Sero-Molecular Epidemiology of Japanese Encephalitis in Zhejiang,an Eastern Province of China

BackgroundSporadic Japanese encephalitis (JE) cases still have been reported in Zhejiang Province in recent years, and concerns about vaccine cross-protection and population-level immunity have been raised off and on within the public health sphere. Genotype I (GI) has replaced GIII as the dominant genotype in Asian countries during the past few decades, which caused considerable concerns about the potential change of epidemiology characteristics and the vaccine effectiveness. The aim of this study was to investigate the prevalence of JE neutralizing antibody and its waning antibody trend after live attenuated JE vaccine immunization. Additionally, this study analyzed the molecular characteristics of the E gene of Zhejiang Japanese encephalitis virus (JEV) strains, and established genetic relationships with other JEV strains.Conclusion/SignificancesJE neutralizing antibody positive rates increase in age ≥10 years old population, likely reflecting natural infection or natural boosting of immunity through exposure to wild virus. JE seropositivity rates were quite low in <35 years old age groups in Zhejiang Province. Waning of neutralizing antibody after live attenuated vaccine immunization was observed, but the clinical significance should be further investigated. Both the peripheral antibody response and genetic characterization indicate that current live attenuated JE vaccine conferred equal neutralizing potency against GI or GIII of wild strains. GI has replaced GIII as the dominant genotype in Zhejiang in the past few decades. Although the chance of exposure to wild JE virus has reduced, the virus still circulates in nature; therefore, it is necessary to implement immunization program for children continually and to conduct surveillance activity periodically.     

Ecological niche modeling to estimate the distribution of Japanese encephalitis virus in Asia

Background

Culex tritaeniorhynchus is the primary vector of Japanese encephalitis virus (JEV), a leading cause of encephalitis in Asia. JEV is transmitted in an enzootic cycle involving large wading birds as the reservoirs and swine as amplifying hosts. The development of a JEV vaccine reduced the number of JE cases in regions with comprehensive childhood vaccination programs, such as in Japan and the Republic of Korea. However, the lack of vaccine programs or insufficient coverage of populations in other endemic countries leaves many people susceptible to JEV. The aim of this study was to predict the distribution of Culex tritaeniorhynchus using ecological niche modeling.

Methods/Principal Findings

An ecological niche model was constructed using the Maxent program to map the areas with suitable environmental conditions for the Cx. tritaeniorhynchus vector. Program input consisted of environmental data (temperature, elevation, rainfall) and known locations of vector presence resulting from an extensive literature search and records from MosquitoMap. The statistically significant Maxent model of the estimated probability of Cx. tritaeniorhynchus presence showed that the mean temperatures of the wettest quarter had the greatest impact on the model. Further, the majority of human Japanese encephalitis (JE) cases were located in regions with higher estimated probability of Cx. tritaeniorhynchus presence.

Conclusions/Significance

Our ecological niche model of the estimated probability of Cx. tritaeniorhynchus presence provides a framework for better allocation of vector control resources, particularly in locations where JEV vaccinations are unavailable. Furthermore, this model provides estimates of vector probability that could improve vector surveillance programs and JE control efforts.     

Genotype v Japanese encephalitis virus is emerging

Japanese encephalitis (JE) is a global public health issue that has spread widely to more than 20 countries in Asia and has extended its geographic range to the south Pacific region including Australia. JE has become the most important cause of viral encephalitis in the world. Japanese encephalitis viruses (JEV) are divided into five genotypes, based on the nucleotide sequence of the envelope (E) gene. The Muar strain, isolated from patient in Malaya in 1952, is the sole example of genotype V JEV. Here, the XZ0934 strain of JEV was isolated from Culex tritaeniorhynchus, collected in China. The complete nucleotide and amino acid sequence of XZ0934 strain have been determined. The nucleotide divergence ranged from 20.3% to 21.4% and amino acid divergence ranged from 8.4% to 10.0% when compared with the 62 known JEV isolates that belong to genotype I-IV. It reveals low similarity between XZ0934 and genotype I-IV JEVs. Phylogenetic analysis using both complete genome and structural gene nucleotide sequences demonstrates that XZ0934 belongs to genotype V. This, in turn, suggests that genotype V JEV is emerging in JEV endemic areas. Thus, increased surveillance and diagnosis of viral encephalitis caused by genotype V JEV is an issue of great concern to nations in which JEV is endemic.     

