IMMUNOGLOBULINSThe results of MAC-ELlSA after treatment to remove nonspecificImmunoglobulins is presented on Appendix 4, The OD values of the problematicsera before and after the various treatments were not different. Therefore suchresults were considered "not conclusive",

1~74.7 DILUTION OF DEN POSITIVE IgM TO DETERMINE THEINFECTING SEROTYPESTable 25 shows the dilution of sample VF 30 which was an example of arecent infection with DEN-2 as demonstrated by IgM capture ELISA. Thecomparative titration against the four serotypes of DENs. indicated that DEN-2 wasthe infectious agent that elicited the IgM antibody by the time of sample collection.

TABLE 25: DILUTION OF DEN POSITIVE IgM (VF30) TODETERMINE THE IN}"ECTING SEROTYPESAMPLEVF30DILUTION DEN-! DEN-2 DEN-3 DEN-41/100 0,746 1.456 0,736 0,64611200 0,635 1.246 0.493 0.4151/400 0,602 1.19 0,304 0,2211/800 0,559 1.124 0,211 0,1541/1600 0376 0,617 0.424 0,1811/3200 0,301 0,448 0,342 0,0771/6400 0.126 0,329 0,216 0,0881112800 0,052 0,189 0,l34 0.0621/25600 0036 0,086 0,067 0,0331151200 0003 0,069 0,052 0,0221/102400 0,011 0,019 0,051 0,001128

4.8 VECTOlUAL STUDIES ON MOSQUITO VECTOR4.8.1 VIRUS ISOLATION FROM MOSQUITOESIncidentally, the two genera of mosquitoes identified were Aedes (295pieces) and Culex (3050) and Mansonia (50 pieces). None of the panel ofarboviruses surveyed was isolated from these mosquitoes.4.8.2 RT-PCR ON MOSQUITOESAlthough no virus was isolated, DEN and WN viral RNAs were detected byRT-PCR from Aedes and Culex species caught from Rain forest and Sahel savanna.Figure 10 and 11 show the optimization of RT- PCR reagents with positive controlviruses using multiplex and single step methods. Maximum level of sensitivity wasobtained using multiplex as compared to single step method. Consequently.multiplex-step was adopted in testing all the mosquitoes as well as the IgM positivesera.129

1Ij!, ':,1," !"Figure 10: FIRST AMPLIFICATION PRODUCTS OF DEN VIRUSES~.' '.. ~ ',I-"' .',, '-~>• "1 ;,.~ t!i, I : I'I, ,,"1"'.!i:I,;:jII130-.(, \':'~~ "_~_-, ,----'---- ----~-------_.~_-----,---_.----------.....,--',l',"'!J"i.\

:.~. , ,, ,'\"i,,~;"'~'.,0,';i~! ,.. ~'+ l~,,):::'>1"t\·." '!.,,:,~',I.).'Figure 11: SEMI NESTED rea FOR DEN-I, DEN-2, DEN-3, DEN-4(MULTIPLE),.',,' ;i}'~', 'l ,'.',;, ,'H""'le'.,' "'.\' ....·;'i~",j'.. ~,131.""',l"".'..---'--" nr-rcn ON AEDES MOSQUITOES FOR DENIn Table 26 the result of RT-PCR 011 Aedes mosquitoes is presented. A totalof I DEN-I, 4 DEN-2, 5 DEN-3 and 4 DEN-4 were detected in both male andfemale Aedes mosquitoes. Table 27 shows the result of RT-PCR with DEN IgMpositive sera. One of the DEN IgM positive sera, showed detectable viral RNA. Itwas interesting to note that one of the DEN IgM negative serum was found positive(DEN-3) by RT-PCR.Figure 12 and 13 show examples of RT-PCR. These are Plates of Aedesmosquitoes for Dengue viruses. In these plates, DEN-!, 2, and 3 were detected inthese mosquitoes.]]2

--_._-~._,--_._-------TABLE 26: THE SUMMARY (RESULT) OF RT-peR ON AEDES SPECIESOSQUlTOI"SPECIES~~edes aegyptibedes aegyptiSEXFemaleMalesNO" OF POOLTESTED277DEN-!kedes species Females12\----- ---------+----,-~edes species Males ~-'---+-~j ~~nidentified NO~known _-~~~~_~_- +-_! TOTAL 59r----~-..--~,oooo----------'----!UT-peR-- ---'----D EN-2 D EN-3 D EN-42 0 41 2 1-- ----0 1 I---- -----1 I 0- -_._-- -------, 0 2 2- --_.-- ------4 6 8_..- .-.---_.__ ...._-.,-,._-----I r'I jif·r,' 4Lhaum 133

4TABLE 27:RESULTS OF RT-PCR WITH DENS IgM POSITIVE SERASINO. SAMPLE NO. RT-PCR IgM! YF30 Negative DEN-22 30 Negative DEN-23 641 Negative DEN-24 2725 DEN-3 DEN-25 334 DEN-3 None6 2463 Negative DEN-!7 667 Negative DEN-!134


';" -,.. ,..... -' ''';''"_~~I "~'~J';~;"'~'Figure 13: SEMI NESTED PCR WITH AEDES MOSQUITOES FOR DEN.",,'~ ,.; ,,: I , RT·PCR FOR WNV IN CULEX MOSQillTOESOut of 62 pools of Culex / Mansonia species, tested WN viral RNA wasdetected in 40 pools of Culex quinquefasciatus (figure 14 and 15). Table 27 showsthe comparison of RT-PCR with WNV IgJ\1 positive. Out of 12 WNV IgM positivesera, 4 (33.3%), had WN viral RNA. However, one WNV IgM negative serum waspositive by RT-PCR and one serum, which was at boarder line by IgM, captureELlSA also showed detectable WN viral RNA (Figure 16). Figure 17 shows thecomparison of the sensitivity between TITAN and RT·PCR, TITAN exhibitedhigher degree of sensitivity and specificity compared with RT·PCR.-", '137

IIFigure 14: RT-PCR ON CULEX MOSQUITOES FOR WNV'":i" "'.;",:,". ',' '.~"''''''l,.}:,~:''~:~,< :~

139, '"i., ',"Figure 15:RT-PCR ON CULEX MOSQUITOES (WNV:1'.I

TABLE 28:COMPARISON OF RT·PCR WITH WNV IgM POSITIVESERASINO SAMPLE NO. RESULT OF RESULT OFRT-PCRIgM1 1243 Positi ve Positive2 3515 Negative Positive3 2172 Negative Positive4 3545 Negative Positive5 3494 Negative Positive6 3679 Negative Positive7 3322 Positive Positive8 2725 Negative Positive9 3460 Positive Positive10 3255 Positive Positive11 3481 Negative Positive12 3500 Negative Positive ~13 3562 Positive Negative14 3222 Positive Border line140

