Change in Japanese Encephalitis Virus Distribution,Thailand

Japanese encephalitis virus (JEV) genotypes in Thailand were studied in pigs and mosquitoes collected near houses of confirmed human JEV cases in 2003–2005. Twelve JEV strains isolated belonged to genotype I, which shows a switch from genotype III incidence that started during the 1980s.

T he origin of Japanese encephalitis virus (JEV) was recognized before 1935, and JEV was isolated in Japan in 1935. The virus has since spread from India to Indonesia and within the past 3 decades has reached previously unaffected parts of Asia and northern Australia (1,2). JEV is one of the most widespread causes of viral encephalitis worldwide; an estimated 3 billion persons are at risk for infection, and 10,000 to 15,000 die annually (3). Although most human infections are asymptomatic (1/1,000), 1/300 infections causes symptomatic infections, and 1/4 patients seeking treatment have symptoms of brain infl ammation, which can lead to permanent neurologic sequelae and a 1/4 death rate (4).
JEV is a fl avivirus transmitted by Culex mosquitoes to birds and pigs; humans are dead-end incidental hosts. On the basis of nucleotide sequencing of capsid/premembrane protein (C/PrM) and envelope (E) genes, 5 virus genotypes have been identifi ed, including genotypes I to III (GI, GII, GIII). These have been found distributed all over southern Asia; a GIV strain was isolated from eastern Indonesia, and an isolate originating in Malaysia may represent a fi fth genotype (5).
Three vaccines, derived from JEV GIII strains, are currently in use. Since the 1960s JEV immunization campaigns have dramatically reduced the effects of the disease in southern and Southeast Asia (6). In Thailand, JEV immunization began as a part of childhood vaccination program in the northern provinces in 1990; this program rapidly expanded to 36 provinces that had reported a persistent incidence of encephalitis (7).

The Study
To study the JEV genotype distribution in Thailand and to eventually detect changes in Japanese encephalitis epidemiologic patterns, we conducted a 3-year survey (2003)(2004)(2005)  Japanese encephalitis were targeted for sample collection. Ten healthy sentinel piglets (10 weeks of age) were surveyed in each province, and blood samples were collected weekly for 14 weeks. Adult mosquitoes were collected on a monthly basis according to the targeted pig farm and availability of breeding sites for vectors (Table 1; Figure 1) by using both the CDC gravid trap (Model 1712) and the CDC light trap (P. Reiter, Centers for Disease Control and Prevention, Fort Collins, CO, USA). Fifty microliters each from pig serum specimens and from fi ltered suspension of crushed mosquitoes were used for virus isolation on C6/36 cells. We tested JEV propagation by immunofl uorescent assay.  and 16 from other Asian countries. A phylogenetic tree was generated, and all 12 JEV new isolates fi t into the same GI cluster, as did 3 other Thai strains previously isolated in 1982, 1984, and 1992 ( Figure 2). Eleven of the newly identifi ed isolates formed a subcluster (Figure 2, GIa) with 2 other strains previously isolated from Chiang Mai and Khon Ken in 1984 and 1982, respectively (B2239NThailand, P19Br NThailand); the remaining new isolate (JE KK 1116NEThailand2005) was associated with another subcluster ( Figure 2, GIb), including strains isolated in 1992 from Chiang Mai (ThCMAr4492) and 3 others isolated from Vietnam, Japan, and Korea (O2VN22; JaNAr0102; and K94P05). Both subclusters were supported by 1,000 bootstrap replications and were consistent with the taxa distance (data not shown) showing introductions of GI in 1982 (within the GIa subcluster followed by a recent dispersion all over the country), and in 1992 for the GIb subcluster followed by local transmission. GI strains appeared to cluster phylogenetically but not geographically, which suggests virus strains were transported over noncontiguous domains at variable geographic distances. Major environmental changes have occurred since the early 1950s with the increase in local and international transportation systems. Some researchers (10) consider the increase of the virus incidence in the human population to be associated with increased commercial activity. However, because of the low level of viremia in humans, traditionally considered dead-end hosts for JEV, it is more likely that the virus was spread within the country and to neighboring countries by migratory birds, infected domestic pigs, or infected mosquitoes (or their eggs) (11,12).
Although GIII strains were historically reported to circulate mostly in northern Thailand in the early 1980s, GI and GIII were found co-circulating from the north to the south; thereafter, only the GI strain was isolated in Thailand (13). The same genotype shift of GIII to GI, dating back to the early 1990s, was reported by several other Asian countries, including Japan and Korea in 1991 and Vietnam in 2001 (14); a steady emergence and dispersion of GI was also noticed in China in 1979, in Taiwan in the 1980s (13), and in Australia in 2000 (2). Altogether, such unique endemic expansion of GI occurred over a 25-year period in several countries of Southeast Asia, replacing the GIII genotype, which was present all over the region since the beginning of the virus genotype identifi cation (prospectively and retrospectively).

Conclusions
In Thailand, the epidemiologic pattern of Japanese encephalitis fi rst showed a visible decline in incidence with the development of immunization programs, but this decline also corresponded to the late 1980s when the practice of raising pigs in the backyard evolved into industrialized pig farming and the high rate of piglet seroconversion showed an intense virus circulation. The dramatic increase of industrial pig farming and trading must have played a major role in the dispersion of JEV genotypes within past decades in Asia. Concurrently with pig farming, the culicid main vectors have changed (14) and such factors as their ecology, trophic preferences, host competence, and virus fi tness could play a role in an evolving rural environment. Moreover, further studies are needed to clarify the expansion of JEV GI strains, including the effi ciency of a human and pig GIII-derived vaccine and the role of potential crossimmunity between another circulating fl avivirus (13).