1 Department of Virology, National Institute of Hygiene and Epidemiology, 1 Yersin Street, Hanoi, Vietnam
2 Department of Virology, Institute of Tropical Medicine, Nagasaki University, Sakamoto 1-12-4, Nagasaki City 852-8523, Japan
3 Division of Epidemiology, Department of Infectious Diseases, Oita Medical University, Hasama-machi, Oita, Japan
4 Department of Vector Ecology and Environment, Institute of Tropical Medicine, Nagasaki University, Sakamoto 1-12-4, Nagasaki, Japan
5 Department of Epidemiology, Entomology Laboratory, National Institute of Hygiene and Epidemiology, 1 Yersin Street, Hanoi, Vietnam
6 CREST, Japan Science and Technology Corporation, Saitama 332-0012, Japan
Correspondence
Kouichi Morita
moritak{at}net.nagasaki-u.ac.jp
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ABSTRACT |
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These authors contributed equally to this work.
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MAIN TEXT |
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Phylogenetic analysis of JEV previously focused on the highly variable prM region, but available sequence data were of limited length (Chen et al., 1990, 1992
; Huong et al., 1993
; Ali & Igarashi, 1997
; Tsuchie et al., 1997
). Recently, the E gene was shown to be a good representative for JEV phylogenetic analysis (Ni & Barret, 1995
; Paranjpe & Banerjee, 1996
; Mangada & Takegami, 1999
; Williams et al., 2000
). To date, five genotypes have been described for JEV: genotype 1 (G1), including isolates from Southeast Asia, Australia, Korea and Japan; genotype 2 (G2), including isolates from Malaysia, Indonesia and Australia; genotype 3 (G3), including isolates from Southeast Asia, Japan, China, Korea, Taiwan and the Central Asia sub-continent; genotype 4 (G4), with isolates only from Indonesia; and genotype 5 (G5), with one isolate from Singapore (Hasegawa et al., 1994
; Williams et al., 2000
; Uchil & Satchidanandam, 2001
; Pyke et al., 2001
). The majority of JEV strains that have been studied belong to G3 (Williams et al., 2000
). Nucleotide sequence analysis of prM or E gene sequences revealed that JEV isolates are grouped in distinct clusters within each genotype, but no clear distinct geographical boundaries appear to exist between the different clusters (Ali & Igarashi, 1997
; Williams et al., 2000
; Holbrook & Barrett, 2002
).
In this study, the E protein gene region of nine JEV isolates from northern Vietnam and seven isolates from Japan was sequenced and compared with a large group of previously published JEV strains. Of nine Vietnamese isolates analysed, two were isolated in 1986 and two in 1989, all four from human brain tissue. One strain was isolated in 2001 from swine blood while the remaining four were isolated in 2002, one from swine blood and three from mosquito pools. Five of the Japanese strains were isolated from a mosquito pool in Nagasaki Prefecture, four in 1990 and one in 2002. Lastly, two strains were isolated from swine blood in 1995, in Oita Prefecture. The capsid-prM sequences of these two strains were published by Ma et al. (2003).
The viruses were propagated in C6/36 cells (Igarashi, 1978). Total RNA was extracted from infected culture fluid and RT-PCR was performed to amplify the complete E gene of JEV. The nucleotide sequences of the purified PCR products were determined using BigDye Terminator Cycle Sequencing reaction kits and ABI 310 Sequencer (Applied Biosystems). Multiple sequence alignments and phylogenetic analysis were done using CLUSTAL W version 1.83 (Thompson et al., 1994
). The phylogenetic tree was constructed by the neighbour-joining method (Saitou & Nei, 1987
) with bootstrap analysis of 1000 replicates and drawn using TreeView software (v.1.1.6) (Page, 1996
). A list of the JEV isolates used for analysis is shown in Table 1
, and a phylogenetic tree drawn from a total of 48 JEV isolates is presented in Fig. 1
. Fig. 2
shows the sampling sites in Vietnam and Japan.
