• View in gallery

    Locations of Tokyo, Oita, Ishikawa, and Osaka in Japan. Twenty three strains of Japanese encephalitis virus analyzed in this study were isolated in Tokyo and Oita. The Ishikawa and JaOArK36-91 strains mentioned in this report were isolated in Ishikawa11 and Osaka, respectively.

  • View in gallery

    Phylogenetic relationship of 30 strains of Japanese encephalitis virus (JEV) predicted from capsid/pre-membrane protein gene sequences. Phylogenetic groupings corresponding to the genotyping classification of Chen and others4,5 and Ali and Igarashi14 are indicated (GI to GIV). The tree was constructed by use of the neighbor-joining method in ClustalW.16 The scale indicates the number of nucleotide substitutions per site. Horizontal branch lengths are proportional to genetic distance, and vertical branch lengths have no significance. The numbers at each branch represent the percentage of 1,000 bootstrap replicate supports. The tree was rooted by use of sequence information of Murray Valley encephalitis (MVE) virus, a member of the JEV serocomplex.

  • View in gallery

    Phylogenetic relationship of the 22 strains of Japanese encephalitis virus (JEV) with selected JEV isolates predicted from capsid/pre-membrane protein gene sequences. MVE = Murray Valley encephalitis. For details of the trees, see Figure 2.

  • 1

    Lindenbach BD, Rice CM, 2001. Flaviviridae: The virus and their replication. Knipe DM, Howley PM, Griffin DE, Martin MA, Lamb RA, Roizman B, Straus SE, eds. Virology. Philadelphia: Lippincott Williams and Wilkins, 991–1125.

  • 2

    Hori H, Morita K, Igarashi A, 1986. Oligonucleotide fingerprint analysis on Japanese encephalitis virus strains isolated in Japan and Thailand. Acta Virol 30 :353–359.

    • Search Google Scholar
    • Export Citation
  • 3

    Banerjee K, Ranadive SN, 1989. Oligonucleotiede fingerprint analysis of Japanese encephalitis virus strains of different geographical origin. Indian J Med Res 89 :201–216.

    • Search Google Scholar
    • Export Citation
  • 4

    Chen WR, Tesh RB, Rico-Hesse R, 1990. Genetic variation of Japanese encephalitis virus in nature. J Gen Virol 71 :2915–2922.

  • 5

    Chen WR, Rico-Hesse R, Tesh RB, 1992. A new genotype of Japanese encephalitis virus from Indonesia. Am J Trop Med Hyg 47 :61–69.

  • 6

    Huong VTQ, Ha DQ, Deubel V, 1993. Genetic study of Japanese encephalitis viruses from Vietnam. Am J Trop Med Hyg 49 :538–544.

  • 7

    Chung YJ, Nam JH, Ban SJ, Cho HW, 1996. Antigenic and genetic analysis of Japanese encephalitis virus isolated from Korea. Am J Trop Med Hyg 55 :91–97.

    • Search Google Scholar
    • Export Citation
  • 8

    Ma SP, Arakaki S, Makino Y, Fukunaga T, 1996. Molecular epidemiology of Japanese encephalitis virus in Okinawa. Microbiol Immunol 40 :847–855.

    • Search Google Scholar
    • Export Citation
  • 9

    Tsuchie H, Oda K, Vythilingam I, Thayan R, Vijayamalar B, Sinniah M, Wada T, Tanaka H, Kurimura T, Igarashi A, 1997. Genotypes of Japanese encephalitis virus isolates in three states in Malaysia. Am J Trop Med Hyg 56 :153–158.

    • Search Google Scholar
    • Export Citation
  • 10

    Jan LR, Yueh YY, Wu YC, Horng CB, Wang GR, 2000. Genetic variation of Japanese encephalitis virus in Taiwan. Am J Trop Med Hyg 62 :446–452.

    • Search Google Scholar
    • Export Citation
  • 11

    Takegami T, Ishak H, Miyamoto C, Hirai Y, Kamimura K, 2000. Isolation and molecular comparison of Japanese encephalitis virus in Ishikawa, Japan. Jpn J Infect Dis 53 :178–179.

    • Search Google Scholar
    • Export Citation
  • 12

    Nam JH, Chae SL, Won SY, Kim EJ, Yoon KS, Kim BI, Jeong YS, Cho HW, 2001. Genetic heterogeneity of Japanese encephalitis virus assessed via analysis of the full-length genome sequence of a Korean isolate. Am J Trop Med Hyg 65 :388–392.

