熱帯医学 Tropical medicine 33(1,2). p15

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1 NAOSITE: Nagasaki University's Ac Title Author(s) Comparative Nucleotide and Amino Ac Encephalitis Virus Strains Isolated Tanaka, Mariko; Aira, Yoshikazu; Ig Citation 熱帯医学 Tropical medicine 33(1,2). p15 Issue Date URL Right This document is downloaded

2 Trop. Med., 33 (1,2), 15-21, June, Comparative Nucleotide and Amino Acid Sequences of Five Japanese Encephalitis Virus Strains Isolated in Japan and China Mariko TANAKA, Yoshikazu AIRA1 and Akira IGARASHI Department of Virology, Institute of Tropical Medicine, Nagasaki University, 12-4, Sakamoto-machi, Nagasaki 852, Japan Abstract: Homology among 5 strains of Japanese encephalitis (JE) virus isolated in Japan and China between 1935 and 1982 was compared by nucleotide sequence of genomic RNAs and deduced amino acid sequences of viral proteins. Both homologies were significantly high (more than 93%) all over the entire genome. The result indicated relative homogeneity of JE virus genome and the homology percentage over 93% as an indicator for the same viral species. Key words: Japanese encephalitis virus, Nucleotide sequence, Amino acid sequence, Homology INTR ODUCTION Japanese encephalitis (JE) virus is a member of the Family Flaviviridae (Westaway et al, 1985) which contains more than 60 members of animal viruses. By classical serology of cross-neutralization using polyclonal antisera, JE virus belonged to one of the 8 subgroups or complexes of flaviviruses, together with West Nile, Murray Valley encephalitis and St. Louis encephalitis viruses (Porterfield, 1980; de Madrid and Porter field, 1974; Calisher et at., 1989). In nature, JE virus is transmitted among susceptible vertebrates by Culex tritaeniorhynchm or related mosquito species whch breed in watered rice fields. Swine and wild birds have been documented as amplifier vertebrate hosts with sufficient viremia to infect vector mosquitoes (Mitamura et al., 1938; Buescher et al, 1959; Scherer et al., 1959a, b). Although JE virus was named by its first isolation in Japan in 1935, the virus and its disease are known to present in wider Asia monsoon area (Miles, 1960). Recent JE epidemics in developing Asian countries have become a great public health problem requring proper control measures (Umenai et al., 1985). Classical serology by the hemagglutination-inhibition or neutralization test with antibody absorption showed at least two different subtypes among JE virus strains (Okuno et Received for Publication, May 22, 1991 Contribution No from the Institute of Tropical Medicine, Nagasaki University. 1 Present address: Department of Parasitology, Aichi Medical College, 21 Karimata, Aza Iwasa, Nagakute-cho, Aichigun, Aichi 480- ll, Japan

3 16 al, 1968; Oda, 1976), which were later confirmed by the cross-neutralization using immune mouse sera (Oya, 1987). Antigenic difference among JE virus strains was further identified by the hemagglutination-inhibition test using monoclonal antibodies (Kobayashi et al., 1984, 1985). Genomic analysis by oligonucleotide fingerprinting showed significant difference among JE virus isolates in geographically distant areas (Hori et al., 1986; Hori, 1986). Relationship among JE virus isolates was recently analyzed by nucleotide sequencing of PreM gene region of viral RNA (Chen et al, 1990). In this report, homology among 5 strains of JE virus isolated in Japan and China between 1935 and 1982 was compared by nucleotide sequence of genomic RNAs and deduced amino acid sequences, in order to get the idea on the degree of strain difference as shown by this parameter. The data was required also for the viral genome detection by polymerase-chain reaction. MATERIALS AND METHODS Sources of the nucleotide and deduced amino acid sequence informations: JE virus Sequence data were obtained from following publications with strain name abbreviation, year, place and source of isolation: JaOArS982 (S9-82, 1982, Osaka, Japan, C. tritaeniorhynchus: Sumiyosh et al, 1987); JaOH0566 (0566, 1966, Osaka, Japan, human brain: Tanaka et al, 1988); Beijing-1 (Beij, 1947, Beijing, China, human brain: Hashimoto et al, 1988); SA14 (SA14, 1954, Si An, China, C. tritaeniorhynchus: Aihara et al, 1991); Nakayama (NAKA, 1935, Tokyo, human brain: McAda et al, 1987) Comparison of nucleotide and amino acid sequences: Genomic RNA sequences were divided into each gene according to Rice et al (1985), excepting ns2a and ns2b genes which were divided as NS2A and NS2B according to Speight et al (1988). Since all JE virus strains possessed the same numbers of nucleotide in each gene, their nucleotide and amino acid sequences were compared without alignment. The comparison was carried out by the Hitachi DNASIS Version 6.0 software system and homology was was presented as simple percentage. RESULTS Comparative nucleotide and amino acid sequences of 5 JE virus strains: Results on the coding region were shown in Figs. 1-3, in which nueleotide sequence homology was shown in left lower falf and amino acid homology in right-upper half, respectively. Nucleotide and amino acid homology of the structural and nonstructural protein gene as well as nucleotide sequence homology of the 5'- and 3'-noncoding regions were shown infig.4. In general, high percentage homologies over 93% were observed for both nucleotide and amino acid sequences in all-over the entire genome region, both in coding and noncoding regions.