"Figure 16; RT-rcn ON WNV POSITIVE IgM SERA, ,...'.\ ., r~,t{;~1"~,1I••, ,;!,~~ ~~. -: .,'.: ''~i>o "", ~''';~''f':-,.,, ::',-,.''iI:,:1141. ,

,"""",....~;~~~~'~~~i·,"~:;/'J,I!Figure 17: COMPARISON OF TITAN AND R RTRT-PCR.'; ,.'142

CHAPTER FIVE5.0 DISCUSSION AND CONCLUSIONFlaviviruses constitute informative natural models of comparative molecularevolution because tbey differ in host, vector and associated disease. (Zanotto, et aI.,1996). Disease description characteristic of dengue datc back to 1779, and the virushas already become a cosmopolitan agent, causing almost simultaneous epidemics inlocations as far apart as Philadelphia, Jakarta, Bavaria and possibly Cairo (Theilerand Downs 1973). Since that time denguc virus population dispersal has probablybeen enhanced by t he expansion 0 f b rccdi ng I' Iaces for m osquitoes and t he m assmovement of non immune human populaiions into areas infested with mosquitoes((Zanolto,ct al., 1996). Another report showed that efficient and rapid mass transporthas favored the introduction and eo- circulation of serotypes around the globe,followed by increase in cases of OF, DHF and DSS (Monath and Trent, 1994). In1997, DEN has been described as the most important mosquito-borne viral diseaseaffectinghumans. Its global distribution is comparable to that of malaria (CDC,2001). That report estimated that 2.5 billion people live in areas at risk for epidemictransmissionWest Nile virus (WNV) is one of the ubiquitous arboviruses occurring over abroad geographical range and a widc diversity of vertebrate host and vector species(Me Lean et aI., 2002). According to these authors, the virus appears to bemaintained in endemic foci on the temperate climates to the north of Europe and tothe south in South Africa. That report further stated that until recently, records ofmorbidity and mortality in wild birds were confined to a small number of cases andinfections causing encephalitis, sometimes fatal in horses were reportedinfrequently. These authors however noted that, in 1996-2001, there was anincreased outbreak of illness due to WNV in animals as well as humans. In addition,they observed that migratory birds are instrumental in disseminating the virus andanti genie variants in different geographical locations. Presently, WNV is consideredan endemic disease in North America including South Dakota. I n these regions itcontinues to threaten birds, horses and most importantly human beings (Kightlinger,2003).Many illnesses in Africa that present with fever are documented as fever ofunknown origin (PUO), e specially if they fail to respond to a nti- microbial drugs14.1

(Nur, et al., 1999). In that report the majority of these Africa remainedundiagnosed. The report also revealed that several arboviruses are frequentlyconsidered in the etiology of acute febrile illness in some African countries. Thesituation in Nigeria seems quite different because most cases of PUOs are hardlyinvestigated for arboviruscs. Two main factors known to favor the emergence ofvector bome diseases are common in the study site. Such factors include highpopulation of mosquito vectors and pool of susceptible individuals. The presence ofthese factors 1ed tot he suspicion t hat a rboviruses could p lay a s igni ficant role insevere febrile illness that are resistant to anti-microbial drugs.In this study, thc activities of Flaviviruscs in Nigeria are eminent. Generally18 (0.6%) of 2921 scrum samples tested had DEN IgM antibodies. Though theprevalence rate of DEN infection as revealed ill this test is low but it confirms theactivities of this virus in Nigeria. This study has revealed that, of the 18 positivecases, 13 (0.67%) of 1948 febrile patients in lour of six ecological zones in Nigeriahad DEN IgM antibodies. The presence of this antibody in Sahel savanna (1.67%)and Rain forest (1.4%) were significantly different from Grass savanna (0.3%) andDeltaic savanna (0.1%) ecological zones. Nevertheless, no patient in Sudan andWoodcd savanna tested positive for DEN IgM antibody. In a previous study(Fagbami, 1977) in Nigeria, denguc v-ncutralizing antibodies was highest inderived savanna (63%), followed by the Rain forest (42%) while the southern guineasavanna and Plateau zones had the lowest. The common feature shared by theprevious and the present studies is that DEN antibody was higher in Rain forestwhile the Guinea savanna had the lowest. However, both studies differed becauseDEN-neutralizing antibody was detected in the previous study while this studymeasured evidence of recent infection. In addition, plaque reduction neutralizationtest (PRNT) and IgM capture ELISA (MAC-EUSA) were the serological techniquesemployed in the previous and present studies respectively. In' the former, higherprevalence rate of DEN-neutralizing antibody were reported as 63% in Derivedsavanna and 42% in Rain forest against 1.67% and 1.4% of DEN IgM in Sahelsavanna and Rain forest respectively revealed in the latter. Also while the formermeasured only DEN IgG, the latter detected primarily DEN IgM, and DEN IgG. Itmay be necessary to mention that the presence of IgG is a measure of past infecticnwhile IgM is a hallmark of recent infection at least three months to the time ofsample collection. This conclusion is supported by a report that although [gM144

antibody positive hy MAC-ELlSA on acute scrum samples arc only provisional andnot necessarily recent infection, but certainly that infection would have occurredsometime in the previous one or two months (WH0,200 l). However, PRNT is morespecific than ELISA because it shows a monotypic reaction to the infecting virusthrough the late convalescent phase of illness (Vordam and Kuno, 1997). It mayshow some degree 0 f heterologous reactivity in scrum taken weeks or months afterthe illness. The previous study reported the prevalence rate of DEN neutralizingantibody as 63% and 42 % in Derived savanna and Rain forest respectively whilethis study showed DE!\ IgM as 1.67%, in Sahel savanna and 1.4% in Rain forest.About 60.0% of the patients in the ecological zones studied had DEN [gGantibodies, which is a clear indication of past exposure to Flaviviruses by long timeresidents in these zones. This observation is supported by a report that the IgGELlSA is very non-specific and exhibits the same broad cross-reactivity amongFlaviviruses as the HI test. Therefore, it cannot be used to identify the infectingflavivirus (WHO, 200 I). Nevertheless, the IgG capture EUSA employed in thisstudy was reported as being 20% more sensitive than HI test in detecting antidengueantibod ies in human serum sampl es (\'orndam and Kuno, 1997)The prevalence rate of DEN IgG antibodies and the ecological zones weresignificantly different with the highest in Sahel savanna (81.7%), Wooded savanna(69.2%), and Rain forest (69.0 0 /,, ) . The least among them were Grass savanna andSudan savanna with 32.6% and 38. I 0!c, respectively. The low percentage of peoplewith DEN 19G antibodies in the two zones is of epidemiological importance. This isbecause any introduction of an epidemic strain or serotypes of any of theFlaviviruses in these zones could result in epidemic due to the presence of highproportion of susceptible host. Vorndam and Kuno (1997) revealed that, because ofthe persistence of IgG antibody and its prevalence in areas of endemic transmission,single serum samples (as in this study) demonstrating the presence of this antibodyhave no clinical significance unless the titcr indicate a secondary serologic response.1n contrary to that report, Thcin (200]), observed that levels of anti dengue IgG inacute phase sera collected from patients during a period of high dengue activitycorrelated with disease severity. This author also reported that sera collected frompatients during the period of moderate tu low dengue activity showed no associationwithdisease severity. In this study, because there is no active surveillance fordengue or other arbovirus activities in Nigeria, it is difficult to di ffercntiate periods._-------------_._--~.14)

