Amino acid (AA) sequences of 4 serotype of dengue viruses deduced from their nucleotide (nt) sequences of genomic RNA were analyzed for each genome segment and each stretch of 10 AA residues. Precursor of membrane protein (pM), and 4 nonstructural proteins (NS1, NS3, NS4B, NS5) were highly conserved, while another nonstructural protein (NS2A) was least conserved among 5 strains of dengue viruses. When homology was compared among heterotypic viruses, type 1 and type 3 dengue viruses showed close relationships, while type 4 dengue virus was relatively remote from other types. When AA sequences were compared for each 10 AA residues, some highly conserved as well as least conserved domains were identified. Significance of these findings for diagnostic virology and epidemiological survey was discussed

Haishi Shozo

Igarashi Akira

Tanaka Mariko

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Short CommunicatuonT rop. Med., 32 (2), 81-87, June, 1990 81Comparative Amino Acid Sequences of Dengue VirusesShozo HAISHI, Mariko TANAKA and Akira IGARASHIDepartment of Virology, Institute of Tropical Medicine,Nagasaki University, 12-4 Sakamoto-machi, Nagasaki 852, JapanAbstract: Amino acid (AA) sequences of 4 serotype of dengue viruses deducedfrom their nucleotide (nt) sequences of genomic RNA were analyzed for each genomesegment and each stretch of 10 AA residues. Precursor of membrane protein (pM),and 4 nonstructural proteins (NS1, NS3, NS4B, NS5) were highly conserved, whileanother nonstructural protein (NS2A) was least conserved among 5 strains of dengueviruses. When homology was compared among heterotypic viruses, type 1 and type 3dengue viruses showed close relationships, while type 4 dengue virus was relativelyremote from other types. When AA sequences were compared for each 10 AAresidues, some highly conserved as well as least conserved domains were identified.Significance of these findings for diagnostic virology and epidemiological survey wasdiscussed.Key words: Dengue virus, Flavivirus, Amino acid sequence, HomologyDengue viruses of 4 different serotypes belong to the family Flaviviridae (Westawayet al.t 1985), and transmitted among humans by mosquitoes, primarily Aedes aegypti (Clarkeand Casals, 1965), thus arthropod-borne viruses (or arvoviruses). Disease manifestationsamongdengue virus-infected humans are various, from mild undifferentiated fever, denguefever to severe dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). Se-quential infection with heterologous type of dengue viruses different from those in theprimary infection has been considered to be related with severe manifestation of thedisease (Russell, 1971; Halstead, 1966, 1980). The latter investigator postulated that an-tibody-dependent enhancement of dengue virus growth in human peripheral monocytes wasunderlying cause of DHF or DSS.Recently, entire nucleotide (nt) sequences of genomic RNA were analyzed for types 2,3, and 4 dengue viruses (Deubel et al., 1988; Hahn et al9 1988; Osatomi, 1988; Mackow etal., 1987; Zhao et al, 1986), while type 1 dengue was partially sequenced (Mason et.al,1987). In order to understand intratypic as well as heterotypic homologies of dengueviruses at a molecular level, we compared deduced AA sequences of 5 dengue virusstrains.Received for Publication, March 13, 1990.Contribution No. 2390 from the Institute of Tropical Medicine, Nagasaki University.82Sequences data were referred from following references: type 1 dengue (Dl: Masonet at., 1987); type 2 dengue Jamaica genotype (D2J: Deubel et al, 1988); type 2 dengue SIcandidate vaccine strain (D2S: Hahn et al, 1988); type 3 dengue (D3: Osatomi, 1988); type 4 dengue(D4: Mackow et al, 1987; Zhao et at., 1986). Viral genomes were divided into ll segments[core protein (C), precursor (pM) of membrane protein (M), envelope glycoprotein (E), andnonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5)] according to Rice etal. (1985) and Speight et al (1988).Since compared 5 dengue virus strains possessed varying numbers of AA residuesfor each genome segment, direct sequence comparison was difficult. Sequences were,therefore, compared after alignment by filling up several residues to obtain maximal mat-ching of the corresponding sequence using DNASIS Version 6.0 software (Hitachi, 1989).For fine comparison, each 10 AA residues were compared and numbers of matching AAresidies were counted which were divided by the length to obtain scores.Analysis for each of the 10 combinations of 5 dengue virus strains was summarizedin Table 1 and Fig. 1. For all combinatis, pM, NS1, NS3, NS4B and NS5 proteins werehighly conserved and NS2A and C proteins were least conserved. Both C and M proteinsshowed high degree of deviation among 5 dengue virus strains. In NS3 protein, an NTP-motif sequence Gly-X-Gly-Lys (G-X-G-K) of ATP-dependent DNA helicase (Gor-balenya et al, 1988) was found for all dengue virus strains as other flaviviruses (Takegami,personal communication). While NS5 protein was considered as a component of viral RNA-dependent RNA polymerase because of its consensus triplet sequence Gly-Asp-Asp(G-D-D) of viral RNA polymerases (Goelet et al, 1982; Kitamura et al, 1981). Both NS3Table 1. Homology % of AAD1/D2J D1/D2S D1/D3 D1/D4 D2J/D2S D2J/D3 D2J/D4 D2S/D3 D2S/D4 D3/D4C 55^4 59.6 66.9 57/7 8OO 4sTs 55^4 5<UJ 50 47.7pM 74.4 75.6 76.7. 