    • Search Google Scholar
    • Export Citation
  • 13

    Pyke AT, Williams DT, Nisbet DJ, Den Hurk AF, Taylor CT, Johansen CA, Macdonald J, Hall RA, Simmons RJ, Mason RJV, Lee JM, Ritchie SA, Smith GA, Mackenzie JS, 2001. The appearance of a second genotype of Japanese encephalitis virus in the Australasian region. Am J Trop Med Hyg 65 :747–753.

    • Search Google Scholar
    • Export Citation
  • 14

    Ali A, Igarashi A, 1997. Antigenic and genetic variations among Japanese encephalitis virus strains belonging to genotype 1. Microbiol Immunol 41 :241–252.

    • Search Google Scholar
    • Export Citation
  • 15

    Saitou N, Nei M, 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4 :406–425.

  • 16

    Thompson JD, Higgins DG, Gibson TJ, 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res 22 :4673–4680.

    • Search Google Scholar
    • Export Citation
  • 17

    Page RDM, 1996. Treeview: an application to display phylogenetic trees on personal computers. Comp Appl Biosci 12 :357–358.

  • 18

    Igarashi A, 2002. Control of Japanese encephalitis in Japan: immunization of humans and animals, and vector control. Curr Top Microbiol Immunol 267 :139–152.

    • Search Google Scholar
    • Export Citation
  • 19

    Matsunaga Y, Yabe S, Taniguchi K, Nakayama M, Kurane I, 1999. Current status of Japanese encephalitis in Japan. Kansenshogaku Zasshi 73 :97–103.

    • Search Google Scholar
    • Export Citation

 

 

 

 

SHORT REPORT: A MAJOR GENOTYPE OF JAPANESE ENCEPHALITIS VIRUS CURRENTLY CIRCULATING IN JAPAN

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  • 1 Division of Epidemiology, Department of Infectious Diseases, Oita Medical University, Oita, Japan; Department of Microbiology, Tokyo Metropolitan Research Laboratory of Public Health, Tokyo, Japan; Department of Virology, Faculty of Medicine, University of the Ryukyus, Okinawa, Japan; Oita Prefectural Institute of Health and Environment, Oita, Japan

A 240-nucleotide sequence of the capsid/premembrane gene region of 23 Japanese encephalitis virus (JEV) strains isolated in Tokyo and Oita, Japan was determined and phylogenetic analyses were performed. All the strains clustered into two distinct genotypes (III and I). All strains isolated before 1991 belonged to genotype III, while those isolated after 1994 belonged to genotype I. In addition, the strains of the genotype I isolated in Japan showed a close genetic relationship with those from Korea and Malaysia.

Japanese encephalitis virus (JEV) is a member of genus Flavivirus, family Flaviviridae. This virus is widely distributed in Asia, including Japan.1 The geographic variation of JEV strains was demonstrated by the studies of RNA fingerprints2,3 and nucleotide sequencing.4–10 The genomic diversity of JEV has been determined by primer extension sequencing of the capsid/pre-membrane (C/prM) gene region.4,5 These analyses indicated that JEV strains could be classified into four geographically distinct genotypes based on the nucleotide differences of more than 12%. Genotype I (GI) consists of the strains from northern Thailand and Cambodia; genotype II (GII) from Indonesia (Sarawak and Java), Malaysia, and southern Thailand; genotype III (GIII) from a broad region encompassing northern Asia (Japan, China, and Taiwan), Vietnam, Nepal, India, and Sri Lanka; and genotype IV (GIV) from eastern Indonesia (Java, Bali, and Flores). However, this classification was determined using JEV strains isolated from 1935 to 1987. In the 1990s, JEV strains showing a discrepancy with the proposed geographic distribution were reported.9,11–13 We also found one JEV strain, JaOArK36-91, which was isolated in Osaka, Japan in 1991, to be a GI strain. It was not clear whether this isolate was one of the minor genotypes of native strains that was isolated by chance or was introduced from other location and became a major genotype in this area. In this paper, we report that the major genotype of JEV circulating in a wide area of Japan had changed from GIII to GI since 1994.