of high and low virus activities. Also, because there was no follow-up on thesecases, correlation of levels of IgG and diseases severity was not applicable in thisstudy. Nevertheless Vaughn et aI., (1999), revealed that the clinical importance ofIgG in diagnosis of DEN infections is its usefulness in distinguishing betweenprimary and secondary dengue infections, with 100% primary and 9G'!!o of secondaryinfections being correctly classified. In support of the report, Innis et a/.( 1989)showed that the ratio of IgM to IgG antibody might be used to differentiate primaryto secondary infections. Anotber study reported that, in secondary Flavivirusinfections, anamnestic IgG response occurs resulting in a rapid rise in titer beginningalmost immediately after onset of illness and attaining almost relatively high levelsin most but not all patients. (Halstead et 11/.1983). Moreover, 'lhein (2003)demonstrated that IgG is the most abundant human immunoglobulin and has thepotential to play a role in both antibody-enhancement and complement activation,whieh is p rineiple behind the development 0 f D HF and D SS (Monath and H einz1996). Based on these reports, from table 9 .md figure G, some suspected cases(samples 1,4, 9, 12 and \3) of anamnestic rcspouse to DEN infections arc shown.These patients were assumed to be at 'he risk of developing DSS. This possibilitywas derived from the report that, the risk of developing DSS following ananamnestic infection was from 82- I03 t irues greater than that 0 I'd eveloping 0 SSfollowing a primary dengue infection (Thein, 20(3). In addition this author observeda significantly higher rate of anamnestic infections with DEN-2 in DSS comparedwith other serotypes. This study has confirmed the endemicity ofDEN-2 in Nigeriaand in between 2003 and 2004 two fatal casc of viral hemorrhagic fever had beenreported in University of Maiduguri Teaching Hospital, Maiduguri (Personalcommunication with the Clinicians), These patients died within few hours afterpresentation to the Clinicians. This implies that these patients did not visit thehospital till at the critical stage of the disease. Therefore, since the patients tested inthis study were not followed up, associating these cases with DSS is beyond thescope of this study. The need for active surveillance and intensive education onarbovirus activities in the environment cannot he emphasized.No significant association was observed between the presence of DEN IgGantibodies and the seasons of the year in Sahc] savanna zone. The highest prevalencerate of DEN IgG was observed during the rainy season (88.')%) compared with hotdry season (83.9'Yc,) and cold harrnattan season (86.0%). However, a significant146

elationship between the prevalence of DEN IgM and the seasons of the year wasobserved in this study with the highest during the rainy season (1.3%) compared todry (0%,) and culd harmatrun season (0.3%) ill Sahel savanna zone in 2001 Table 11and figure 4 displays the di fference. This findillg compared Iavorably with the reportof Moore et al. (1975) who revealed thar arboviral activity was highest during t1]1~rainy season with the peak in the month or .Junc, July and August and lowest in thedry month of January and February, in Nigeria. In addition, Akhtar and Ebi (200])reported that DEN transmission occurs year round in tropical arC3S but has seasonalpeaks in most countries during the months with high rainfall and humidity. ;\ reportshowed that the bulk of the yearly rainfall [nvt-r 90%) in Maiduguri, the study area,is concentrated between the months of June and September (Gimand Associates,2002). That report f1lJ1hcr stated I hat t hc montl: 0 fA ugust marks the peak 0 f therainy season with the mean rainfall amount of 21J5.9mm. In addition, these authorsreported that, the months ofJuly to September in the study area are associated withhigh relative humidity of more than 60%. This implies that the month ofJuly duringwhich the highest prevalence rate of DEN Igl'v1 was obtained had high rainfall am;relative humidity in conformation with Gimand Associates' (2002) report. It hasbeen determined that warmer temperatures reduce larval size of Ae. Aegypti, andthis usually result in a smaller adult size (Reuda, 1990). Smaller adult femalemosquitoes have been found to feed more frequently to nourish the developing eggs(Reita, 1988), which increase the possibility of transmission. The positiverelationship between biting rates and temperature has been supported in field studiesin Bangkok. A second consideration in DEN virus transmission is the report that theextrinsic incubation period for Adese Aegvpu decreased from twelve to scven dayswhen mosquitoes were kept at 32-35"C instead of 30"e (Watts et 01.. 1987).Koopman et 01. (1991) therefore concluded that median temperature during the rainyseason was the strongest predictor of DEN infection. Other authors's reports arguedthat temperature affects the rate of mosquito larval development, adult survival,vector size and gonotrophic cycle as well as the extrinsic incubation period of thevirus in the vector (Focks et. ai, 1993). In contrast to that report, Akhtar and Ebi(2001) showed that transmission intensity for dengue VIruses in tropical endemiccountries is limited primarily by herd immunity but not temperature. These authorstherefore postulated that temperature increase is not likely to affect transmissionsignificantly. This finding compared favorablv with the report of Moore et aI.,147..--------------~~~-- -------

