AD-A261 707

AD____

ARMY PROJECT ORDER NO: 89PP9961

TITLE: ANALYSIS OF DENGUE VIRUS ENHANCING EPITOPES USINGPEPTIDE ANTIGENS DERIVED FROM THE ENVELOPE GLYCOPROTEINGENE SEQUENCE

PRINCIPAL INVESTIGATOR: May C. Chu, Ph.D.

CONTRACTING ORG: Division of Vector-Borne Infectious DiseasesCenter for Infectious DiseasesCenters for Disease ControlAtlanta, Georgia 30333

REPORT DATE: November 29, 1991 MAR09 1993

TYPE OF REPORT: Final Report

PREPARED FOR: U.S. ARMY MEDICAL RESEARCH AND DEVELOPMENT COMMANDFort Detrick, Frederick, Maryland 21702-5012

DISTRIBUTION STATEMENT: Approved for public release;distribution unlimited

The findings in this report are not to be construed as anofficial Department of the Army position unless so designated byother authorized documents.

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I. AGENCY USE ONLY (Leave blank) 2. REPORT DATE 3. REPORT TYPE AND DATES COVERED

29 Nov 91 Final Report (9/1/89 - 11/30/91)4. TITLE AND SUBTITLE Ana ysis or Dengue Vnrus nancing 5. FUNDING NUMBERS

Epitopes Using Peptide Antigens Derived From the Army Project OrderEnvelope Glycoprotein Gene Sequence 89PP9961

6. AUTHOR(S) 61102AMay C. Chu, Ph.D. 3M161102BS12.AB.192

WUDA320711

7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) B. PERFORMING ORGANIZATION

Division of Vector-Borne Infectious Diseases REPORT NUMBER

Center for Infectious DiseasesCenters for Disease ControlAtlanta, Georgia 30333

9 SPONSORING MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSORING MONITORING

U.S. Army Medical Research & Development Command AGENCY REPORT NUMBER

Fort DetrickFrederick, Maryland 21702-5012

11. SUPPLEMENTARY NOTES

12a DISTRIBUTION AVAILABILITY STATEMENT 12b DISTRIBUTION CODE

Approved for public release; distribution unlimittd

13 ABSTRACT ,3xm,,.n; 200 words)

The biological events leading to the development of severe diseasemanifestations of dengue infections (dengue hemorrhagic fever/dengueshock syndrome, DHF/DSS) has been the focus of many investigations.Though DHF/DSS cases occur during primary dengue infections, the major-ity of severe dengue disease has been associated with persons experi-encing a secondary heterotypic dengue infection. What do we know aboutthe pathogenic processes that result in the exacerbation of denguesymptoms? Can these factors be identified in vitro and then correlatedwith clinical disease? The results of previous studies suggest thatpre-existing DEN antibodies circulating in a person may be a riskfactor in the development of DHF/DSS. However, other host and viralfactors must be involved since only a small proprotion of the populatioresiding in endemic dengue disease regions develop severe denguedisease.

14 SJB,ECT •;' • 15 N_%*ý Fý Cý ;-A, Eý

Dengue Fever; Nucleotide Sequencing; Peptides;Enhancing Epitopes; RA I; PO; Antigens; Dengue Virus; 16 PRICE COD;Analysis17 SoCuk_ CA'½ i :CAmiON tj SEcAT c"; ..J2 'R ,S.~A ~ :

OF UcaOsie OF THU l PAas O• A i•F dUACT

Unclassified Unclassified Unclassified Unlimited

FOREWORD

Citations of commercial organizations and trade names inthis report do not constitute an official Department of the Armyendorsement or approval of the products or services of theseorganizations.

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TABLE OF CONTENTS

REPORT DOCUMENTATION PAGE .

FOREWORD ii

TABLE OF CONTENTS ................ .......................

