Japanese Encephalitis Virus Genotype Research Articles

Envelope protein gene based molecular characterization of Japanese encephalitis virus clinical isolates from West Bengal, India: a comparative approach with respect to SA14-14-2 live attenuated vaccine strain.

BackgroundIncreasing virulence of Japanese encephalitis virus (JEV), a mosquito-borne zoonotic pathogen is of grave concern because it causes a neurotrophic killer disease Japanese Encephalitis (JE) which, in turn, is responsible globally for viral acute encephalitis syndrome (AES). Despite the availability of vaccine, JE/AES cases and deaths have become regular features in the different rural districts of West Bengal (WB) state, India, indicating either the partial coverage of vaccine or the emergence of new strain of JEV. Therefore, a study was undertaken to characterize and compare the complete envelope (E) protein gene based molecular changes/patterns of JEVs circulating in WB.MethodsTotal of 98 AES case-patients’ samples were tested to detect the presence of JEV specific immunoglobulin M (IgM) antibody by Mac-ELISA method. Only JEV IgM negative samples with a history of ≤3 days’ illness were screened for virus isolation and RT-PCR. E gene sequences of JEV isolates were subjected to molecular phylogeny and immunoinformatics analysis.ResultsPresent study confirmed JEV etiology in 39.7% and 29.1% of patients presenting ≤15 days’ febrile illness, as determined by Mac-ELISA and RT-PCR respectively. Phylogenetic analysis based on complete E gene sequences of JEV isolates showed the co-circulation of JEV genotype I (GI) with genotype III (GIII). This study also demonstrated that isolate-specific crucial amino acid substitutions were closely related to neurovirulence/neuroinvasiveness of JE. On the basis of immunoinformatics analysis, some substitutions were predicted to disrupt T-cell epitope immunogenicity/antigenicity that might largely influence the outcome of vaccine derived from JEV GIII SA14-14-2 strain and this has been observed in a previously vaccinated boy with mild JE/AES due to JEV GI infection.ConclusionsBased on molecular evolutionary and bioinformatic approaches, we report evolution of JEV at a local level. Such naturally occurring evolution is likely to affect the disease profile and the vaccine efficacy to protect against JEV GI may demand careful evaluation.

Nonsuppurative Encephalomyelitis in a Calf in Japan and Isolation of Japanese Encephalitis Virus Genotype 1 from the Affected Calf

Japanese encephalitis virus (JEV) was isolated from the cerebrum of a calf which showed severe neurological symptoms in late September 2009, and the JEV isolate was revealed to be of genotype 1. This is the first report describing the isolation of genotype 1 JEV from cattle.

Genetic Diversity of Japanese Encephalitis Virus Isolates Obtained from the Indonesian Archipelago Between 1974 and 1987

Five genotypes (GI-V) of Japanese encephalitis virus (JEV) have been identified, all of which have distinct geographical distributions and epidemiologies. It is thought that JEV originated in the Indonesia-Malaysia region from an ancestral virus. From that ancestral virus GV diverged, followed by GIV, GIII, GII, and GI. Genotype IV appears to be confined to the Indonesia-Malaysia region, as GIV has been isolated in Indonesia from mosquitoes only, while GV has been isolated on three occasions only from a human in Malaysia and mosquitoes in China and South Korea. In contrast, GI-III viruses have been isolated throughout Asia and Australasia from a variety of hosts. Prior to this study only 13 JEV isolates collected from the Indonesian archipelago had been studied genetically. Therefore the sequences of the envelope (E) gene of 24 additional Indonesian JEV isolates, collected throughout the archipelago between 1974 and 1987, were determined and a series of molecular adaptation analyses were performed. Phylogenetic analysis indicated that over a 14-year time span three genotypes of JEV circulated throughout Indonesia, and a statistically significant association between the year of virus collection and genotype was revealed: isolates collected between 1974 and 1980 belonged to GII, isolates collected between 1980 and 1981 belonged to GIV, and isolates collected in 1987 belonged to GIII. Interestingly, three of the GII Indonesian isolates grouped with an isolate that was collected during the JE outbreak that occurred in Australia in 1995, two of the GIII Indonesian isolates were closely related to a Japanese isolate collected 40 years previously, and two Javanese GIV isolates possessed six amino acid substitutions within the E protein when compared to a previously sequenced GIV isolate collected in Flores. Several amino acids within the E protein of the Indonesian isolates were found to be under directional evolution and/or co-evolution. Conceivably, the tropical climate of the Indonesia/Malaysia region, together with its plethora of distinct fauna and flora, may have driven the emergence and evolution of JEV. This is consistent with the extensive genetic diversity seen among the JEV isolates observed in this study, and further substantiates the hypothesis that JEV originated in the Indonesia-Malaysia region.