Minocycline neuroprotects, reduces microglial activation, inhibits caspase 3 induction, and viral replication following Japanese encephalitis

Minocycline is broadly protective in neurological disease models featuring inflammation and cell death and is being evaluated in clinical trials. Japanese encephalitis virus (JEV) is one of the most important causes of viral encephalitis worldwide. There is no specific treatment for Japanese encephalitis (JE) and no effective antiviral drugs have been discovered. Studies indicate that JE involves profound neuronal loss as well as secondary inflammation caused because of cell death. Minocycline is a semisynthetic second-generation tetracycline that exerts anti-inflammatory and antiapoptotic effects that are completely separate from its antimicrobial action. Because tetracycline treatment is clinically well tolerated, we investigated whether minocycline protects against experimental model of JE. Intravenous inoculation of GP78 strain of JEV in adult mice results in lethal encephalitis and caused primarily because of neuronal death and secondary inflammation caused because of cell death. Minocycline confers complete protection in mice following JEV infection ( p  < 0.0001). Neuronal apoptosis, microglial activation, active caspase activity, proinflammatory mediators, and viral titer were markedly decreased in minocycline-treated JEV infected mice on ninth day post-infection. Treatment with minocycline may act directly on brain cells, because neuronal cell line Neuro2a were also salvaged from JEV-induced death. Our data suggest that minocycline may be a candidate to consider in human clinical trials for JE patients.     

A Novel Immunochromatographic Test Applied to a Serological Survey of Japanese Encephalitis Virus on Pig Farms in Korea

Among vertebrate species, pigs are a major amplifying host of Japanese encephalitis virus (JEV) and measuring their seroconversion is a reliable indicator of virus activity. Traditionally, the hemagglutination inhibition test has been used for serological testing in pigs; however, it has several limitations and, thus, a more efficient and reliable replacement test is required. In this study, we developed a new immunochromatographic test for detecting antibodies to JEV in pig serum within 15 min. Specifically, the domain III region of the JEV envelope protein was successfully expressed in soluble form and used for developing the immunochromatographic test. The test was then applied to the surveillance of Japanese encephalitis (JE) in Korea. We found that our immunochromatographic test had good sensitivity (84.8%) and specificity (97.7%) when compared with an immunofluorescence assay used as a reference test. During the surveillance of JE in Korea in 2012, the new immunochromatographic test was used to test the sera of 1,926 slaughtered pigs from eight provinces, and 228 pigs (11.8%) were found to be JEV-positive. Based on these results, we also produced an activity map of JEV, which marked the locations of pig farms in Korea that tested positive for the virus. Thus, the immunochromatographic test reported here provides a convenient and effective tool for real-time monitoring of JEV activity in pigs.     

Historical review and surveillance of Japanese encephalitis, Republic of Korea, 2002–2004

Japanese encephalitis virus (JEV), transmitted by culicine mosquitoes, is endemic throughout much of South‐East Asia, extending to the Korean Peninsula. The zoonotic cycle is from large water birds to culicine mosquitoes, with swine as an amplifying host and man as an incidental host. Culex tritaeniorhynchus, the primary JEV vector in the Republic of Korea, populations peak in late August through to early September when most cases of Japanese encephalitis (JE) are reported. Cx. tritaeniorhynchus were observed near the Demilitarized Zone in each of the years that mosquitoes were assayed for JEV. Each year that vector mosquitoes were assayed for JEV, minimum field infection rates (number of JEV positive mosquites/1000 Cx. tritaeniorhynchus assayed) ranged from 0.31 to 3.27. The epidemiology of JE has been recorded in Korea for more than half a century, from 1949 to 2005. During a major epidemic in 1949, there were 5616 cases and 2729 deaths reported, with levels persisting near epidemic levels of 1000 cases annually thereafter until 1969. Following the introduction and government mandated mass immunization in 1971, JE decreased dramatically. Since 1984, 0–6 cases of JE have been reported each year. However, continued evidence of mosquitoes positive for JEV indicates that JE continues to be a civilian and military health threat to immunocompromised persons in Korea, as well as non‐immune US soldiers, civilians and their family members.     