INTRODUCTION ............................................. 1Summary of previous accomplisments for FY 1990 ... ..... 1

ACCOMPLISHEMENTS FOR FY 1990................. 2Examination of FcR-expression in HL-CZ promonocytic and

enhanced virus replication ........... ............ 2Reactivities of anti-DEN-2 E glycoprotein synthetic

peptide antibodies with DEN-i and DEN-2 virus . . . 2Cloning and sequencing the nucleotides encoding the

non-structural DEN-I CV1636/77 genes .......... 3Antibody preparation for use in a standardized ADE 3

DISCUSSION ........ ................................ 4Relevance of epitope mapping using anti-peptide

antibodies ............................ 4Analyses of the genetic relatedness of DEN viruses 4Future studies ................... ...................... 5

REFERENCES ........................ ......................... 6

APPENDIX 1 .................. ........................ TABLES

APPENDIX 2 .................. ........................ FIGURES

APPENDIX 3 .................. ....................... ABSTRACTS

APPENDIX 4 ................ ...................... PUBLICATIONS

PEiSiiL

INTRODUCTION

The biological events leading to the development of severedisease manifestations of dengue infections (dengue hemorrhagicfever/dengue shock syndrome, DHF/DSS) has been the focus of manyinvestigations (1,8). Though DHF/DSS cases occur during primarydengue infections, the majority of severe dengue disease has beenassociated with persons experiencing a secondary heterotypic dengueinfection (6,7). What do we know about the pathogenic processesthat result in the exacerbation of dengue symptoms? Can thesefactors be identified in vitro and then correlated with clinicaldisease? The results of previous studies suggest that pre-existingDEN antibodies circulating in a person may be a risk factor in thedevelopment of DHF/DSS (9). However, other host and viral factorsmust be involved since only a small proportion of the populationresiding in endemic dengue disease regions develop severe denguedisease.

Antibody-dependent enhancement (ADE) of dengue virus (DEN)replication in vitro has been proposed as a pathogenic mechanism inthe development of DHF/DSS. We believe that enhanced virusreplication is an important part of DEN pathogenesis and that ADEis the result of the interplay of host cells, antibody and virusstrain. We investigated DEN virus replication in order to definethe parameters that could lead to enhanced growth in the presenceof antibodies to flaviviruses.

SUMMARY OF PREVIOUS ACCOMPLISHMENTS FOR FY 1990

0 Developed a sensitive ADE assay to examine the interplay ofantibody, virus and host cells in DEN replication. Humanpromonocytic HL-CZ cells supported enhanced DEN replicationbetter than K562 or U937 human cells at low MOIs (104-106).

M Determined that the sensitivity of the HL-CZ cells was relatedto the number of Fc-receptors (FcRs) and the FcR types (FcR II> FcR III > FcR I) expressed on the cell surface sinceblocking the FcRs by specific FcR monoclonal antibcdies (MAbs)abrogated virus replication.

M Characterized the abilities of MAbs 4G2 and 3H5 to mediatevirus neutralization and enhanced replication. Determinedthat the MAbs recognizing DEN envelope (E) glycoproteinepitopes were involved in NT and ADE and confirmed theobservation that these activities were concentration relatedrather than epitope-directed.

M Compared the growth profiles of selected DEN virusesrepresenting epidemiologically important genotypes using ourADE system. Viruses from endemic dengue region (Thailand)were more antibody-dependent than that of viruses from thedengue epidemic region (Caribbean).

1

M] Attempted to define the antigenic epitopes that mediate ADEand NT by using antibodies raised against DEN-2 E glycoproteinsynthetic peptides. We were able to determine that someantipeptide antibody pools were reactive in ADE and/or NTtests, though reactions appeared much weaker than the MAbcontrols.

M] Continued to determine the nucleotide sequence encoding thenon-structural (NS) regions of DEN-l CV1636/77 virus. Of theregions examined thus far, it appeared that DEN-4 Dominica andDEN-l CV1636/77 were very similar in the NS region, suggestingthat the viruses have evolved closely or that there may begenomic recombination.