(1975) who demonstrated that DEN antibodies were highest during the rainy seasoncompared with other seasons of the year. In another report, mosquito-borne diseasetransmission is climate sensitive for several reasons which include the fact thatmosquitoes require standing water to breed, a warm ambient temperature which iscritical to adult feeding behavior and mortality, the rate of larval development, andthe feed of viral replication (Patz cl al., 199~). Therefore, if the climate is loo cold,viral development is slow and mosquitoes arr unlikely to survive long enough tobecome infectious (Hail'S et al., 2002).Cross reactivity within DEN serotypes and other Flaviviruscs is a commonphenomeno in most serological techniques. Cliunge et al. (198%), reported that thecross reactivity of anti-dengue 19M is variable depending 011 the antigens used andthe test procedure. In this study it was observed that the optical density (OD) of aparticular dengue serotype was higher than others. In such cases the serotype orflavivirus with highest OD value was considered the infecting virus at that time. Forexample, Table 25, shows the OD values at I: 100 dilution of the serum VF30 asfollows: - DEN1= 0.746. DEN-2 = 1.456, DFN3 =0.735 and DEN-4 was 0.046 Inthat example DEN-2 was considered as the infecting serotype. This was .nconsonance with the previous report (Chungc Cl "I., 1989a), which showed that theearly IgM response might be dengue serotype specific in ELlSAs in some primarybut less frequently in secondary infections. In that same example (Table 25), thisstudy shows that comparative titrations could di ITerentiate among dengue serotypesas well as other Flaviviruses. For sample VE 30.'DEN-I, 3, and 4 had OD value of0.301,0.342, and 0.372 respectively with the tiler of 1:1600 while DEN-2 had ODvalue of 0.329 with a titer of I: 3200, Similarly. this result agreed favorably with thereport that despite the cross reactivity with other Flaviviruses, such as JE, and SLEbut comparative endpoint titration may be able 10 distinquish anti-DFiN 19M fromthat of other Flaviviruscs. (Innis, et al., 1989). Table 24 shows the cross reactivitywith other FJaviviruscs and how the differences in the OD value for each genuscould to some extent distinguish between these viruses.In agreement with Vasconcelos et al. (I ()'J8), no signi ficant relationship wasobserved between the prevalence of DEN Igivl and the agc and gender of thepatients. Nevertheless, 4 (1.8'/:,), of children aged 0-9 had DEN IgM antibody. Suchchildren are at a highcr risk of developing DIIF/OSS compared with other agegroups as reported by Dietz et al., (19%.). In addition virus strain, the immune14844J

status (that is having previous dengue infections), age, and genetic background ofthe human host, are the main important risk factors for developing DHF (eDC,200 I). To further support the above reports, it was demonstrated that proportionatelymore of cases below 15 years of age tended 10 result in DHF but the adult are morelikely to develop symptoms characteristic of DF with fewer subclinical infections(Halstead and Deen 2002; Ooi et al., 200)). Numerically, infections were moreamong age groups 20-29 than others. Therefore, these people arc more likcly todevelop symptomatic DF than OHF according to the above reports,This study also shows that 12 (1.2%) of the 973 sera tested had WNV IgMantibodies by MAC-EUSA. This serologic test (MAC-ELISA) has been consideredas the most efficient diagnostic method for dctecting IgM antibody to WNV inserum or cerebral spinal fluid (CS F). Collected within eight clays of illness.(Kightlinger, 2003). That report was further supported by MeLean et al., (2002),who revealed that MAC· ELlSA is ill currcnt use for WNV antibody confirmationand frequently replace Ill, CFT, and NT ill many situations. The endemicity ofWNV in Sahel savanna (semi- arid zone), as revealed ill this study is comparablewith previous report which showed that WNV transmission usually occurs in coastalplains, river delta areas forests, semi-arid areas and highland. plateaus ecologicalhabitats (McLean et aI., 2002). Prcviousely, Olalcye, et al., (1990), demonstrated HIantibody to WNV in human and animal sera from lbadan (Rain forest) andMaiduguri (Sahel savanna) respectively. Tile previous and the present studiesemployed different serological techniques which differ both in sensitivity andspecificity. T he HI generally m easures both IgM and IgG and requires a cute andconvalescent sera to establish the presence of recent or past infections while MAC­ELISA used in this test, measures primary in lcctions even with acute sera. Bothstudies however established the endemicity of WNV infections in Nigeria includingMaiduguri, the study area of this report. Earlier study also showed that 10 viral typesincluding WNV and other arboviruses were isolated from cattle, sheep, and goatsand camels in northern, Nigeria. (Kemp, et al., 1973). Lack of consistentsurveillance for these viruses tend to allow misdiagnosis of such cases in agreementwith Monath et al,(2001) who observed that West Nile fevcr (WNF), a self limitedsyndrome is so nonspecific that it often escapes medical attention and it has not beenreported often in USA or Europe. lt may he necessary to note that Central nervoussystem infection (aseptic meningitis, meningncneephalitis, transverse myelitis, optic.14')

neuritis or polyradiculitis) occurs in a small proportion of persons infected withWNV. (Monath, et al.,2001). In the study site (Maiduguri), there had heen case ofunexplained encephalitis and mcningoencephalitis but because of lack of adequatediagnostic facilities In the area, such cases often attract inappropriatediagnosis/treatment. In view of this Kightlingcr (2003), suggested Ih"t WNV shouldbe considered in all persons with unexplained encephalitis and meningitis.No significant relationship IV as 0 bservcd b etwccn the P rcvalcncc 0 f W NV[gM and the age ofthc patients in this study. IIowcver, a female patient aged 8 yearshad WNV IgM antibody. This 11nding compared with a rcport that children andinfants do not appear In he at greater risk than adult for becoming ill with Vvl\lV.(New York City Health, 2004). That report noted the fact that children can becomeinfected with the virus if bitten by an infected mosquito and may need an adult'shelp in taking precaution against mosquito bill'S. Age has been identified as one ofthe factors influencing the degree of the virulence of the virus (Haves, 1'J8'J; Komar2000). However, Monath et al., (2001) reported that central nervous system (eNS)infection due to WNV occurs principally and is most severe in the elderly. Insupport of that report, during an outbreak of WNV infection in Israel, the infectionratio was 1:24 in aged persons with a case-fataiity rate of33% (Monath et al., 2001).These authors attributed the increased susceptibility of elderly persons to lowerimmune responsiveness. Also among cancer patients who were experimentallyinfected with WNV, 11% developed encephalitis (Southam and Maore 1954). It wasconcluded that the high disease incidence in these group of patients was probablydue to immune suppression (Monath et al., 2(01). Incidentally none of the WNVIgM positive patients was up to 50 years old. Numerically WNV IgM appearedhighest within the age groups 20~49 years (4.8'1'0). This could he attributed to highrates ofc xposure to mosquito bites during outdoor activities such as fanning andfishing which are the major occupation of the Residents of the study area and socialgatherings. Large-scale tree planting is a common practice in Sahcl savanna zone tocombat high degree of dcscrtification and erosion. It is worth mentioning that, it isalso the habits of groups of people in the environment, to gather under shadesprovided by these trees to socialize and discuss issues of common interest Suchpractice is common among the adults than children or infants. This practice couldpromote the exposure of adults of the st'ltcd age group to mosquito bites. It wasinteresting to note that the mosquito vector (Culex quinqucfasci.uus) which yieldedI'll)