Estimating the Burden of Japanese Encephalitis Virus and Other Encephalitides in Countries of the Mekong Region

Diverse aetiologies of viral and bacterial encephalitis are widely recognized as significant yet neglected public health issues in the Mekong region. A robust analysis of the corresponding health burden is lacking. We retrieved 75 articles on encephalitis in the region published in English or in French from 1965 through 2011. Review of available data demonstrated that they are sparse and often derived from hospital-based studies with significant recruitment bias. Almost half (35 of 75) of articles were on Japanese encephalitis virus (JEV) alone or associated with dengue. In the Western Pacific region the WHO reported 30,000–50,000 annual JEV cases (15,000 deaths) between 1966 and 1996 and 4,633 cases (200 deaths) in 2008, a decline likely related to the introduction of JEV vaccination in China, Vietnam, or Thailand since the 1980s. Data on dengue, scrub typhus and rabies encephalitis, among other aetiologies, are also reviewed and discussed.Countries of the Mekong region are undergoing profound demographic, economic and ecological change. As the epidemiological aspects of Japanese encephalitis (JE) are transformed by vaccination in some countries, highly integrated expert collaborative research and objective data are needed to identify and prioritize the human health, animal health and economic burden due to JE and other pathogens associated with encephalitides.     

乙型脑炎病毒及其疫苗的研究进展

流行性乙型脑炎是由乙型脑炎病毒引起的、经蚊虫传播的严重危害中枢神经系统的人畜共患急性传染病,其重症病死率高,易造成永久性的神经系统后遗症,严重威胁着人类的健康。目前尚无特效的治疗流行性乙型脑炎的方法,控制蚊虫传播和免疫接种是当前的主要防御手段。简要综述了乙型脑炎病毒的基因组结构、结构蛋白与非结构蛋白功能、基因分型,以及流行性乙型脑炎疫苗的研究进展。     

Pathogenic and Genotypic Characterization of a Japanese Encephalitis Virus Isolate Associated with Reproductive Failure in an Indian Pig Herd

Background

India is endemic to Japanese encephalitis virus (JEV) and recurrent outbreaks occur mainly in rice growing areas. Pigs are considered to be the amplifying host for JEV and infection in gestating pigs results in reproductive failure. Most studies conducted on JEV infection in Indian pigs have been serological surveys and very little is known about JEV genotypes circulating in pigs. So the potential risk posed by pigs in JEV transmission and the genetic relationship between viruses circulating in pigs, mosquitoes and humans is poorly understood.

Methodology/Principal Findings

This study was conducted in pigs with a history of reproductive failure characterized by stillborn piglets with neuropathological lesions. Japanese encephalitis (JE) suspected brain specimens inoculated intracerebrally into mice and Vero cells resulted in successful isolation of JEV/SW/IVRI/395A/2014. Clinicopathological observations in infected mice, demonstration of JEV antigen in brain, and analysis of the envelope protein identified the swine isolate as being neurovirulent. Phylogenetic analysis based on prM and E gene sequences showed that it belonged to genotype III. This swine isolate was closely related to JEV associated with the 2005 outbreak in India and JaoArS982 from Japan. Phylogenetic analysis of JEV strains collected between 1956 and 2014 in India categorized the GIII viruses into different clades blurring their spatial distribution, which has been discernible in the previous century.

Conclusions/Significance

Isolation of JEV from stillborn piglets and its close genetic relationship with viruses detected at least three decades ago in humans and mosquitoes in Japan suggests that the virus may have been circulating among Indian pigs for several decades. The close similarity between the present swine isolate and those detected in humans affected in the 2005 outbreak in Uttar Pradesh, India, suggests the need for more intensive surveillance of pigs and implementation of suitable strategies to control JE in India.     

The current burden of Japanese encephalitis and the estimated impacts of vaccination: Combining estimates of the spatial distribution and transmission intensity of a zoonotic pathogen