4 17 C protein PrM protein Mprotein E protein Fig. 1. Nucleotide and amino acid sequence homology of C, PrM, M, and E proteins Figures in the left lower half show nucleotide and in the right upper half amino acid sequence homology, respectively. NS1 protein NS2A protein Fig. 2. Nucleotide and amino acid sequence homology of NS1, NS2A, NS2B, and NS3 proteins Figures in the left lower half show nucleotide and in the right upper half amino acid sequence homology, respectively.

5 18 NS4A protein NS4B protein S Beij SA14 NAKA S BeiJ SA14 NAKA S S qq yy-o o Beij Beij SA SAM NAKA NAKA NS5 protein Fig. 3. Nucleotide and ammoacid sequence homology of NS4A, NS4B, and NS5 proteins Figures in the left lower half show nucleotide and in the right upper half amino acid sequence homology, respectively. Structural protein Non structural protein Fig. 4. Nucleotide sequence homology of 5'- 3'-noncoding regions and amino acid sequence homology of structural and nonstructural proteins. \ Figures in the left lower half show nucleotide and in the right upper half; amino acid sequence homology, respectively.

6 19 DISCUSSION High degree of homology above 93% observed in this study may be related with the fact that all these 5 strains were isolated in Japan and China. Previous oligonucleotide fingerprint analysis showed that the strains from the same or nearest geographical areas were relatively similar each other, while those from remote areas were different (Hori et al, 1986; Hori, 1986). Similarity ration of the specific long Oliogonucleotides was even amongjapanese isolates, while the ratio between a Chines strain, Beijing- 1, and Japanese strains was Further studies will be required to clarify the difference between the similarity ratio and the nucleotide or amino acid percent homology. Sequence data on 240 nucleotides in the PreM region of 46 JE virus strains classified them into 3 genotypes, one from northern Thailand and Cambodia, a second from southern Thailand, Malaysia, Sarawak and Indonesia, and the remainder in the thrid group (Chen et al., 1990). By their calculation, percentage homology between JE virus strains ranged from 2 to 16%, and the difference between a Japanese strain, JaOArS982, was 2-4.6% for 6 other Japanese strains and 2-3.3% for 3 Chines strains. These figures agree with our present calculation. Homology between 2 dengue type 2 virus strains were 80-96% by our previous report (Haishi et al, 1990). Present result provides sound bases to the viral genome detection by polymerase-chain reaction using universal primers to amplify JE virus genome of different strains. Further studies will be required to substantiate strain difference among JE virus isolates from geographically different areas. ACKNOWLEDGMENTS The study was supported by a Grant-in Aid for Scientific Research No from the Ministry of Education, Science and Culture of Japan, and also by a Grant-in Aid for nona-nonbhepatitis from the Minsitry of Health and Welfare of Japan. Excellent technical help by Ms. C. Uchida and Ms. K. Jodai is appreciated. REFERENCE S 1 ) Aihara, S., Chunming, R., Yu, Y-X., Lee, T., Watanabe, K., Kamiya, T., Sumiyoshi, H., Hashimoto, H. & Nomoto, A. (1991): Identification of mutation that occurred on the genome of Japanese encephalitis virus during the attenuation process. Virus Genes, 5 (in press). 2) Buescher, E. L., Scherer, W. F., Rosenberg, M. Z., Gresser, I., Hardy, J. L. & Bullock, H. R. (1959): Ecologic studies of Japanese encephalitis virus in Japan. II. Mosquito infection. Am. J. Trop. Med. Hyg., 8, ) Calisher, C. H., Karabatsos, N., Dalrymple, J. M., Shope, R. E., Porterfield, J. S., Westawya, E. G. & Brandt, W. (1989): Antigenk relationships between flaviviruses as determined by cross-neutralization tests with polyclonal antisera. J. Gen. Virol., 70,

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