The viruses used in this study are listed in Table 1. A total of 23 JEV strains, 14 from Tokyo and 9 from Oita, were analyzed in this study. Oita is located approximately 800 km southwest of Tokyo (Figure 1). Among these 23 strains, 22 strains were isolated from swine sera, and one (JaTAr1/75) was isolated from a mosquito pool. The RNA of JEV-infected C6/36 cells was amplified by a reverse transcriptase-polymerase chain reaction with a pair of primers (JE317 and JE821R),14 followed by sequencing as previously described.8 Phylogenetic analyses were then performed on sequence information for a 240-nucleotide region of the C/prM gene.4,5 A phylogenetic tree was constructed by the neighbor-joining (NJ) method of Saitou and Nei.15 The Murray Valley encephalitis virus genome (NC_000943) was used as an out-group in all analyses. ClustalW16 was used for the construction of the NJ tree. All trees were constructed by the bootstrap analysis of 1,000 replicates. The tree was drawn using TreeView software.17 The nucleotide sequence data in this paper will be found in the DDBJ/EMBL/GenBank nucleotide sequence databases with the access numbers AB099851-AB099874.

The results of phylogenetic analyses of 23 JEV strains isolated both in Tokyo and Oita with seven reference strains representing four genotypes are shown in Figure 2. Strains from Tokyo and Oita formed two distinct genetic groups. One group was composed of the strains isolated before 1991 (GIII). The other group was composed of the strains isolated after 1994 (GI). These strains clustered based on the year of isolation, rather than the location. The genetic distance between these two groups was 15.1% by the unweighted pair group method using arithmetic average, which satisfied the original genotype criteria.

To evaluate the genetic relationship of the recent Japanese GI strains, 11 GI strains from Japan were compared with four GI strains isolated in Korea, Malaysia, and Thailand in the 1990s. One GIII strain (JaOArS982) was added as a reference. All 11 Japanese GI strains, the Korean strain, and the Malaysian strain showed a close genetic relationship with the Thailand strains (Figure 3).

Our results clearly show that GIII was the major genotype of JEV, circulating in Tokyo and Oita until 1991, but it has since changed to GI. It has been reported that one GI strain was isolated in 1994 in Ishikawa, which is located approximately 300 km west of Tokyo, whereas, two GIII strains were isolated in 1998 in the same area.11 Thus, the swift change of the genotype during this period appears to be a common phenomenon in a wide area of Japan. It is noteworthy that one GI strain was also isolated in 1994 in Korea,12 whereas GIII strains continued to be isolated in Taiwan and constituted a major genotype on this island.10 The reason why GIII has been replaced by GI since the mid 1990s in Japan has not been determined. The annual number of human cases of Japanese encephalitis has remained less than 10 since 1992.11,18,19 However, during the summer, the positivity rate of antibody to JEV in swine sera has been shown to increase to more than 50% in many parts of Japan.19 To our knowledge, there is no report of clinical changes in these animals before and after the middle 1990s. Further studies are needed to determine to what extent the GI strain of JEV has expanded in Japan.

Table 1

Strains of Japanese encephalitis virus and Murray Valley encephalitis (MVE) virus used in this study

StrainYearLocationSource*GenotypeAccession number
* CSF = cerebrospinal fluid.
† MVE virus was used as an outgroup in all phylogenetic analyses.
Nakayama1935Tokyo, JapanHuman CSFIIIS75726
MVE-1-511951AustraliaHuman brainNC_000943
JaGAr#011959Gunma, JapanCulex tritaeniorhynchusIIID00961
JaTAn1/651965Tokyo, JapanSwine serumIIIAB099851
M-8591967CambodiaCulex gelidusID00984
JaTAn1/721972Tokyo, JapanSwine serumIIIAB099852
Th23721972ThailandHuman brainID76424
JaTAn1/751975Tokyo, JapanSwine serumIIIAB099853
JaTAr1/751975Tokyo, JapanMosquito poolIIIAB099854
80P1361980Oita, JapanSwine serumIIIAB099855
JaTAn1/801980Tokyo, JapanSwine serumIIIAB099856
81P2411981Oita, JapanSwine serumIIIAB099857
81P2441981Oita, JapanSwine serumIIIAB099858
JKT84421980IndonesiaCulex tritaeniorhynchusIVL42159
JaOArS9821982Osaka, JapanMosquito poolIIINC_001437
B-22391984ThailandSwine bloodID00993
JaTAn1/841981Tokyo, JapanSwine serumIIIAB099859
B-25821985ThailandSwine bloodID00985
JaTAn1/871987Tokyo, JapanSwine serumIIIAB099860
89C491989Oita, JapanSwine serumIIIAB099861
89P1411989Oita, JapanSwine serumIIIAB099862
JaTAn1/901990Tokyo, JapanSwine serumIIIAB099863
JaOArK36-911991Osaka, JapanMosquito poolIAB099864
JaTAn1/911991Tokyo, JapanSwine serumIIIAB099865
JaTAn2/911991Tokyo, JapanSwine serumIIIAB099866
ThCMAr44921992ThailandCulex tritaeniorhynchusID45360
ThCMAr67931993ThailandCulex tritaeniorhynchusID45361
Ishikawa1994Ishikawa, JapanSwine mononuclear cellsIAB051292
JaTAn1/941994Tokyo, JapanSwine serumIAB099867
JaTAn2/941994Tokyo, JapanSwine serumIAB099868
K94P051994KoreaCulex tritaeniorhynchusIAF045551
MAPAV2941994MalaysiaSwine bloodID83922
MaSAr019941994MalaysiaCulex tritaeniorhynchusIID83940
95-911995Oita, JapanSwine serumIAB099869
95-1671995Oita, JapanSwine serumIAB099870
97-821997Oita, JapanSwine serumIAB099871
JaTAn1/981998Tokyo, JapanSwine serumIAB099872
10-1731998Chiba, JapanPorcine tonsilIAF226682
JaTAn1/991999Tokyo, JapanSwine serumIAB099873
TS002000AustraliaSwine serumIAF289814
TS41522000AustraliaMosquitoIAF318290
2001-232001Oita, JapanSwine serumIAB099874
Figure 1.
Figure 1.