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Data on molecular epidemiology of JEV in Vietnam are very limited. Huong et al. (1993) conducted a phylogenetic analysis on 16 JEV isolates from Northern and Southern Vietnam covering a time span of 25 years (19641988). In their study, all Vietnamese isolates examined through the capsid-prM region were closely related to the Japanese Nakayama isolate and the two Chinese strains Beijing-1 and SA-14, which belong to G3. In addition, Chen et al. (1990)
and Williams et al. (2000)
examined two isolates from the same country, Saigon (1962) and VN-118 (1979), and both strains were reported as belonging to G3. In this study, we report that Vietnamese JEV strains isolated between 1986 and 1989 clustered with isolates belonging to G3. However, all strains isolated in 2001 and 2002 in Vietnam belonged to G1. Together, these facts suggest that G3 had been the major epidemic JEV type in Vietnam, at least until 1989. Moreover, in this report we present for the first time the emergence and preponderance of JEV G1 in Vietnam, a previously undetected genotype in this country and probably introduced within the last decade.
Hori et al. (1986) examined JEV isolates from Japan and Thailand by oligonucleotide fingerprints and concluded that mutations and selections of the JEV genome would have progressed independently in these two geographical areas. Considering, on the one hand, that JEV G1 had probably been recently introduced into Vietnam as well as East Asia, and on the other hand, that Vietnamese G1 isolates clustered together in a distinct subcluster, phylogenetic analysis supports both long distance transfer of G1 and also local evolution in different geographical areas. Furthermore, temporal correlation with selection of the JEV genome was observed.
Japan and Korea have recently reported the introduction of JEV G1 (Takegami et al., 2000; Nam et al., 1996
; Ma et al., 2003
). Ma et al. (1996)
reported that JEV strains in Japan isolated from 1968 to 1992 belong to G3. Most recently, Ma et al. (2003)
stated that Japanese JEV strains, mostly from swine serum, isolated between 1965 and 1991 clustered within G3, but all strains isolated from 1994 to 2001 clustered within G1. Furthermore, Takegami et al. (2000)
reported that one Japanese JEV strain isolated in 1999 from mosquito belongs to G1, as does the Ishikawa strain isolated in 1994 from swine mononuclear cells. Finally, we report that Japanese JEV strains isolated in 1990 belonged to G3 whereas strains isolated in 1995 and 2002 belonged to G1. Recently, Solomon et al. (2003)
have suggested that the Southeast Asian region may be an important region for emerging pathogens. Our results strongly support the hypothesis that JEV G1 was introduced from Southeast Asia into Korea, Japan and Vietnam, probably in the early 1990s. Therefore, although there is no information on JEV molecular epidemiology in other Southeastern or East Asian countries, such as China, G1 might have emerged there as well.
JEV has spread greatly during the past few decades (Mackenzie et al., 2002). Although the reasons for JEV's spread to new territories are unknown, they are thought to be associated with bird migration, new irrigation projects and increasing animal husbandry (Innis, 1995
; Tsai, 1997
). An issue to consider is that active vaccination, using inactivated JEV of either Nakayama and Beijing-1 or JaGAr-01 serotypes, which belong to G3, has been used to control JE in Japan, China, Korea, Taiwan, India, Thailand, Russia and Vietnam. As a result of this, a reduction of human and swine JE was observed in Japan, China and Korea, changing its geographical distribution and epidemiology (Weaver et al., 1999
). Recently, in Japan and Korea, new strains have been isolated and they are closely related to Thai strains, which belong to G1 and have been shown to be genetically and antigenically different from those isolated in previous decades (Chung et al., 1996
; Ma et al., 1996
, 2003
; Nam et al., 1996
; Takegami et al., 2000
; Ali & Igarashi, 1997
). In this study, all Vietnamese strains isolated in the years 2001 and 2002 belong to this same genotype. Besides, animals involved in the JEV natural cycle in countries like Vietnam, Korea and Japan, where G3 was the only circulating JEV genotype until the late 1980s or early 1990s, must have become naturally immunized against this genotype. Therefore, this could somehow facilitate the introduction and spread of G1 in these countries.