WNV RNA were C~ll[; in areas of large-scale tree planting nearhuman habitations. Therefore, tree-planting which serves a useful purpose could alsopose serious threat to public health. This su!:gestion was supported by a report thatweeds, tall grasses and bushes provide outdoor resting places for WNV mosquitovectors.Gender dist ribul ion of positive sera in this study showed that ].5% of malesand] .6'1., of females had WNV [gM antibodies. No significant difference betweenthe presence of this lUltihody and the se.' of the patients was observed in agreementwith Olaleye cl al., (1990). This is pl'llh:ibly because the hungry mosquito vectorhas no respect for sex in its quest for blood meals.In addition, the presence 01' ]gM antibody for WNV and the seasons 01' theyear were significantly different. The [Iighest prevalence rate was observed duringthe cold season (2.9%) compared to dry (0.1 I';!',) and rainy (0.1'%) seasons. The peakof WNV activity as revealed in this study was in November 2001. In an agreementWith this observation. Ben- Naihan and Fcucrstein (I ')90) suggested that physical ornon-physical stress situations enhance WNV encephalitis by accelerating virusproliferation and increase mortality in mice. Another report showed that a midwinterisolate from a hawk suggests tb:it chronic infection and vertebrate- tovertebratetransmission contributes to winter survival (Garmcndia et al..2000). In thenortheastern United States, the higbest prevalence of WNV activity was mid Augustto early October (winter). Moreover. during the ]999 outbreak ofVlNV infections inthe New York City, WNV-infected mosquitoes and dead birds were found duringthe cold winter mouths (Nasci era!.. 2002; Garrncndia et al., 2000). The abovereports were supported by Kightlingcr, 2111n who observer! that the peak period ofWNV activity in South Dakota (North America), was the last week in August andthe first week of September. These authors suggested that during the cold wintermonths, WNV aver-winters in infected adult mosquitoes. It was interesting to notethat McLean cl a! .. (2000), expressed their surprise to sec WNV become establishedso quickly in the temperate climate of New York City. With all these favorablereports to the linding of this study, it is possible that cold weather promotes thetransmission of WNV.The seasons of the year and the prevalence of [gC; to WNV wer«significantly different. Other studies have demonstrated that the presence of WNVIIgG suggests exposure to Flaviviruscs such as W NV, SLE, YFV, DEN, and .IE\'..1.\ I

152(Nur et al., 1999; Kightlinger, 2003;).The high prevalence ofWNV IgG (81l.16%)obtained in this study indicates tlie endemicity of any of these Flavivil1lses in theenvironment due to higlidcgree ofcross-reactivity among flavivirusesTwo sera from the febrile patients tested positive for WNV and DEN !pMDantibodies while one patient had both WNV and YFV. This observation agreed withprevious studies, one of which reported the possibility of WNV following infectionwith another Flaviviruscs, and resulting in equivocal IgM levels by MAC-EUSA.(MeLean et al..20(2). That report further revealed that in areas in which more thanone Flaviviruses occur, that in the event of au infection by one Flavivirus and latcr asecond Flavivirus, the antibody titcr in the serum taken at the time of secondinfection may be higher for the virus which caused the first infection than for thatcausing the current infection (original antigenie sin). For example sample 3515 had11OD value of 0.352 and 0.254 for DEN and WNV !gM antibodies respectively whilesample 2725 with OD value of 0.23 for DEN and 0.32 for WNV IgM antibodies.These two cases were considered mixed infections of both viruses with thepossibility that sample 3515 had DEN before WNV infections while sample 2725probably contracted WNV before DEN (Table 25). This suggestion was furthersupported by the report of Nur, et al., (1999) who demonstrated low-titcr IgMantibodies against WNV in a DEN 1gM positive patient. Another report in whichIgM captured EUSA was compared with a commercial rapidimmunochromatographic care! test and IgM micro well ELISA for the detection ofantibodies to DEN, also revealed mixed infections of DEN and WNV (Sathish etal.,20(2). That report further stated that MAC-ELISA had high sensitivity (96%)compared with micro well EUSA (72°/,,) and specificity rates detecting not onlyDEN but also mv, and WNV !gM antibodies in a single run. According to theseauthors, the test (MAC-ELlSA) uses specific viral antigens to dctcct IgM antibodiesto commonly encountered Flaviviruses in the environment. Mixture of two virulentviruses in a patient could be devastating and requires persistent surveillance andadequate laboratory diagnostic facilities for proper treatment. Since these cases werenot followed up, they were probably missed out having failed to response lo antmicrobialdrugs. Moreover, another report stated that simultaneous presence of morethan one species of flavivirus is known to occur in the vector Aedes aegypti (Gubler,1985). This report confirms that mixed infections of different Flaviviruses in a

patient is not uncommon if it could occur in the vector even though the cases aboveIinvolved DEN (transmitted by Ae species) and WNV, by Culex mosquitoes.Although \TV IgM was not detected in all the 973 sera tested, except a caseof mixed infection with WNV, a significant number (86.9°,{,) of these patients hadIgG antibodies to Yf'V. Like DEN ami W:"lV, the presence of YF IgG and theseasons of the year were significantly different. As a result ofintcrmittent outbreaksof YF in Nigeria, most part of the country are considered "zone of emergence"(WHO, 1986). 1nl 989, t here was an outbreak w hich the present Yohr- State wasinvolved and in 2000 another ontbreak occurred in Kano northwestern part ofNigeria, Although Ihe presence of IgG is not Flavivirus genus- specific, but tnerehad been many outbreaks of YF in Nigeria since 1973, which could have left manypeople in the environment exposed. Therefore, the high level of YFV IgG obtainedin this study indicates the endemicity of this virus in the study area.Surprisingly 110nc of the 70 arboviruses sought for in field-caught mosquitovectors from Ruin forest and Sahcl savann.i was isolated. Probably storage andtransport conditions were not adequate for the rescue of infectious virus in thesevectors. Most of the mosquitoes were stored at .zo-c for a year with frequent powerfailures that occasionally lasted for more than 72 hours before being transportedusing wet ice 10 IPD, Senegal within 48 hours for analysis. Also, themorphologically damaged culex or Aedes mosquitoes prevented the identification ofall the mosquitoes to species level. This w,as due to the fact that morphologiccharacteristics essential for accurate species identification were often damagedthrough the collection and transportation process and as part of natural aging of themosquitoes. Consequently, many specimens were initially identified only to thelevel of genus or to species. Lanciotti, cl 1/1.. (1992) had identificd some of theseproblems which arc commonly associated to virus isolation to include the fact that ittakes from days to weeks, and is not always successful because of small amount ofviable virus in the inocula, virus -antibody complexes and inappropriate handling ofsamples. Consequently, a clear need existed for an assay that can be performedrapidly and is sufficiently sensitive and specific to he clinically andepidcmiologically useful. These authors concluded that dengue viruses for instance,could be accurately detected and typed by RT-PCR from vircrnic human scrum. Inanother study, it was proposed that virologic surveillance by RT-peR for detectingl'i 1