68.9 88-9 71-1 68.9 71.1 70.0 74-4M 70.0 68.8 82.5 61.3 96.3 68-8 65.0 67.5 65.0 58.8E 64.6 65.8 70.4 57.7 92.9 60.2 58.5 61.0 58.7 56.2average 64.8 65.7 71.7 59.3 90.7 60.4 59.8 61.0 60-2 57.1NS1 72.6 71.7 78.3 67.7 96.3 72.6 70.6 72.0 70.3 70.9NS2A 88.3 26.7 25.4 26.3 26.7 30.0NS2B 93.1 56.2 50.8 53.8 50-8 53.8NS3 96.2 79.0 75.7 78.4 74.8 76.7NS4A 98.0 62.7 64.0 60.7 63.3 60.0NS4B 93.2 71.6 73.2 72.8 74.8 72.0NS5 95.6 77.6 71.6 78.1 72.5 74.3average(nonstructural) 95.3 70.5 67.2 70.3 67-5 68.6average(fulllength) 94.0 67.9 65.3 67.9 65.6 65.7Abbreviation of virus name and genome segment is shown in text<<V»X>1O)_0OEoo<£83à"D2J/D2Sà"D2J/D3à"D2J/D4D2S/-D3à"D2S/D4D3/D4C pfl M E NSl N2A N2B NS3 NS4ANS4B NS5Genome segmentFig. 1. Graphic presentation of AA homology % in each genome segment.Abbreviation of virus name and genome segment is shown in text.and NS5 proteins could be important for the survival and replication of the virus and thushighly conserved (Rice et at., 1985, 1986; Strauss and Strauss, 1986).Intratypic homology between D2J and D2S was higher (91%) than heterotypichomology (57-51 %), although these 2 strains possessed different isolation and passagehistories. Among heterotypic combinatis, homology between Dl and D3 (72%) was higherthan other combinations, indicating closer relation between Dl and D3, which is compati-ble with the observation by monoclonal antibodies (Henchal et al., 1982). Homology of D4to other types of dengue viruses was lower than other combinations (Fig. 2), as illustratedin 3 dimensional scheme of Fig. 3. This scheme was obtained by comparing total score ofstructural protein region and each distance among 5 viruses was compared with thedistance between D2J and D2S which was taken as 1.0.In order to find out the highest and lowest homology regions, more fine analysis ofeach 10 AA sequences were performed and the result was shown in Fig. 4. Homologytendency among dengue viruses was almost similar to those of other flaviviruses, withhighly conserved regions at almost similar regions (Haishi, 1990).In C protein region a hexapeptide sequence of Asn-Met-Leu-Lys-Arg-Gly wasseen in all dengue viruses, similar to other mosquito-borne flaviviruses, except tick-borneencephalitis (TBE) virus (Haishi, 1990). Similar sequence was reported in some othernucleic acid-binding proteins (Heinkoff et al., 1983; Delcuve et al., 1980; Brauer and Rom-ing, 1979). On the other hand the least conserved region was AA No. 2325-2345 in NS4Aof dengue and other flaviviruses (Fig 5). Since this region appears to be type (or strain)specific, it would be a suitable sewuence for serotyping of viral isolates or assaying type-specific antibodies.84100>O)2oEoJC#40D2J/D2S D1 /D2S D2S/D3 D2S/D4 D1 /D4 D3/D4D1 /D3 D1 /D2J D2J/D3 D2J/D4Virus nameFig. 2. Graphic presentation of AA homology % of whole virus genome.Abbreviation of virus name is shown in text.D4Fig. 3. Three dimensional configuration of dengue virus homology Abbreviation ofgenome segment is shown in text.60->D)o 50-oEO 40-30-20-10-0-85Genome segmentFig. 4. Graphic presentation of AA average homology % of D2J, D2S, D3, D4 ineach 10 AA residues.Abbreviation of virus name is shown in text.AANoVIRUS 2325 2335 2345D2J TK KDLGLGNIATQQPESNILDI DLD2S TK KDLGLGSITTQESESNILDI DLD3 TK RDLGMSKEPGWSPTSYLDV DLD4 TK TDFGFYQVKTETTILDV DLWN TK NDIGSLLGHRPEARETTLGVESFLL DLJE TK ADLKSMFVGKTQASGLTGLPSMAL DLKUN TK SDISGLFGQRIETKENFSIGEFLL DLTBE TK ADLSTALWSEREEPRPWSEWTNY DIYF TK EDLFGKKNLIPSSASPWSWPDL DLFig. 5. The least conserved regions in flaviviruses.Abbreviation of virus name is shown in text and follow-ings: JE: Japanese encephalitis, KUN: Kunjin, TBE:tick-born encephalitis, WN: West Nile viruses, YF:yellow fever. AA names were shown by single lettercodes.ACKNOWLEDGMENTSWe would like to express thanks to Ms. K. Jodai for her helping in sequence dataprocessing. This study was supported by a Grant-in-Aid for Scientific Research No.62480161 from the Ministry of Education, Science and Culture of japan.86REFERENCES1 ) Brauer, J. F. & Roming, R. (1979): The primary structure of protein S3 from the small ribosomalsubunit of Escherichia coll FEES Letters, 106, 352-357.2) Clarke, D, H. & Casals, J. (1965): Arboviruses; group B. pp606-658. In F. L. Horsfall, Jr., I.Tamm(ed.). Viral and Rickettsial Infections of Man. J. B. Lippincott Co. Philadelphia, Montreal.3 ) Delcuve, G., Downing, W., Lewis, H. & Dennis, P. P. 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Comparative Amino Acid Sequences of Dengue Viruses

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Comparative Amino Acid Sequences of Dengue Viruses

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