To date, how and why JEV G1 expanded its area of distribution to more temperate regions such as Northern Vietnam and Eastern Asia within the last decade remains unknown. It is conceivable that wind-blown mosquitoes caught in air currents during the typhoon season may play a role (Mackenzie et al., 2001; Ritchie & Rochester, 2001
). It is known that Culex tritaeniorhynchus can disperse over long distances under natural conditions and can be carried for a very long distance by the wind, at least in some circumstances (Asahina & Noguchi, 1968
; Wada et al., 1969
; Asahina, 1970a
, b
; Ree et al., 1975
). Studies on dispersal of Culex tritaeniorhynchus reported that this mosquito could be captured over the Yellow Sea at a distance of 250 km from land, at 380 km southeast of the Lyaodong Peninsula (near the ChinaKorea border) and at 500 km south of Shionomisaki Cape in Japan (Asahina & Noguchi, 1968
; Asahina, 1970a
). Considering that south to north winds are common during summer in this region, a contribution of wind-blown mosquitoes to the dispersal of JEV G1 would be expected in the area comprising East Coastal ChinaKoreaJapan, (Asahina & Noguchi, 1968
). On the other hand, the presence of a significant number of infected mosquitoes in ships or airplanes between Southeast Asia and East Asia might also contribute (Laird, 1948
; Russell, 1987
; Reiter, 1998
). However, it is possible that JEV G1 acquired increased infectivity for birds, which in turn could influence the spread of the virus to new territories, according to their migratory pattern. This was also pointed out for West Nile virus (Lanciotti et al., 1999
). In the case of JEV, migrating birds are thought to be important in its dispersion to new geographical areas (Innis, 1995
; Solomon et al., 2003
). Vector studies conducted by Buescher et al. (1959)
on several ardeid birds inhabiting areas of Japan showed that ardeids were regularly and silently infected with JEV during the summer and that viraemia almost invariably followed avian infection. In addition, the virus titre and duration of viraemia they observed was sufficient to infect colonized Culex tritaeniorhynchus. Furthermore, every year, two to three million birds migrate back and forth along the East AsianAustralasian flyway (Fig. 2
) (WWF, 2000
; APMWCC, 2001
; Williams, 1994
). Birds migrate from the Malay Archipelago and Oceania, passing through Southeast Asia on to Taiwan. They then continue either into East China, Korea, Japan via Ryukyu Islands or they even proceed further northward (Fig. 2
) (China Birding, 2003
; Takano, 1984
). Therefore, spread by viraemic migratory birds may provide a sound explanation to the widening distribution of JEV genotypes. This is further supported by results from phylogenetic analysis in which, not only for G1 but also for G3, temporally proximal isolates either from Vietnam, Korea or Japan were closely related (Fig. 1
).
Until now, based on prM or E gene analysis, there are seven strains within G1 isolated from human brain (Chen et al., 1990; Williams et al., 2000
). They were all isolated between 1979 and 1984 in Thailand. Phylogenetic analysis shows they cluster with strain B-2239 (swine, 1984) from the same country and with an older strain from Cambodia (mosquito, 1967). This cluster, in our study containing only isolate Th2372 (1979), was clearly distinct from that containing G1 strains isolated during the 1990s, all from swine or mosquito, which included the isolates presented herein (Fig. 1
). Could G1 have lost its capacity to efficiently infect humans?
Further studies are needed to confirm the infectivity and pathogenecity of JEV G1 isolates, and their contribution to the spread of this genotype to new territories. We recommend continuous comprehensive surveys of JEV, precise evaluation of JEV isolates, particularly newly introduced genotypes, and monitoring of cross-protection of the virus against widely distributed vaccination.
Finally, our results on the spread of JEV to new territories may have potential implications for the future spread of West Nile virus (WNV); if WNV reaches Southeast Asia, bird migration might contribute to its rapid dispersal throughout East Asia.
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ACKNOWLEDGEMENTS |
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Received 12 November 2003;
accepted 21 January 2004.
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