Circulation of Japanese encephalitis virus in pigs and mosquito vectors within Can Tho city, Vietnam.

Japanese encephalitis virus (JEV) is a mosquito-borne, zoonotic flavivirus causing encephalitis in humans and reproductive disorder in pigs. JEV is present in large parts of Asia, where urbanization is high. Households within and outside Can Tho city, South Vietnam, were selected to monitor circulation of JEV. A nested RT-PCR was established to detect the presence of JEV in mosquitoes whereas sera from pigs belonging to households within the province were analyzed for the presence of antibodies to JEV. A total of 7885 mosquitoes were collected and divided into 352 pools whereof seven were JEV-positive, six of which were collected within the city. Fragments from four pools clustered with JEV genotype III and three with genotype I. Of the 43 pigs sampled inside the city 100% had JEV antibodies. Our study demonstrates exposure to JEV in pigs, and co-circulation of JEV genotype I and III in mosquitoes within an urban environment in South Vietnam. Thus, although JEV has mainly been considered a rural disease, the potential for transmission in urban areas cannot be ignored.

Complete Genome of a Genotype I Japanese Encephalitis Virus Isolated from a Patient with Encephalitis in Vientiane, Lao PDR.

Japanese encephalitis virus (JEV) (Flaviviridae, Flavivirus) is an arthropod-borne flavivirus transmitted by Culex species mosquitoes. We report here the complete genome of the JEV genotype I strain JEV_CNS769_Laos_2009 isolated from an infected patient in Vientiane, Lao People’s Democratic Republic (PDR) (Laos).

Molecular Detection and Genotyping of Japanese Encephalitis Virus in Mosquitoes during a 2010 Outbreak in the Republic of Korea

Japanese encephalitis virus (JEV), a mosquito-borne zoonotic pathogen, is one of the major causes of viral encephalitis. To reduce the impact of Japanese encephalitis among children in the Republic of Korea (ROK), the government established a mandatory vaccination program in 1967. Through the efforts of this program only 0–7 (mean 2.1) cases of Japanese encephalitis were reported annually in the ROK during the period of 1984–2009. However, in 2010 there was an outbreak of 26 confirmed cases of Japanese encephalitis, including 7 deaths. This represented a >12-fold increase in the number of confirmed cases of Japanese encephalitis in the ROK as compared to the mean number reported over the last 26 years and a 3.7-fold increase over the highest annual number of cases during this same period (7 cases). Surveillance of adult mosquitoes was conducted during the 2010 outbreak of Japanese encephalitis in the ROK. A total of 6,328 culicine mosquitoes belonging to 12 species from 5 genera were collected at 6 survey sites from June through October 2010 and assayed by reverse-transcription polymerase chain reaction (RT-PCR) for the presence of JEV. A total of 34/371 pooled samples tested positive for JEV (29/121 Culex tritaeniorhynchus, 4/64 Cx. pipiens, and 1/26 Cx. bitaeniorhynchus) as confirmed by sequencing of the pre-membrane and envelope protein coding genes. The maximum likelihood estimates of JEV positive individuals per 1,000 culicine vectors for Cx. tritaeniorhynchus, Cx. pipiens, and Cx. bitaeniorhynchus were 11.8, 5.6, and 2.8, respectively. Sequences of the JEV pre-membrane and envelope protein coding genes amplified from the culicine mosquitoes by RT-PCR were compared with those of JEV genotypes I-V. Phylogenetic analyses support the detection of a single genotype (I) among samples collected from the ROK in 2010.