Locations of Tokyo, Oita, Ishikawa, and Osaka in Japan. Twenty three strains of Japanese encephalitis virus analyzed in this study were isolated in Tokyo and Oita. The Ishikawa and JaOArK36-91 strains mentioned in this report were isolated in Ishikawa11 and Osaka, respectively.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 2; 10.4269/ajtmh.2003.69.151

Figure 2.
Figure 2.

Phylogenetic relationship of 30 strains of Japanese encephalitis virus (JEV) predicted from capsid/pre-membrane protein gene sequences. Phylogenetic groupings corresponding to the genotyping classification of Chen and others4,5 and Ali and Igarashi14 are indicated (GI to GIV). The tree was constructed by use of the neighbor-joining method in ClustalW.16 The scale indicates the number of nucleotide substitutions per site. Horizontal branch lengths are proportional to genetic distance, and vertical branch lengths have no significance. The numbers at each branch represent the percentage of 1,000 bootstrap replicate supports. The tree was rooted by use of sequence information of Murray Valley encephalitis (MVE) virus, a member of the JEV serocomplex.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 2; 10.4269/ajtmh.2003.69.151

Figure 3.
Figure 3.

Phylogenetic relationship of the 22 strains of Japanese encephalitis virus (JEV) with selected JEV isolates predicted from capsid/pre-membrane protein gene sequences. MVE = Murray Valley encephalitis. For details of the trees, see Figure 2.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 2; 10.4269/ajtmh.2003.69.151

Authors’ addresses: Shao-Ping Ma and Yoshihiro Makino, Division of Epidemiology, Department of Infectious Diseases, Oita Medical University, Hasamamachi, Oita 879-5593, Japan. Yasuko Yoshida, Department of Microbiology, Tokyo Metropolitan Research Laboratory of Public Health, 3-24-1, Hyakunincho, Shinjukuku, Tokyo 169-0073, Japan. Masayuki Tadano, Department of Virology, Faculty of Medicine, University of the Ryukyus. 207 Uehara, Nishihara, Okinawa 903-0215, Japan. Testuro Ono and Masao Ogawa, Oita Prefectural Institute of Health and Environment, 2-51, Hougawaradai, Oita, Oita 870-0948 Japan.

Acknowledgment: We thank Dr. Yoshinobu Okuno (Osaka Prefectural Institute of Public Health, Osaka, Japan) for providing one isolate of Japanese encephalitis virus (JaOArK36-91).

REFERENCES

  • 1

    Lindenbach BD, Rice CM, 2001. Flaviviridae: The virus and their replication. Knipe DM, Howley PM, Griffin DE, Martin MA, Lamb RA, Roizman B, Straus SE, eds. Virology. Philadelphia: Lippincott Williams and Wilkins, 991–1125.

  • 2

    Hori H, Morita K, Igarashi A, 1986. Oligonucleotide fingerprint analysis on Japanese encephalitis virus strains isolated in Japan and Thailand. Acta Virol 30 :353–359.