dengue virus ill infected Aedes mosquiloes ill the field might serve as all earlywarning monitoring system for DEN outbreaks. (Vincent, et al., 1YY8),It was therefore interesting to note that. where virus isolation failed in thisstudy, RT-pCR became useful by detecting RNA to both Df'N and WNV inagreement with Lanciot.i, et Id.(IYn) and Vincent, et (11.,(1998). The results of thisstudy have shown the advantagc of RT-f'CR over virus isolation as suggested byLanciotti, et al. (1992). Another report identified a significant advantage ofRT-PCRover virus isolation to be particularly in Africa, where outbreaks occur in areaswhere cold chain is not always available and in situations where the duration of thetransport is usually long (Sail, cl (l1.,20()2). This observation was further' supportedby Miagostovich et (11.,( I()'!7) , who described RT-pCR as a useful tool 1

eports (Gublcr 1997, 1998b), which identified movement of new dengue virusstrains and serotypes between countries as one of the major factors causingepidemics. For example, a report showed thal DEN-J strains imported from Africaor Asia or Pacific Island was responsible for an outbreak in 1996-1998 at Guatemcla(Japan) (Usuku et al.,2001).In this study the only onc IgM negative serum included in the lest had DEN­J virall{NA while all the DEN IgM positive sera tested negative. The comparison ofthe sensitivity and the spccificity of EUSA ami RT-PCR are not applicable in thisstudy because all the DEN IgM positive and negative sera were not tested by RTpeR.Unfortunately, because the facility for RT-PCR is usually very expensive andnot easily alfordahlc by Ill;lllY laboratories. it \\;IS not practically feasible to test allthe IgM negative sera (2903) It 111ay be necessary to mention that only six out of18of DEN IgM positive sera were tested by RT-I'CR because others were exhausteddue to repeated ELISAs. Nevertheless, RT-I'CR was performed on the fgM positivebecause Vorndam in dengue laboratory, CDC (unpublished observation) proposedthat virus identification could be increased up to three-fold in sera containingdetectable IgM antibody. While the specificitv of RT-PCR appeared to be highenough for diagnostic purposes, the sensitivity even though acceptable would needto be improved. (Sall et al.,2002). The observation that one IgM negative serum waspositive by RT-PCR suggests that most of the IgM negative samples could havebeen false results. This probably contributed 10 the low prevalence rate of DENinfections obtained in this study. 11 may be necessary to note that it was not withinthe scope of this study to consider the time of onset of symptoms and samplecollection. Such consideration would have given precise information DU the status ofthese patients with regards to recent DEN infections in Nigeria. This is because areport has shown that specimens taken earlier t)Wl six days after .onset would have avariable percentage of false negatives due to insufficient time for antibodydevelopment (Vorndarn and Kuno, 1997). 1'h;1I report further stated that a smallpercentage of patients have detectable IgM antibodies on the day that symptomsbegin and most patients become positive by the sixth day afler onset. Therefore theIgM positive sera in this case yielded 110 viral RNA probably because the time ofsample collection did not Iavor RT-Pf.R result. IIowever, the detection of viral RNAin IgM negative s erum cornpared f avorably IV iih P revious s ludy (M iagostovicl: etal., J 9')7), which delllonstrMed that R1'-I'CR was most useful when anti-dengueI SS

antibodies were undetected. These possibilities therefore necessitate the need toemploy the two techniques (MAC-EUSA and RT-PCR) in proper diagnosis of DENinfections In this way, it is envisaged that the high sensitive RT-PCR, wouldcomplement the low specifieity and sensitivity of Mi\C-ELISA (Sathish et al.,ZU(2).This study revealed a conflicting case of being IgM positive for DEN-2(OD valuc=O.23) but RT-PCR positive for DEN-3 01, WNV IgM positive sera, only 4 (33.3% were RT­PCR) were RT-peR positive. It was interesting to note that cases ofWNV IgMnegative and boarder linc by EUSA (Tahle 29) had WN viral RNA. Thisobservation implied that as in DEN cases, all tbe IgM negative sera for WNVprobably contained false negative results. As mentioned earlier for DEN, it was notfeasible to test all the [gM negative sera (9GO) Ior WNV viral RNA due 10 high costof the reagents. The proportion of PCR..positive to IgM positive (Sof 13) was notsignificantly higher (X' = 0.4nS714) than the proportion of rCl~-posilive 10 [gMnegativesamples (Iof 1). Therefore in agreement with Sail ct al.,(2002), IgM156

detection is a serologic method that coulrl complement RT-PCI< tor the diagnosis ofFlavivirus infectionsThe important fuctors to be considered in the choice of diagnostic procedureto adopt for dengue and other arbovirus infl.etions are the time 0 I'onset of symptomsand sample collection, It may be neccssar to mention that the commonest clinicalf~aturcin all the piltiCllts tested in this stlldv was fcbrile illness, The time of onset ofthe illness was not given a priority because the primary objective was to determinethe role of these lIaviviruscs in febrile cases suspected of malaria or typhoid, with aview to establishing proper diagnostic facilities for these viruses in healthInstitutions in the country. Reports haw shown that, as with many virus infections,dengue virus for example replicates and reaches peak titers in the blood before thepatient becomes sick enough to present to a physician (Vorndam and Kuno 1997).Therefore, the diagnostic laboratory frequently has \0 dcal with rapidly decliningviraemia levels in attempting a diagnosis by virus isolation. In such a casecirculating virus remains readily detectable in the blood UJl to five days after onset ofsymptoms, which could reach levels up to XloglO ID50 per milliliter. (Gubler et al.,1981; Miagostovich et (/1., 199.1), and then is rapidly cleared with the appearance ofspecific antibody, Consequently, isolation of' virus is generally not practical withsamples taken six or more days after onset of symptoms. As mentioned earlier,specimens for IgM, earlier than six days arc likely to have Cl variable percentage offalse negatives due to iusufficient time for antibody development. For this reason,negative results on Cl single scrum sample in this time period should be reported as'indeterminate' and a second convalescent serum sample should be requested(Vorndam and Kuno ]997). These author-. further suggested that after sixth day,negative results should he reported negative. From this reports, it may he advisable,to complement IgM testing with RT-I'CF, so that early secondary infection withnegative or low IgM antibody could be detectedThe detection 01' viral RNA in male Aedes and Culex .\PP in this study is anindication of transovar:a! infection, This postulation is supported by a report thatvertical transmission from female arthropod to her progeny is an importantmechanism for ovcrwinier survival of certuin mosquito and tick- borne Flaviviruses(Monath and Heinz, 1996). In addition. vcncrealtransmission 01' Flaviviruscs frommale to female has been demonstrated. (Roscn, 1987a). In the study area, it is acommon occurrence that the population (If' mosquitoes is at peak during the rainyI 'i 7