Reduced neutralizing antibody titer against genotype I virus in swine immunized with a live-attenuated genotype III Japanese encephalitis virus vaccine

A shift in prevalence from Japanese encephalitis virus (JEV) genotype III (GIII) to GI virus has been observed in several Asian countries. Genotype I virus was first detected in Taiwan in 2008, and became the dominant JEV island-wide within a year. We conducted a serosurvey using swine serum specimens from multiple counties in Taiwan following the transmission season in 2009 and results showed 67-100% of JEV seropositive swine showed evidence of GI virus infection. The envelope (E) protein is a structural protein that elicits protective neutralizing antibodies (Nt Ab). The GIII at222 (a live-attenuated swine vaccine) virus E protein differs at eight amino acid residues (E-123, E-129, E-138, E-176, E-209, E-222, E-327 and E-366) from that of the GI TC2009-1 strain (isolated in Taiwan in 2009). Twenty piglets were vaccinated with two doses of at222 vaccine, and serum specimens were collected to evaluate the strain-specific Nt Ab titer against GIII at222, GIII CJN, and GI TC2009-1 viruses. Seropositivity rates (Nt Ab titer≥1:10) and geometric mean titers (GMT) were similar against at222 and CJN viruses. However, sera from swine vaccinated with at222 were least potently neutralizing against GI TC2009-1 virus. The estimated protective threshold against GI virus was observed only when the PRNT50 against at222 virus was ≥1:320. Thus, our current study indicates that the live-attenuated at222 swine vaccine can be partially protective against GI virus, and suggests that the efficacy of GIII swine vaccines currently used may require a comprehensive reevaluation in the field.

Surveillance of Japanese Encephalitis Virus Infection in Mosquitoes in Vietnam from 2006 to 2008

Japanese encephalitis virus (JEV) infection in mosquitoes was monitored in Vietnam from 2006 to 2008. A total of 15,225 mosquitoes, identified as 26 species in five genera were collected and 12,621 were grouped into 447 pools for examination of JEV infection by assays for cytopathic effects in C6/36 cells and by RT-PCR to detect flavivirus RNA. Three JEV strains were isolated from Culex tritaeniorhynchus Giles collected in northern and southern Vietnam and two JEV strains were isolated from Culex vishnui Theobald collected in the highlands of Vietnam. Genetic and phylogenetic analyses, based on complete E gene nucleotide sequences, revealed that the five JEV strains were classified into the genotype I group and six amino acid differences were found in these five strains. These results indicated that multiple JEV genotype I populations are circulating countrywide in Vietnam, transmitted by bites of their Cx. tritaeniorhynchus and Cx. vishnui.

Production and Characterization of Monoclonal Antibodies to Japanese Encephalitis Virus

In this study, eighteen monoclonal antibodies (MAbs) to recent Japanese encephalitis virus (JEV) genotype I were produced and characterized by plaque reduction neutralization test, western blot analysis, indirect immunofluorescence assay and enzyme-linked immunosorbent assay. All MAbs recognized only envelope (E) protein or conformational epitope of E and precursor membrane proteins. Two MAbs (7E5 and 3-3H8) possessed virus-neutralization activity, and their escape mutants possessed a change of glutamine to histidine at the position of 52 of E protein, suggesting that these neutralizing MAbs recognize the domain I-II hinge region of E protein. Five MAbs recognized all examined flaviviruses, and two were specific to JEV. These MAbs may be useful for differentiation and diagnosis of flaviviruses.