ACCOMPLISHEMENTS FOR FY 1991

Examination of FcR-expression in HL-CZ promonocytic and enhancedvirus replication. Dr. Wu Tse Liu of the National Yang MingMedical School, Taipei, Taiwan provided us with several lines ofHL-CZ cells (14). We compared the virus yields of the uncloned,clone 3 and clone CCC-5 cells. The results of the enhanced growthprofile of DEN-2 16681 virus were variable thus suggesting that thepermissiveness of the cells to support enhanced virus replicationcould be intrinsically different. We investigated the discrepancyof virus yield of these cultures and determined that if the ADEresults were grouped by >20% FcR-expression (rosetting) andcompared with virus yield (>10' pfu/ml) , the presence of rosettingcells was a factor in cell permissiveness (Table 1) as we haddemonstrated before when comparing virus growth in U937, K562 orHuPBL cells. However, in a few cases, neither high nor low numberof rosettes correlated with virus yield, thus implying that someother factors also affect virus replication. Therefore, in ouranalyses of ADE results, we only accepted the tests in which 1)virus control cultures (no antibody) contained no visible growthand 2) positive ADE controls of cultures containing antibodyproduced 103 pfu/ml virus yield.

Reactivities of anti-DEN-2 E glycoprotein synthetic peptideantibodies with DEN-l and DEN-2 virus. We have previouslydetermined the reactivities of mouse antibodies immunized with DEN-2 E glycoprotein peptides in PRNT and ADE assays. Though we wereable to demonstrate some activity in sera obtained from miceimmunized by i.m. and s.c. routes, there was variation betweenantibody specimens directed against the same peptide fromheterologous mouse strains (19) . To normalize antibody reactivity,sarcoma-primed BALB/C mice were immunized with individual DEN-2 Eglycoprotein synthetic peptides and the resulting mouse ascitesfluids (MAFs) were assessed (Table 2). To compare the anti-peptideantibodies on an equal basis, immune mouse ascites fluids (MAFs)were purified by elution from protein A columns and standardized to

2

1 mg/ml and examined by ELISA. The reactivities of the purifiedIgs were not appreciably different from crude MAFs suggesting thatbinding to virus antigen or peptide was related to the originalstrength of the MAF and was not affected by increased/decreasedaffinity of the antibody to the peptide.

in addition to testing each of the anti-peptide antibodiesindividually, we attempted to reconstitute antigenic binding sitesby mixing the antibodies together (Table 3). Seven anti-peptideantibodies were mixed in various combinations and each tested forbinding to antigen, for ability to neutralize virus and formediating enhanced virus growth. Binding of MAFs, 1 Ag and 10 Agof anti-peptide antibodies to DEN-2 Jamaica virus antigen wereexamined by ELISA, and these results reflect original MAF titers.HL-CZ cells were infected with DEN-I 16681 virus at MOI of 105.Where the positive ADE and background culture controls cells metcur acceptance criteria, the enhanced virus yields in the presenceof 1 •Ig of anti-peptide antibodies was not consistent (Table 3).Neutralization activity of the anti-peptide antibodies againstDEN-2 16681 virus was examined. One Mg of an antibody was mixedwith 1000 pfu of virus and incubated at 37 C for 1 hour. Thesurviving virus was ascertained by testing the virus/Ab mixes inthe BHK-21 plaque assay (16) and the results reported as percentageof reduction of input virus (Table 3). Both anti-peptide mix 21(antibodies to peptides 06, 240, 274, 17 and 361) and mix 22 (anti-peptides 240, 274, 17, and 361) neutralized 53% of the input virus,and anti-peptide mix 42 (antibodies to peptides 240 and 274)neutralized 100% of the input virus.

Cloning and sequencing the nucleotides encoding the non-structuralDEN-I CV1636/77 genes. The genome encoding the structural genes ofDEN-I CV1636/77 have been previously published (3). Last year wecompleted the nucleotide sequences encompassing NS4a, NS4b, and NS5genomic regions of DEN-i CV1636/77 and compared the results withthe published sequences of DEN-2 (5), DEN-3 (18), and DEN-4 (15).We have completed the sequencing of the rest of the non-strucuturalnucleotide regions for NSI, NS2a, NS2b, NS3 and the final 800 basepair sequence of the 3'-end of the viral RNA (Figure 1).