Japanese encephalitis virus (JEV) is a major cause of neurological disability in Asia and causes thousands of severe encephalitis cases and deaths each year. Although Japanese encephalitis (JE) is a WHO reportable disease, cases and deaths are significantly underreported and the true burden of the disease is not well understood in most endemic countries. Here, we first conducted a spatial analysis of the risk factors associated with JE to identify the areas suitable for sustained JEV transmission and the size of the population living in at-risk areas. We then estimated the force of infection (FOI) for JE-endemic countries from age-specific incidence data. Estimates of the susceptible population size and the current FOI were then used to estimate the JE burden from 2010 to 2019, as well as the impact of vaccination. Overall, 1,543.1 million (range: 1,292.6-2,019.9 million) people were estimated to live in areas suitable for endemic JEV transmission, which represents only 37.7% (range: 31.6-53.5%) of the over four billion people living in countries with endemic JEV transmission. Based on the baseline number of people at risk of infection, there were an estimated 56,847 (95% CI: 18,003-184,525) JE cases and 20,642 (95% CI: 2,252-77,204) deaths in 2019. Estimated incidence declined from 81,258 (95% CI: 25,437-273,640) cases and 29,520 (95% CI: 3,334-112,498) deaths in 2010, largely due to increases in vaccination coverage which have prevented an estimated 314,793 (95% CI: 94,566-1,049,645) cases and 114,946 (95% CI: 11,421-431,224) deaths over the past decade. India had the largest estimated JE burden in 2019, followed by Bangladesh and China. From 2010-2019, we estimate that vaccination had the largest absolute impact in China, with 204,734 (95% CI: 74,419-664,871) cases and 74,893 (95% CI: 8,989-286,239) deaths prevented, while Taiwan (91.2%) and Malaysia (80.1%) had the largest percent reductions in JE burden due to vaccination. Our estimates of the size of at-risk populations and current JE incidence highlight countries where increasing vaccination coverage could have the largest impact on reducing their JE burden.     

A single intramuscular injection of recombinant plasmid DNA induces protective immunity and prevents Japanese encephalitis in mice

Plasmid vectors containing Japanese encephalitis virus (JEV) premembrane (prM) and envelope (E) genes were constructed that expressed prM and E proteins under the control of a cytomegalovirus immediate-early gene promoter. COS-1 cells transformed with this plasmid vector (JE-4B clone) secreted JEV-specific extracellular particles (EPs) into the culture media. Groups of outbred ICR mice were given one or two doses of recombinant plasmid DNA or two doses of the commercial vaccine JEVAX. All mice that received one or two doses of DNA vaccine maintained JEV-specific antibodies 18 months after initial immunization. JEVAX induced 100% seroconversion in 3-week-old mice; however, none of the 3-day-old mice had enzyme-linked immunosorbent assay titers higher than 1:400. Female mice immunized with this DNA vaccine developed plaque reduction neutralization antibody titers of between 1:20 and 1:160 and provided 45 to 100% passive protection to their progeny following intraperitoneal challenge with 5,000 PFU of virulent JEV strain SA14. Seven-week-old adult mice that had received a single dose of JEV DNA vaccine when 3 days of age were completely protected from a 50, 000-PFU JEV intraperitoneal challenge. These results demonstrate that a recombinant plasmid DNA which produced JEV EPs in vitro is an effective vaccine.     

The Effect of Vaccination Coverage and Climate on Japanese Encephalitis in Sarawak,Malaysia

Background

Japanese encephalitis (JE) is the leading cause of viral encephalitis across Asia with approximately 70,000 cases a year and 10,000 to 15,000 deaths. Because JE incidence varies widely over time, partly due to inter-annual climate variability effects on mosquito vector abundance, it becomes more complex to assess the effects of a vaccination programme since more or less climatically favourable years could also contribute to a change in incidence post-vaccination. Therefore, the objective of this study was to quantify vaccination effect on confirmed Japanese encephalitis (JE) cases in Sarawak, Malaysia after controlling for climate variability to better understand temporal dynamics of JE virus transmission and control.

Methodology/principal findings

Monthly data on serologically confirmed JE cases were acquired from Sibu Hospital in Sarawak from 1997 to 2006. JE vaccine coverage (non-vaccine years vs. vaccine years) and meteorological predictor variables, including temperature, rainfall and the Southern Oscillation index (SOI) were tested for their association with JE cases using Poisson time series analysis and controlling for seasonality and long-term trend. Over the 10-years surveillance period, 133 confirmed JE cases were identified. There was an estimated 61% reduction in JE risk after the introduction of vaccination, when no account is taken of the effects of climate. This reduction is only approximately 45% when the effects of inter-annual variability in climate are controlled for in the model. The Poisson model indicated that rainfall (lag 1-month), minimum temperature (lag 6-months) and SOI (lag 6-months) were positively associated with JE cases.