Reverse Transcription Loop-Mediated Isothermal Amplification for Rapid Detection of Japanese Encephalitis Virus in Swine and Mosquitoes

Japanese encephalitis (JE) can infect many agriculturally important animals and humans, and has a high incidence in Asia. One of the natural hosts of the mosquito-borne JE virus (JEV) is domestic pigs, which act as amplifier hosts. Porcine infection results in fatal encephalitis, abortion, and stillbirth in pregnant sows, and hypospermia in boars. In this study, a rapid JEV detection method for swine and mosquitoes was developed based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) targeting the nucleocapsid (E) genes of JEV genotype I (lineage K94PO5), and genotype III (lineage SA14-14-2). Fifty-six swine blood samples and 20,000 mosquitoes were used to evaluate the method, compared to conventional RT-polymerase chain reaction (PCR) and real-time RT-PCR. RT-LAMP had detection limits of 2.57 and 2.34 copies/μL for JEV I and III, respectively. Assay sensitivity was similar to real-time RT-PCR, but was 10-fold higher than conventional RT-PCR. Assay specificity was high, showing no cross-reactivity to other flaviviruses. The results of virus isolation and identification of swine blood samples and mosquito samples were fully consistent with RT-LAMP. Finally, the JEV RT-LAMP assay was simpler and less time consuming than conventional RT-PCR or real-time RT-PCR, since the amplification step could be completed in a single tube within 50 min at 63°C. In conclusion, the newly-developed RT-LAMP assay is an accurate and convenient method for rapid and sensitive diagnosis of JEV in swine and mosquitoes, and may prove to be a practical molecular tool for surveillance and epidemiological investigations.

Molecular evidence for the occurrence of Japanese encephalitis virus genotype I and III infection associated with acute encephalitis in patients of West Bengal, India, 2010.

BackgroundJapanese encephalitis virus (JEV), a mosquito-borne zoonotic pathogen, is the sole etiologic agent of Japanese Encephalitis (JE); a neurotropic killer disease which is one of the major causes of viral encephalitis worldwide with prime public health concern. JE was first reported in the state of West Bengal, India in 1973. Since then it is being reported every year from different districts of the state, though the vaccination has already been done. Therefore, it indicates that there might be either partial coverage of the vaccine or the emergence of mutated/new strain of JEV. Considering this fact, to understand the JEV genotype distribution, we conducted a molecular epidemiological study on a total of 135 serum/cerebrospinal fluid (CSF) samples referred and/or collected from the clinically suspected patients with Acute encephalitis syndrome (AES), admitted in different district hospitals of West Bengal, India, 2010.FindingsJEV etiology was confirmed in 36/135 (26.6%) and 13/61 (21.3%) 2–15 days’ febrile illness samples from AES cases by analyzing Mac-ELISA followed by RT-PCR test respectively. Phylogenetic analysis based on complete envelope gene sequences of 13 isolates showed the emergence of JEV genotype I (GI), co-circulating with genotype III (GIII).ConclusionThis study represents the first report of JEV GI with GIII, co-circulating in West Bengal. The efficacy of the vaccine (derived from JEV GIII strain SA-14-14-2) to protect against emerging JEV GI needs careful evaluation. In future, JE outbreak is quite likely in the state, if this vaccine fails to protect sufficiently against GI of JEV.

North American Birds as Potential Amplifying Hosts of Japanese Encephalitis Virus

Japanese encephalitis virus (JEV) is an emerging arbovirus, and inter-continental spread is an impending threat. The virus is maintained in a transmission cycle between mosquito vectors and vertebrate hosts, including birds. We detected variation in interspecies responses among North American birds to infection with strains of two different JEV genotypes (I and III). Several native North American passerine species and ring-billed gulls had the highest average peak viremia titers after inoculation with a Vietnamese (genotype I) JEV strain. Oral JEV shedding was minimal and cloacal shedding was rarely detected. The majority of birds, both viremic (72 of 74; 97.3%) and non-viremic (31 of 37; 83.8%), seroconverted by 14 days post-inoculation and West Nile virus-immune individuals had cross-protection against JEV viremia. Reservoir competence and serologic data for a variety of avian taxa are important for development of JEV surveillance and control strategies and will aid in understanding transmission ecology in the event of JEV expansion to North America.