season and lowest during the dry hot season. This observation correlates with thepeak and lowest incidence of flavivirus infections as measured by IgM to DRN andWNV in this study. It is therefore assumed that, vertical transmission of' theseviruses in mosquitoes may he an important survival mechanism ior the mamtcnanceof these viruses in the environment.5,1 SUMMARY AND CONCLUSIONIn this study modern yet sensitive speci fie diagnostic methods were used todetermine the activities of Dengue and West Nile virus infections in Nigeria. Thesetechniques include 19M capture ELlS/\ (MAC-ELlS/\), Jmrnunofluoresenceantibody technique (1FA) and Rcvcrsc'Iruuscriptio- Polymerase Chain Reaction(RT-PCR. It has been clearly demonstrated thnt dengue and West Nile viruses arcendemic in Nigeria. These viruses contribute to febrile illness commonly limited tomalaria and typhoid diagnosis and treatment. Sucb conditions could result in highmorbidity, complication and mortality duc to these virusesSecondly, the study has confirmed the circulation of DEN-l and 2 in Nigeria.It also revealed that new serotypcs of the virus, DEN-3 and 4 had been introduced inthe country between 1970s when studies on dengue viruses were carried out andnow. The implication of this trend is the possibility of the emergence of DHFIDSSby a phenomenon called Antibody-dependent enhancement (ADE). The sequentialheterologous infection with dengue has been a fundamental principle behind theestablishment of the severe form of dengue infections. Generally DEN-2 appearedthe most prevalent serotypes in the country.Thirdly, the presence of DEN IgM and IgG antibodies were significantlydifferent from the ecological zones studied, with the virus activities more in Rainforest, Sahcl savanna than in Deltaic and Grass savanna. No DEN 19M wasdetected in Wooded and Sudan savanna and Ilavivirus activities as evidenccd byDEN 19G wcrc lowest in these two zones. The epidemiological implication is thatany introduction of an epidemic strain of thC';c Flaviviruses in these zones couldresult in outbreak due \0 hig,l\ percentage of susceptible human hosts.Fourthly, DEN and WNV infections were seasonally related. DEN infectionswere favorcd by the raiuv season with it., peak ill the month of July while WNVpreferred the cold h:l\maIL\I\ period with the pcuk of its 3ctivities ill the lIlolllh of15K

November. This is because mosquito density, activity and survival is related tovarious weather conditions.Fi(thly, the detection of viral RNA in Aedes and Culex species showed thesignificant role played by mosquitoes in the maintenance of DENs and WNVrespectively in the environment. The hii2h prevalent of mosquito vector andsusceptible vertebrate hosts calls for constant surveillance of arboviruses in febrilecases in the country.In addition, large-scale tree planting (0 combat high degree of desertilieationand erosion could pose health hazard to human population. This is because the treesprovide breeding paces Cor the mosquito vectors that transmit these viruses Theprincipal vector, Culex quinquefasciatus, [) [. WNV, which yielded the viral RNAwere caught in area with Illany trees near human habitations.In addition mixed infections of [)I':N and WNV were oh served in few cases.It could be very devastating for two virulent viruses to eo-infect an individualFinally several nrboviruses arc etio]()gie agents of acute febrile illness Mostof these viruses cause fever only or with hcmorrhagcs or rncninguencephalitis orrash. In Nigeria, febrile cases which rue resistant to anti-microbial are usuallydocumented as PUGs. Worst still, thc carlvsymptorns ofarbovirus infections mimicmalaria, measles and influenza, thereby rendering the diagnosis of these cases veryconfusing., In such situations, arbovirus infections arc quite ollen misdiagnosed andso, inappropriately treated. Consequently, these cases often result in high rate ofmorbidity, complications and mortality. It is therefore imperative to include theseviruses in the differential diagnosis of fehrile illnesses.Other studies had revealedthat Virological and Vectoral surveillance serves as early warning system for anyoutbreak of arbovirus disease.J 5')

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APPENDI X I:INOCULATION OF MICE WITH SEED VIRUSDENGUE 1DAYS 0 1 2 3 4 5 6 7 8DATE 22/8 23/8 24/8 25/8 26/8 2718 28/8 29/8 30/8BOX 1 10 9 9 9 9 9 9 9SickBOX 2 10 7 7 7 7 7 7 7BOX 3 10 2 2 2 2 2 2 2andBOX4 10 6 6 6 6 6 6 6wereBOX 5 10 2 2 2 2 2 2 2BOX6 10 4 4 4 4 4 4 4 harvestedDENGUE2DAYS 0 1 2 3 4 5 0 1 2 3 4 5DATE 20/8 21/8 22/8 23/8 24/8 25/8 26/8 27/8 28/8 29/8 30/8 3118- --BOX 1 10/10 10/10 10110 10 1 10 3 3 3 3 AllBOX2 10/10 8/10 8/10 8 2 10 5 5 5 5 wereBOX3 10/10 8/10 8/10 8 3 10 6 6 6 6 sickBOX4 7110 7110 7110 6 4 10 8 6 6 6 aridBOX 5 6/10 5/10 5/10 5 5 10 7 7 7 5 harvestedBOX 6 4/10 4110 4/10 4 6 10 2 2 2 2BOX7 5/10 5/10 5/10 5 7 10 2 2 2 2DENGUE 3DAYS 0 1 2 3 4 5 6 7DATE 22/8 23/8 24/8 25/8 26/8 27/8 28/8 29/8BOX 1 10/104 3 3 3 ~ 3BOX 2 10/10 9 9 9 9 9 9 SickBOX 3 10/10 9 9 9 9 9 9BOX4 10/10 7 7 7 7 7 7 andBOX5 ~~ 6 6 6 6 6BOX6 10/10 1 1 1 1 1 1 wereBOX7 10/10 8 8 8 8 8 8BOX8 10/10 6 6 6 6 6 6 harvestedBOX 9 10/10 5_~ 5.-_ 5 5 5- -19')

DENGUE 4DAYS 0 1 2 3 4 5DATE 22/8 23/8 24/8 25/8 26/8 27/8BOX 1 10110 4

APPENDIX 2PREPARATION OF REAGENTS1. 0.51\1 borate buffer (pH9.0130.0g H3Bo3, lIiter distilled waterNaCL 1.5M87.6 g NaCI, 1 liter ofdistilled waterNaOH 1.0N (40g/l)Ig NaOH, 25ml distilled water2. Borate buffer (pII9.0)80ml NaCI (1.5M), 100ml H3B03 (O.5M),24.0ml NaOH (l.ON), 1000ml distilled water3. TRIS BUFFER 11\1 rH 8.5 at ZIoC121.lg Tris base, I OOOml distilled water4. Preparation of deoxynucleotide triphosphate (dNTPS)The originaldeoxynuc1eotide triphosphateA at IOOmM, C at 100mM, G at I OOmM, T at 100mMThe working solution was 2.5mM of A,C,G, and T100125 =1/40, The total volume required was 100,tl = 100/40 =2511Therefore 25/-l1 each of A,C,G,T was mixed with 900/ll of PCR water5. Preparation of electrophoresis buffer (T.A.E) :This consisted of 242grams of Tris .base (Sigma T-1503), 57.lml of aceticacid (90% prolab), 100ml ofEDTA (0.5M, pH 8.0), The mixture was madeup to I liter, This resulted in final concentration of50x.6. Preparation of the Blue0.2%xylene,0.1 % blue bromophenol,0.5%glycerole, Iml T.A.E, lOOm]distilled water7. Ethidium bromide0.7mg/ml- Reference Gepbotoz 5ml8. Substrate: O-toIidine5mg O-tolidine, 250111 Dimethyl-forrnamide,30JlI Citrate buffer (pH 4.0),12111 H 202 10 Vol9. Carbonate buffer: .201