Conclusions/significance

This study provides the first improved estimate of JE reduction through vaccination by taking account of climate inter-annual variability. Our analysis confirms that vaccination has substantially reduced JE risk in Sarawak but this benefit may be overestimated if climate effects are ignored.     

Detection of Flaviviruses by Reverse Transcriptase-Polymerase Chain Reaction with the Universal Primer Set

Using a universal primer set designed to match the sequence of the NS1 gene of flaviviruses, the virus RNA of dengue (DEN), Japanese encephalitis (JEV), powassan and langat of Flaviviridae were successfully amplified by polymerase chain reaction (PCR) via cDNA; and with different internal primers, the serotypes of the dengue viruses were identified. Of the 78 clinically diagnosed dengue fever patients, 18 patients were positive for DEN 1, 48 patients for DEN 2 and 8 patients concurrently infected with DEN 4. Of the 52 patients admitted with Japanese encephalitis (JE), 45 were determined to be JEV infections. By nested PCR, we completed the identification of flaviviruses within 2 days. The results show that seven primers have a potential value for rapid clinical diagnosis of flavivirus infections.     

Axl Alleviates Neuroinflammation and Delays Japanese Encephalitis Progression in Mice

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus,which causes the most commonly diagnosed viral encephalitis named Japanese encephalitis (JE) in the world with an unclear pathogenesis.Axl,a receptor tyrosine kinase from TAM family,plays crucial role in many inflammatory diseases.We have previously discovered that Axl deficiency resulted in more severe body weight loss in mice during JEV infection,which we speculate is due to the anti-inflammatory effect of Axl during JE.Currently,the role of Axl in regulating the neuroinflammation and brain damage during JE has not been investigated yet.In this study,by using Axl deficient and heterozygous control mice,we discovered that Axl deficient mice displayed accelerated JE progression and exacerbated brain damage characterized by increased neural cell death,extended infiltration of inflammatory cells,and enhanced production of pro-inflammatory cytokines,in comparison to control mice.Additionally,consistent with our previous report,Axl deficiency had no impact on the infection and target cell tropism of JEV in brain.Taken together,our results suggest that Axl plays an anti-inflammatory and neuroprotective role during the pathogenesis of JE.     

Evaluation of mouse brain-derived, inactivated Japanese encephalitis vaccine

Japanese encephalitis (JE) is a serious encephalitis caused by JE virus. Approximately 20% of JE patients die and 50% patients recover with neuro-psychiatric sequelae. In Japan, the number of JE patients was over 1000 per year in 1960s; however, the number decreased dramatically and has been less than 10 since 1990. Ministry of Health, Labour and Welfare suspended the strong recommendation for vaccination with the mouse brain-derived JE vaccine, because of cases who developed acute disseminated encephalomyelitis (ADEM) after vaccination with JE vaccine. However, it has not been fully confirmed on scientific bases that ADEM was caused by mouse brain-derived JE vaccine. Tissue culture derived-JE vaccine is under development. It is expected that this new vaccine will come to the market soon and that the recommendation of universal vaccination with JE vaccine will be implemented at the earliest occasion.     

Japanese encephalitis virus infects neural progenitor cells and decreases their proliferation

Japanese encephalitis virus (JEV), a common cause of encephalitis in humans, especially in children, leads to substantial neuronal injury. The survivors of JEV infection have severe cognitive impairment, motor and behavioral disorders. We hypothesize that depletion of neural progenitor cells (NPCs) by the virus culminates in neurological sequelae in survivors of Japanese encephalitis (JE). We utilized both in vivo model of JEV infection and in vitro neurosphere cultures to study progressive JEV infection. Cellular infection and cell death was determined by flow cytometry. BrdU administration in animals and in neurospheres was used to determine the proliferative ability of NPCs. JEV leads to massive loss of actively proliferating NPC population from the subventricular zone (SVZ). The ability of JEV infected subventricular zone cells to form neurospheres is severely compromised. This can be attributed to JEV infection in NPCs, which however do not result in robust death of the resilient NPC cells. Instead, JEV suppresses the cycling ability of these cells, preventing their proliferation. JEV primarily targets at a critical postnatal age and severely diminishes the NPC pool in SVZ, thus impairing the process of recovery after the insult. This arrested growth and proliferation of NPCs might have an effect on the neurological consequences in JE survivors.     