Identification of genotype III Japanese encephalitis virus isolated in Zhejiang province

To investigate the molecular characteristics and genotype of Japanese encephalitis virus (JEV) found in vector mosquitoes in Zhejiang province from 1982 to 1983. A total of 3188 mosquitoes were collected in Dinghai district and Yiwu city in Zhejiang province, during year 1982 and 1983. The virus was isolated by C6/36 cell, and then identified by hemagglutination inhibition test. The isolated strains were activated in year 2011, and plaque forming unit (PFU) were applied to test the virus titer. The suckling rats were tested under intracranial inoculation, where PrM and E genes were amplified and sequenced. Their nucleotide and amino acid sequences were analyzed and compared with the JEV vaccine strain SA14-14-2 and the JEV isolated in Zhejiang province during 2007 and 2010; and phylogenetic tree were constructed by bioinformatic software. From the 3188 mosquitoes captured, eleven virus strains were isolated and found to be able to cause cytopathogenic effect (CPE) in C6/36 cells within 72 hours. Virus titer ranged from 2.5 to 6.47 lg PFU/ml. The suckling rats would die within 72 hours since the inoculation. The phylogenetic analysis with the PrM and E genes showed that the JEV isolated in Zhejiang during 1982 and 1983 belonged to genotype III; while the JEV isolated in Zhejiang during 2007 and 2010 belonged to genotype I. The analysis of E genes from 5 isolated strains found that the homology of nucleotide sequence was over 98.9%, and the homology of amino acid sequence was over 99.8%. The compare between the 5 virus strains and the vaccine strain SA14-14-2 found 10 common amino acid variation sites, and showed that the homology of nucleotide sequence was over 97.7%, and the homology of amino acid sequence was 99.2%. The compare between the 5 virus strains and the JEV isolated in Zhejiang during 2007 and 2010 found a 87.7% - 87.9% nucleotide homology and an over 98.8% amino acid homology. The JEV isolated from the mosquitoes in Dinghai district and Yiwu city between year 1982 and 1983, were genotype III.

Japanese encephalitis and Japanese encephalitis virus in mainland China

Japanese encephalitis (JE), caused by Japanese encephalitis virus (JEV) infection, is the most important viral encephalitis in the world. Approximately 35,000-50,000 people suffer from JE every year, with a mortality rate of 10,000-15,000 people per year. Although the safety and efficacy of JE vaccines (inactivated and attenuated) have been demonstrated, China still accounts for 50% of the reported JE cases worldwide. In this review, we provide information about the burden of JE in mainland China and the corresponding epidemiology from 1949 to 2010, including the morbidity and mortality of JE; the age, gender, and vocational distribution of JE cases; its regional and seasonal distribution; and JE immunization. In addition, we discuss the relationships among vectors, hosts, and JEV isolates from mainland China; the dominant vector species for JEV transmission; the variety of JEV genotypes and the different biological characteristics of the different JEV genotypes; and the molecular evolution of JEV.

Broad-Spectrum Antiviral Activity of RNA Interference against Four Genotypes of Japanese Encephalitis Virus Based on Single MicroRNA Polycistrons

Japanese encephalitis virus (JEV), a neurotropic mosquito-borne flavivirus, causes acute viral encephalitis and neurologic disease with a high fatality rate in humans and a range of animals. Small interfering RNA (siRNA) is a powerful antiviral agent able to inhibit JEV replication. However, the high rate of genetic variability between JEV strains (of four confirmed genotypes, genotypes I, II, III and IV) hampers the broad-spectrum application of siRNAs, and mutations within the targeted sequences could facilitate JEV escape from RNA interference (RNAi)-mediated antiviral therapy. To improve the broad-spectrum application of siRNAs and prevent the generation of escape mutants, multiple siRNAs targeting conserved viral sequences need to be combined. In this study, using a siRNA expression vector based on the miR-155 backbone and promoted by RNA polymerase II, we initially identified nine siRNAs targeting highly conserved regions of seven JEV genes among strains of the four genotypes of JEV to effectively block the replication of the JEV vaccine strain SA14-14-2. Then, we constructed single microRNA-like polycistrons to simultaneously express these effective siRNAs under a single RNA polymerase II promoter. Finally, these single siRNAs or multiple siRNAs from the microRNA-like polycistrons showed effective anti-virus activity in genotype I and genotype III JEV wild type strains, which are the predominant genotypes of JEV in mainland China. The anti-JEV effect of these microRNA-like polycistrons was also predicted in other genotypes of JEV (genotypes II and IV), The inhibitory efficacy indicated that siRNAs×9 could theoretically inhibit the replication of JEV genotypes II and IV.