I.59g Sodium carbonate (Na2COj), 2.93g Sodium hydrogen carbonate(NaHCO]), 1000011 Distilled water10. Phosphate buffer sa'liue (PUS) rH 7.28g NaCI, 1.15g Na2HP04.0.02g KH 2I'0 4.IOOOmldistilled water.11. Citrate buffer rH 4.011.77g Citric acid I H20, 4.48g NaOlI, DissolveNaOH in 200ml ofdistilled water,Add the citric acid, Add 4001111ofdistilled water, Adjust the -H to 4.0 withIN HCl, Complete to lliter with distilled water12. Capture anti-human IgMGoat Affinity purified Ab to human, IgM (5FC~1) - 2MG,Catalog 11 55073, Lot Number, 0123313. Diluent for ELISA:PBS- 0.05% Twecn 20,1 % Skim milk,14. Washing buffer in ELISA:PBS-O.5% Twcen20: (Polyoxyethylcnc-sorbitan monolaurate)( Add 0.5ml Tween 20 to 1 Iiter of I'Ds)15. Conjugate for IgM capture ELlSA.Cappel Peroxidase- conjugated Sheep, lGG Fractionto Mouse lGG (NoX Human) -2ml, Catalog 1155558, Lot 11 4067116. Conjugate for IgG capture ELISACappel peroxidase- conjugated Goat, F (AB') 2 Fragment to Human I GG,•(Whole molecule) -2m!, Catalog # 55244, Lot Number: 39240, TotalProtein: 17.9mglml, Titer 1: I05,200202

•17. Preparation of PrimersPrimers Original concentrationRequiredDSI 3.83flg/~t1 IOOngi}t1DS2 4.41~lg/fll IOOng/fllTSI 2.83flg/~t1 IOOng/fllTS2 3.22flg/ftl IOOng/fllTS3 3.37~lg/Fl ](IOng/fllTS4 3.37flg/~t1 1GOng/fllDSlwas diluted 1:38.3, DS2 wasdiluted 1:44.1,TSI-I:28.3, TS2-1:32.2, TS3-1:33.7, TS4-1:33.720J

APPENDIX3POOLED MOUSE ASCITIC FLUID FOR DIFrERENT AIWOVIRUSESAPPENDIX 2POOLED MOUSE ASCITIC FLUID (IMMNUNE) FOR DIFFERENT ARBOVIRUSESPool 1BabankiMiddelburgBunyamweraSimbuOrungoPalyamTataguineMossurilBwambaO'NyongNyongPool 2Semllki ForestNdumuWessefsbronBagazaUganda SDengue 2Dengue 4West NileKedollgouUsuluPool 3ChikunqunyaYellow FeverHeshaMpokoOkolaRift Valley FeverNyandoEret 147BozoIgbo OraPool 4PerinetBolekeGermistonBoubouiZlkaNgariPongalaSaboyaBiraoShokwePoolSSpondweniYokaOubiTangaBanguiYalaOubanquiAcadoOakar BatPool 6NkolbissonPool 7KamesePool8AkabanePool 9ArumowolAkabaneOdrenisrouGomokaTaiPataKindiaAndasibeNdelieBangoranAr Mg 966BobiaDabakalaNgoupeSomoneAr MMP 1SeAr D 66707ButtonwillowSaboSangoShanandaShuni8athuperiEubenanqeeOdrenisrouGabek ForestGordilSaint FlorisRift Valley FeverKameseBangoranMg 966GomokaNala204

APPENDIX 4IgM EUSA RESULTS AFTER TilE TREATMENT OF PROBLEMATICSERABy Dilution Method667 VF 30D1 DZ D3 D4 D1 D21 Z 3 4 5 6 7 8 9 10 11 12A 1.566 1.596 1.371 1.349 1.417 1.431 1.181 1.355 0.983 0.237 1.678 0.222B 0.174 1.093 1.276 1.237 1.171 1.219 1.207 1.1 71 0.855 0.22 1,456 0.21C 0.044 1.023 1.144 1.027 0.99 1.007 1.178 1.043 0.85 0.248 1.372 0.182D 0.917 0.898 0.94 0.854 0.819 0.896 0.9 0.829 0.796 0.237 1 331 0.207 'E 0.883 0.84 0.856 0.779 0.737 0.767 1.113 0.694 1.028 0.293 0.976 '0.33F 0.796 0.728 0.726 0.677 0.674 0.638 0.803 0.645 0.931 0.438 0.853 0.438G 0.738 0.744 0.706 0.656 0.594 0.593 0.781 0.606 0.808 0.504 0.689 0468H 0.69 0.687 0.737 0.633 0.576 0.432 0.717 0.628 0.808 0.597 0.63 0.476D3 D4 ,-lBy Adsorption with tile Normal An tlq en1 2 3 4 5 6 7 8A 0.738 0.716 0.34 0.339 0.433 0.427 0.26 0.208B 0.424 0.391 0.471 0.161 0.418 0.419 0.523 0.552C 0.602 0.61 0.44 0.445 0.446 0.432 0.344 0.36o 0.325 0.2330.5130.513 0.540.5661.4891.545E 0.325 0.321 0.201 0.18 0.593 0.612 0.641 0.626F 0.232 0.217 0.214 0.207 0.342 0.332 0.262 0.248G 0.23 0.232 0.285 0.253 0.234 0.246 0.412 0.431H 0.295 0.298 0.566 0.562 0.34 0.32 0.502 0.517By Absorption with Rheumatoid Factor Reagent1 2 3 4 5 618 LEVF30 2.577 0.116 2.247 0.13 2.071 0.22257 0.487 0.3 0.346 0.292 0.187 0.125667 3.134 3.136 2.81 2.827 1.779 1.554FH 3.321 3.085 3.003 3.023 2.902 2.5369 10 11 120.699 0.72 0.403 0.3970.374 0.36 0.341 0.2710.71 0.736 0.34 o 319'0.281 0.283 0.132 0.141.081 1.16 0.106 0.109-0.712 0.771 0.249 0.270.263 0.252 0.358 0.3781.084 1.115 0.31 0.312--l,' ,_ 205

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