    • Search Google Scholar
    • Export Citation
  • 3

    Banerjee K, Ranadive SN, 1989. Oligonucleotiede fingerprint analysis of Japanese encephalitis virus strains of different geographical origin. Indian J Med Res 89 :201–216.

    • Search Google Scholar
    • Export Citation
  • 4

    Chen WR, Tesh RB, Rico-Hesse R, 1990. Genetic variation of Japanese encephalitis virus in nature. J Gen Virol 71 :2915–2922.

  • 5

    Chen WR, Rico-Hesse R, Tesh RB, 1992. A new genotype of Japanese encephalitis virus from Indonesia. Am J Trop Med Hyg 47 :61–69.

  • 6

    Huong VTQ, Ha DQ, Deubel V, 1993. Genetic study of Japanese encephalitis viruses from Vietnam. Am J Trop Med Hyg 49 :538–544.

  • 7

    Chung YJ, Nam JH, Ban SJ, Cho HW, 1996. Antigenic and genetic analysis of Japanese encephalitis virus isolated from Korea. Am J Trop Med Hyg 55 :91–97.

    • Search Google Scholar
    • Export Citation
  • 8

    Ma SP, Arakaki S, Makino Y, Fukunaga T, 1996. Molecular epidemiology of Japanese encephalitis virus in Okinawa. Microbiol Immunol 40 :847–855.

    • Search Google Scholar
    • Export Citation
  • 9

    Tsuchie H, Oda K, Vythilingam I, Thayan R, Vijayamalar B, Sinniah M, Wada T, Tanaka H, Kurimura T, Igarashi A, 1997. Genotypes of Japanese encephalitis virus isolates in three states in Malaysia. Am J Trop Med Hyg 56 :153–158.

    • Search Google Scholar
    • Export Citation
  • 10

    Jan LR, Yueh YY, Wu YC, Horng CB, Wang GR, 2000. Genetic variation of Japanese encephalitis virus in Taiwan. Am J Trop Med Hyg 62 :446–452.

    • Search Google Scholar
    • Export Citation
  • 11

    Takegami T, Ishak H, Miyamoto C, Hirai Y, Kamimura K, 2000. Isolation and molecular comparison of Japanese encephalitis virus in Ishikawa, Japan. Jpn J Infect Dis 53 :178–179.

    • Search Google Scholar
    • Export Citation
  • 12

    Nam JH, Chae SL, Won SY, Kim EJ, Yoon KS, Kim BI, Jeong YS, Cho HW, 2001. Genetic heterogeneity of Japanese encephalitis virus assessed via analysis of the full-length genome sequence of a Korean isolate. Am J Trop Med Hyg 65 :388–392.

    • Search Google Scholar
    • Export Citation
  • 13

    Pyke AT, Williams DT, Nisbet DJ, Den Hurk AF, Taylor CT, Johansen CA, Macdonald J, Hall RA, Simmons RJ, Mason RJV, Lee JM, Ritchie SA, Smith GA, Mackenzie JS, 2001. The appearance of a second genotype of Japanese encephalitis virus in the Australasian region. Am J Trop Med Hyg 65 :747–753.

    • Search Google Scholar
    • Export Citation
  • 14

    Ali A, Igarashi A, 1997. Antigenic and genetic variations among Japanese encephalitis virus strains belonging to genotype 1. Microbiol Immunol 41 :241–252.

    • Search Google Scholar
    • Export Citation
  • 15

    Saitou N, Nei M, 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4 :406–425.

  • 16

    Thompson JD, Higgins DG, Gibson TJ, 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res 22 :4673–4680.

    • Search Google Scholar
    • Export Citation
  • 17

    Page RDM, 1996. Treeview: an application to display phylogenetic trees on personal computers. Comp Appl Biosci 12 :357–358.

  • 18

    Igarashi A, 2002. Control of Japanese encephalitis in Japan: immunization of humans and animals, and vector control. Curr Top Microbiol Immunol 267 :139–152.

    • Search Google Scholar
    • Export Citation
  • 19

    Matsunaga Y, Yabe S, Taniguchi K, Nakayama M, Kurane I, 1999. Current status of Japanese encephalitis in Japan. Kansenshogaku Zasshi 73 :97–103.

    • Search Google Scholar
    • Export Citation

Author Notes

Reprint requests: Shao-Ping Ma, Division of Epidemiology, Department of Infectious Diseases, Oita Medical University, Hasamamachi, Oita 879-5593, Japan, Telephone: 81-97-586-5735, Fax: 81-97-586-5739, E-mail: masp@oita-med.ac.jp.
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