Emergence of Japanese encephalitis virus genotype V in the Republic of Korea

BackgroundJapanese encephalitis virus (JEV) genotype V reemerged in Asia (China) in 2009 after a 57-year hiatus from the continent, thereby emphasizing a need to increase regional surveillance efforts. Genotypic characterization was performed on 19 JEV-positive mosquito pools (18 pools of Culex tritaeniorhynchus and 1 pool of Cx. bitaeniorhynchus) from a total of 64 positive pools collected from geographically different locations throughout the Republic of Korea (ROK) during 2008 and 2010.FindingsTwo regions of the JEV genome were sequenced from 19 pools; the envelope gene and the nonstructural protein 5 (NS5)/3'-untranslated region (UTR). Eighteen pools of Culex tritaeniorhynchus and one pool of Cx. bitaeniorhynchus were positive for genotype I and genotype V, respectively. Sequence alignment of the complete E gene from Cx. bitaeniorhynchus showed high amino acid similarity (98.8%) to the Muar strain, characterized as the first report of genotype V, isolated from an encephalitis patient in Malaysia in 1952.ConclusionThis study represents the first report of JEV genotype V in the ROK. The reemergence of genotype V in Asia (China and ROK) after more than a half-century and its discovery in Cx. bitaeniorhynchus, a mosquito species previously unknown to carry JEV in the ROK, emphasizes the need for enhanced JE surveillance to monitor the dynamics of JEV strains within the region. Future findings may have implications with regard to JEV vaccination/prevention strategies.

A specific and sensitive antigen capture assay for NS1 protein quantitation in Japanese encephalitis virus infection

Japanese encephalitis virus (JEV) is a human pathogenic, mosquito-borne flavivirus that is endemic/epidemic in Asia. JEV is rarely detected or isolated from blood or cerebrospinal fluid (CSF), and detection of IgM is generally diagnostic of the infection. The flavivirus nonstructural glycoprotein NS1 is released transiently during flavivirus replication. The aim of this study was to set up a quantitative JEV NS1 antigen capture assay. A soluble hexameric form of JEV NS1 protein was produced in a stable Drosophila S2 cell clone and purified from supernatant fluids. Two IgG1 monoclonal antibodies (MAbs) with high affinity against two different epitopes of JEV NS1 antigen were used to develop an antigen-capture assay with a limit of detection of 0.2ngml(-1) NS1. Up to 1μgml(-1) JEV NS1 protein was released in supernatants of mammalian cells infected with JEV but <10ngml(-1) was released in sera of virus-infected mice before the onset of encephalitis and death. Moreover, NS1 protein was detected at low levels (<10ngml(-1)) in 23.8% of sera and in 10.5% of CSF of patients diagnosed as IgM-positive for JEV. This quantitative test of NS1 protein is proposed for highly specific diagnosis of acute infection with JEV genotypes I to IV.

Molecular phylogenetic and evolutionary analyses of Muar strain of Japanese encephalitis virus reveal it is the missing fifth genotype