Serendipitous identification of natural Intergenotypic recombinants of hepatitis C in Ireland

Background

Encephalitis caused by flaviviruses, Japanese encephalitis virus (JEV) and West Nile virus (WNV) is responsible for significant morbidity and mortality in many endemic countries. Dengue-2 (Den-2) virus is a recent addition to the list of encephalitogenic viruses, after its Central Nervous System (CNS) invasion capability has been established. There is a wide array of laboratory tools that have helped us not only in the diagnosis of these conditions but also in understanding their pathogenesis and pathology. However, there are no reports of Shell Vial Culture (SVC), a centrifuge enhanced tissue culture assay that has revolutionized viral culturing in terms of rapidity and sensitivity being optimized for these flaviviral encephalitic conditions. The present study is an attempt to standardize and evaluate the usefulness of SVC for the laboratory diagnosis of JE, WN and Den-2 encephalitis cases and to compare it with Indirect Immunofluorescence (IIF) technique that detects cell associated virus antigen. Analysis of the various clinical parameters with respect to viral etiology has also been carried out.

Results

Pediatric patients constituted the major group involved in the study (92%). Etiological diagnosis of viral encephalitis could be established in twenty nine (58%) patients. JE encephalitis was the commonest with 19 (39%) cases being positive followed by, WN (9 cases-18%) and Den-2 (one case). IIF test could detect antigens of JE, WN and Den-2 viruses in 16(32%), 7(14%) and 1 case respectively. Shell vial culture assay picked up all cases that were positive by IIF test. In addition, SVC assay could detect 3 and 2 more cases of JE and WN encephalitis respectively, that were negative by the IIF test.

Conclusion

Shell vial culture is a rapid and efficient tool for the etiological diagnosis of JE, WN and Den-2 encephalitis cases. Early, prompt collection, transport and processing of the CSF samples, would make SVC a better method for the rapid diagnosis of these flaviviral infections.     

Japanese Encephalitis��A Pathological and Clinical Perspective

Japanese encephalitis (JE) is the leading form of viral encephalitis in Asia. It is caused by the JE virus (JEV), which belongs to the family Flaviviridae. JEV is endemic to many parts of Asia, where periodic outbreaks take hundreds of lives. Despite the catastrophes it causes, JE has remained a tropical disease uncommon in the West. With rapid globalization and climatic shift, JEV has started to emerge in areas where the threat was previously unknown. Scientific evidence predicts that JEV will soon become a global pathogen and cause of worldwide pandemics. Although some research documents JEV pathogenesis and drug discovery, worldwide awareness of the need for extensive research to deal with JE is still lacking. This review focuses on the exigency of developing a worldwide effort to acknowledge the prime importance of performing an extensive study of this thus far neglected tropical viral disease. This review also outlines the pathogenesis, the scientific efforts channeled into develop a therapy, and the outlook for a possible future breakthrough addressing this killer disease.     

Molecular Epidemiology of Japanese Encephalitis Virus in Mosquitoes in Taiwan during 2005–2012

Japanese encephalitis (JE) is a mosquito-borne zoonotic disease caused by the Japanese encephalitis virus (JEV). Pigs and water birds are the main amplifying and maintenance hosts of the virus. In this study, we conducted a JEV survey in mosquitoes captured in pig farms and water bird wetland habitats in Taiwan during 2005 to 2012. A total of 102,633 mosquitoes were collected. Culex tritaeniorhynchus was the most common mosquito species found in the pig farms and wetlands. Among the 26 mosquito species collected, 11 tested positive for JEV by RT-PCR, including Cx. tritaeniorhynchus, Cx. annulus, Anopheles sinensis, Armigeres subalbatus, and Cx. fuscocephala. Among those testing positive, Cx. tritaeniorhynchus was the predominant vector species for the transmission of JEV genotypes I and III in Taiwan. The JEV infection rate was significantly higher in the mosquitoes from the pig farms than those from the wetlands. A phylogenetic analysis of the JEV envelope gene sequences isolated from the captured mosquitoes demonstrated that the predominant JEV genotype has shifted from genotype III to genotype I (GI), providing evidence for transmission cycle maintenance and multiple introductions of the GI strains in Taiwan during 2008 to 2012. This study demonstrates the intense JEV transmission activity in Taiwan, highlights the importance of JE vaccination for controlling the epidemic, and provides valuable information for the assessment of the vaccine''s efficacy.