The similarity of the non-structural gene regions between the4 DEN viruses are summarized in Figure 2. DEN-l, DEN-2, and DEN-3share between 63%-68% similarity by nucleotide sequence comparisonand 65%-78% similarity by their deduced amino acid sequences.overeach of the non-strucutural gene regions. DEN-I and DEN-4 howeverare very similar in NS3, NS4a, and NS4b regions (95%-98%); this iscontrasted by a comparison of DEN-I and DEN-4 over the NS5 regionwhere similarity extends to only 78%. When the combined nucleotideand deduced amino acid sequences over NS3, NS4a, NS4b, and NS5regions are examined, the similarity of 90% remains between DEN-Iand DEN-4 (Figure 3,4).

3

DISCUSSION

Studies to identify genomic correlates of DHF/DSS haveprimarily depended mouse, monkey and other laboratory experiments.Animals infected with DEN will respond by seroconversion to theinfected virus but do not develop DHF/DSS. Therefore most diseaseparameters of infected humans are based on clinical observations,and epidemiological assements. The availability of genomicsequences of flavi- and dengue viruses, the synthesis of peptidescorresponding to deduced amino acid sequences, and the developmentof molecular techniques, make it possible to begin studies tocorrelate biological activities with genomic variation.

Relevance of epitope mapping using anti-peptide antibodies. Withthe standardized ADE assay, we have been able to examine divergentaspects of the roles of antibody, virus and host cells. From thesestudies, we could determine that each component share roles invirus replication and that the ADE system developed could isolatemany of these factors for further examination. In our attempts toidentify the genomic regions that encode antigenic epitopesinvolved in biological responses using anti-peptide antibodies, wecould identify the epitopes that mediate neutralization of thevirus. However, the enhancing activity could not be located withina specific region using the anti-peptide antibodies. This does notmean ADE activity cannot be found, the non-or broad reactivity mayonly be a matter of using sub-optimal antibody concentrations, theinappropriate mix of the anti-peptides, the virus strainvariability or the susceptibility of the host cell stage to virusreplication. It is likely that in using anti-peptides to mapstrategic epitopes, a reactive result implies reconstitution ofepitopes, whereas non-reactivity implies only that epitopes werenot reconstituted.

Using anti-peptide sera directed against selected DEN-2 Eglycoprotein regions, we have been able to demonstrate that some ofthe E regions will elicit antibody that will mediate enhanced virusreplication, neutralization and enhancement, and neutralizationalone. Though this series of experiments need to be repeated withother antibody mixes and other DEN viruses, the neutralizationepitope defined by amino acids 240-274 is is the first directassociation of specific E-glycoprotein regions with biologicalfunctions.

Analyses of the genetic relatedness of DEN viruses. The similarityof the non-structural genomic sequences of DEN-l and DEN-4 viruseswas unexpected. Published sequences of NS regions of DEN-2 andDEN-3 viruses led us to expect only 63%-68% homology among the DENviruses. DEN-i and DEN-4 viruses, however, appeared to be nearlyidentical from NS1 through NS5 and again at the 3'-end, suggestingthat there may be a double-crossover genetic recombination. Topreclude that possibility that the DEN stock cultures may have beencontaminated with one or the other viruses, we plaque-pickedpurified virus from our CV1636/77 stock and found that they all

4

contained the same characteristics (data not shown). In addition,we are examining, by PCR amplification and sequencing, of otherDEN-I and DEN-4 strains in the 3'-end region to analyze therelationship.

Future studies. During this study, we were not able to achieve allthe goals set forth in the original proposal. We would liked tohave been able to get anti-DEN-l-peptide serum to analyze and workas we did with DEN-2 peptides. However, we were delayed by theunexpected complication of the DEN-i sequence homology with DEN-4and felt that was important finding to follow-up and analyze. Weare planning to do work on examining patient serums that wouldrecognize DEN-2 peptide mix 240/274 and to identifiy whether thisregion of the E-glycoprotein is a useful diagnostic tool.

5

REFERENCES

1. Brandt WE. 1990. Development of dengue and Japaneseencephalitis Vaccines. J Infect Dis 162:577-83.

2. Brandt WE, McCown JM, Gentry MK, and Russell PK. 1982.Infection enhancement of dengue type 2 virus in the U937 humanmonocyte cell line by antibodies to flavivirus cross-reactivedeterminants. Infect Immun 36:1036-41.