Japanese encephalitis virus (JEV) is the most important cause of epidemic encephalitis worldwide but its origin is unknown. Epidemics of encephalitis suggestive of Japanese encephalitis (JE) were described in Japan from the 1870s onwards. Four genotypes of JEV have been characterised and representatives of each genotype have been fully sequenced. Based on limited information, a single isolate from Malaysia is thought to represent a putative fifth genotype. We have determined the complete nucleotide and amino acid sequence of Muar strain and compared it with other fully sequenced JEV genomes. Muar was the least similar, with nucleotide divergence ranging from 20.2 to 21.2% and amino acid divergence ranging from 8.5 to 9.9%. Phylogenetic analysis of Muar strain revealed that it does represent a distinct fifth genotype of JEV. We elucidated Muar signature amino acids in the envelope (E) protein, including E327 Glu on the exposed lateral surface of the putative receptor binding domain which distinguishes Muar strain from the other four genotypes. Evolutionary analysis of full-length JEV genomes revealed that the mean evolutionary rate is 4.35 × 10 −4 (3.4906 × 10 −4 to 5.303 × 10 −4) nucleotides substitutions per site per year and suggests JEV originated from its ancestral virus in the mid 1500s in the Indonesia-Malaysia region and evolved there into different genotypes, which then spread across Asia. No strong evidence for positive selection was found between JEV strains of the five genotypes and the E gene has generally been subjected to strong purifying selection.

Isolation and Genetic Characteristics of Human Genotype 1 Japanese Encephalitis Virus, China, 2009

BackgroundSeveral studies have shown that the predominant genotype of Chinese Japanese encephalitis virus (JEV) is evolving from genotype 3 to genotype 1. However, in recent years, almost all genotype 1 isolates were from mosquitoes, and genotype 1 has been less associated with human disease than genotype 3. This study reports the isolation of human genotype 1 JEV and its genetic characteristics to provide additional insights into human JE pathogens that are currently circulating in China.Methods and ResultsIn 2009, 31 cerebrospinal fluid samples were collected from patients living in Yunnan and Shanxi provinces and were used to inoculate Aedes albopictus C6/36 cells for virus isolation. The JEV strains were identified using immunofluorescent assays and the reverse transcription-polymerase chain reaction. Phylogenetic analyses based on the partial capsid/pre-membrane and full envelope (E) sequences were performed using Clustalx 1.8 software. Three JEV isolates were obtained from a 4-year-old girl and a 2-year-old boy living in Yunnan and an 82-year-old woman in Shanxi. The boy had been immunized with one dose of JE live attenuated vaccine. New isolates were grouped into genotype 1. Amino acid sequence for the viral E protein indicated 95% to 100% identity with each other and with other JEV strains. When compared with a consensus sequence of E protein, two amino acid substitutions were found: SerE-123-Asn in the two Yunnan isolates and LysE-166-Arg in the Shanxi isolate.ConclusionsOur findings indicate that the genotype 1 of JEV is causing human infections in China. Our observation of a previously vaccinated boy developing JE from genotype 1 virus infection also calls for more detailed studies, both in vitro and in vivo neutralization tests as well as active surveillance, to examine the possibility of a lack of complete protection conferred by the live attenuated JE vaccine against genotype 1 virus.

Study on the protective efficacy of SA14-14-2 attenuated Japanese encephalitis against different JE virus isolates circulating in China

Prior to 1976 only Japanese encephalitis virus (JEV) genotype III could be detected in China. Recently, numerous genotype I JEV strains have been isolated from JE patients, mosquitoes and pigs while genotype III strains remain present. Two kinds of JEV vaccines are currently used in China for the prevention disease: the JE live attenuated vaccine (LAV) SA14-14-2 virus and the inactivated P3 strain (IPV) vaccine. The SA14-14-2 and P3 viral strains were isolated in the year of 1953 and 1949 respectively and both belonged to the JEV genotype III. In order to evaluate the protective efficacy of both vaccines against the JEV genotype I isolates we conducted vaccination-challenge protection assays in mice. These data demonstrated that both LAV (≥234pfu virus) and IPV (1:5 dilution) vaccines effectively conferred protection against all 16 isolates tested following intraperitoneal (i.p.) challenge. However, when vaccinated mice were challenged via intracerebral (i.c.) injection, ≥60% LAV vaccinated animals were protected against challenge with most JEV isolates but only ≤40% protection was observed following vaccination with IPV. These results indicated that JE vaccines used in China still protected effectively against both JEV genotypes now prevalent in China and that the LAV formulation conferred higher levels of protection compared to the protection conferred by IPV.