3. Chu MC, O'Rourke EJ, and Trent DW. 1989. Genetic relatednessamong the structural genome of dengue-1 viru strains. J GenVirol 70:1701-1712.

4. Deubel V, Kinney RM, and Trent DW. 1986. Nucleotide sequenceand deduced amino acid sequence of the structural proteins ofdengue type 2, Jamaica genotype. Virology 155:365-77.

5. Deubel V, Kinney RM, and Trent DW. 1988. Nucleotide sequenceand deduced amino acid sequence of the non-structural proteinsof dengue type 2 virus, Jamaican genotype: comaparativeanalysis of the full-length genome. Virology 165:234-44.

6. Gubler DJ, Reed D, Rosen L, and Hitchcock JG. 1978.Epidemiologic, clinical and virologic observations on denguein the kingdom of Tonga. Am J Trop Med Hyg 27:581.

7. Guzman MG, Kouri GP, Bravo J, Soler M, Vazquez S, and MorierL. 1990. Dengue hemorrhagic fever in Cuba. 1981: aretrospective seroepidemiologic study. Am J Trop Med Hyg42:179-84.

8. Halstead SB. 1988. Pathogenesis of dengue: challenges tomolecular biology. Science 239:476-81.

9. Halstead SB, and O'Rourke EJ. 1977. Dengue viruses andmononuclear phagocytes. I. Infection enhancement by non-neutralizing antibody. J Exp Med 146: 218-29.

10. lenchal EZ, Gentry MK, McCown JM, and Brandt WE. 1982. Denguevirus-specific and flavivirus group determinants identifiedwith monoclonal antibodies by indirect immunofluorescence. AmJ Trop Med Hyg 31:830-6.

11. Kerschner JH, Vorndam AV, Monath TP, and Trent DW. 1986.Genetic and epidemiological studies of dengue type 2 virusesby hybridization using synthetic deoxyoligonucleotides asprobes. J Gen Virol 67:2645-61.

12. King SD, Rose E, Bancroft WH, McCown JM, Woodall J, and SatherG. 1979. The laboratory diagnosis of dengue in Jamaica, 1977.PAHO Scientific Publication 375:153-8.

6

13. Littaua R, Kurane I, and Ennis FA. 1990. Human IgG Fc receptorII mediates antibody-dependent enhancement of dengue virusinfection. J Immunol 144:3183-6.

14. Liu WT, Chen SC, Lee N, Tan SK, Liu SF, Dunn P and Chang KSS.1989. Establishement and characterization of a cell line ofmonocytic origin, HL-CZ, from human leukemia. J Biomed Lab Sci4:284-92.

15. Mackow E, Makino Y, Zhao B., Zhang YM, Markoff L, Buckle-WhiteA, Guiler M, Chanock R, and Lai CJ. 1987. The nucleotidesequence of dengue type 4 virus: analysis of genes coding fornon-structural proteins. Virol 159:217-28.

16. Morens DM, Halstead SB, Repik PM, Putvatana R, and RaybourneN. 1985. Simplified plaque reduction neutralization assay fordengue viruses by semimicro methods in BHK-21 cells:comparison of the BHK suspension test with standard plaquereduction neutralization. J Clin Microbiol 22:250-4.

17. Morens DM, Marchette NJ, Chu MC, Burke DS, and Halstead SB.Growth in human peripheral blood leukocytes of dengue type 2isolates correlates with severe and mild dengue disease. AmJ Trop Med Hyg 1991 (in press).

18. Osatomi K and Sumiyoshi H. 1990. Complete nucleotide sequenceof dengue type 3 virus genome RNA. Virol 176:643-7.

19. Roehrig JT, Johnson AJ, Hunt AR, Bolin RA, and Chu MC. 1990.Antibodies to dengue 2 Jamaica E-glycoprotein syntheticpeptides identify antigenic conformation. Virology 177:668-75.

20. Unkeless JC, Scigliano E, and Freedman VH. 1988. Structure andfunction of human and murine receptors for IgG. Ann RevImmunol 6:251-81.

7

Tables

Table 1 Chi-square comparison of the relationship between rosetteexpression and virus replication in HL-CZ cells

Table 2 Reactivity of the mouse ascites fluids obtained from miceimmunized with DEN-2 E glycoprotein synthetic peptides

Table 3 Mixtures of purified anti-DEN-2 synthetic peptideimmunoglobulins and PRNT/ADE results

Figures

Figure 1 Location of CV1636/77 clones sequenced and che diagram ofthe similar regions between DEN-l and DEN-4.

Figure 2 Percentage of homology of the non-structural regionnucleotide and deduced amino acid sequences of DENviruses.

Figure 3 Comparison of the nucleotide sequences comprising thenon-structural genome regions of DEN-l, DEN-2, DEN-3 andDEN-4 viruses.

Figure 4 Comparison of the deduced amino acid sequences of thenon-structural regions of the four DEN viruses.

TABLE 1Chi-square comparison of the relationship between rosette

expression and virus replication in HL-CZ cells

PFU VIRUS/% ROSETTE > 20% ROSETTE < 20% ROSETTE

* 103 VIRUS/ML 6* 1

* 10' VIRUS/ML 1 4*p <0.001

TABLE 2 REACTIVITY OF ANTI-PEPTIDE ANTIBODIES AGAINST HOMOLOGOUSPEPTIDE, DENGUE-2 AND DENGUE-1 VIRUSES

PEPTIDE" ANTIGEN USED IN ELISA6PEPTIDE DENGUE-2 DENGUE-1

1-2* 6400 1280 320

35 102400 128000 4000

3-8/1 100 <10 <10

4-6 400 40 10

04* 1600 160 10

142* 3200 640 10

167* 100 10 <10

06* 25600 5120 40

240# 1600 1280 80

274* 100 10 <10

16* 800 160 80

17# 3200 160 <10

361* 12800 640 80

437* 3200 20 80

+control -- >12800 >12800

-control -- <10 <10

Roehrig et al (19)."Each well of the ELISA plates were coated with either 1 ug ofpeptide, or gradient-purified virus antigen. The antibodytiters as reported as positive were adjusted by subtracting 2standard-deviations above background reactions.

* Synthetic peptides which are distinct by deduced amino acidsequence from DEN-2 Jamaica.

# Synthetic peptides that are not distinct from DEN-2 Jamaicavirus (>90% homology) but are in the NT/ADE region mapped byanti-peptide antibodies.

TABLE 3 ANTIBODY-DEPENDENT ENHANCEMENT (ADE) AND PLAQUE REDUCTIONNEUTRALIZATION (PRNT) RESULTS

ANTI-PEPTIDE ANTIBODIES 100 NG;' PRNTb ADE'

1-2 35 3/8-1 4-6 06 240 274 17 361 DEN1 DEN2 pfu/ml

___ ___ ___ 20 35 2150

__ ____ 15 41 < 7

19 32 < 7

m 24 35 < 7

____ ______ ___ 15 32 >8300

24 31 280

_-----" 31 32 4600

S27 36 >8300

28 46 < 7

36 42 3700

42 56 260

36 42 1400

22 53 400

16 56 < 7

4 38 15008m m 35 4 000S35 100 800

•16 27 5500

0 0 10 3600

4G,2 M•*, flavivirus group reactive 96 97 >8300

3H5 M•*, dengue-2 specific 12 100 8300

,Virus control@ t0 0 < 7

Legend for TABLE 3

Purified anti-DEN-2 peptide antibodies, used 100 ng of eachanti-peptide in the mixes. Filled boxes indicate the anti-peptide serum used.PRNT by BHK-21 semi-micro assay. Average plaques of DEN virusin each 24-well = 26. Serum and virus were incubated at 37 Cfor 1 hour. Percentage of neutralization shown here. >50 %reduction shown in bold face.ADE assay with HL-CZ cells and MOI of 10s. Cells wereinfected and incubated for 4 days at 37 C in 5% COQ. Virusreplication was assessed by plaque titration in BHK-21 semi-micro assay. Results are the average of 3 experiments.

* The amount of 4G2 used for PRNT = 10 ug, for ADE = 1 ug. 3H5used for PRNT = 1 ug, for ADE = 100 ng.Virus control = only virus without antibody for PRNT and ADE.

ABSTRACTASTMH 1991206 THE NON-STRUCTURAL GENOME OF DENGUE-I VIRUS CV1636/77:

COMPARISON OF THE NUCLEOTIDE AND DEDUCED AMINO ACID SEQUENCESWITH THE OTHER DENGUE SEROTYPES.Chu MC, Putvatana R, and Trent DW. Division of Vector-Borneinfectious Diseases, National Center for Infectious Diseases,Centers for Disease Control, Ft. Collins, Colorado 80526

The determination of the entire nucleotide and deduced aminoacid sequences of the DEN-i Jamaica CV1636/77 virus togetherwith the known sequences of DEN-2, DEN-3 and DEN-4 virusesprovide a complete sequence database of the DEN serotypecomplex. We have previously presented analyses of thenucleotide sequences encoding the structural and the non-structural NS3-NS4-NS5 regions. Sequence analyses of theentire genomic RNA and cloned cDNA of DEN-i reveal that thegenomic RNA contains 10,641 nucleotides and encodes an openreading frame of 10,224 nucleotides that translate into 3408amino acid residues. A comparison of the DEN-i sequence withDEN-2 (Jamaica; Deubel et al., 1988), and DEN-3 ýH87; Osatomiet al., 1990) reveal that the genomic sequences encoding thestructural (C-prM-M-E) and some of the non-structural ýNSI,NS3, and NS5) regions are conserved (>70% homology). The3'non-coding regions of DEN-i, DEN-2, DEN-3, and DEN-4(Dominica; Mackow et al., 1987) are of different nucleotidelengths comprising 387, 455, 448, and 385 base pairsrespectively. The entire non-structural sequences for DEN-iand DEN-4 share >90% identity. These results reveal a closeevolutionary relationship between Caribbean DEN-i and DEN-4viruses which cannot be detected by conventional serologicalmethods. Thus the sequence database of all the DEN virusserocomplex will be a useful tool in studying the geneticvariation and evolution of these viruses.

Prepared for ASTMH 1991

ABSTRACTAntibody-dependent enhancement (ADE) of dengue (DEN) virusinfection in human mononuclear cells in vitro has been standardizedusing a human promonocytic cell line HL-CZ, purified monoclonalantibodies (MAbs) , and select DEN viruses. Characterization of theFc-receptors (FcRs) expressed on HL-CZ cells have indicated thatsubsets of FcR mediate ADE better than others. Using thisstandardized system, we have compared the ability of mouse anti-DEN2 envelope (E) peptides to elicit virus neutralization and ADE.Peptides 1-2, 437 appear to elicit ADE activity in contrast toother peptides that appear to elicit neutralization but not ADE.Though these assays need to be repeated, it appears thatdifferential functions may be attributed to particular E genomicregions. The comparison of the nucleotide sequences of DEN-i RNAencoding the non-structural proteins to the other DEN sequences hasrevealed that DEN-4 and DEN-i share > 90% similarity in NS3 andNS4a, 4b genome regions. DEN-3 and DEN-i have a deletion in in NS5that is conserved in other DEN-i and DEN-3 isolates. These genomicsequence comparisons indicate that non-structural regiondifferences need to be studied as well for our understanding of DENreplication and pathogenesis.

PUBLICATIONS

'I. Chu MC, Huang GH, Collins ND, and Trent DW. An experimentalmodel for analyzing antibody-dependent enhanced growth ofdengue virus in human promonocytic HL-CZ cells. Submitted forpublication.

Chu MC, Putvatana R, Huang GH, Roehrig JT, and Trent DW.Analyses of the reactivities of anti-dengue-2-E glycoproteinantibodies to dengue-1 and dengue-2 viruses. Manuscript inpreparation.

3. Chu MC, Putvatana R, Repik P and Trent DW. Similarities ofthe dengue-l CV1636/77 and dengue-4 Caribbean viruses:genomic sequence comparison and oligonucleotidefingerprinting. Manuscript in preparation.

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