Search Results (38)

Japanese encephalitis virus (JEV) is a mosquito-borne orthoflavivirus and a major cause of human encephalitis throughout Asia, although it is currently not reported in Europe. To assess the potential impact of climate change, such as increased temperatures, and the potential for native Cx. pipiens to transmit JEV genotype I in the United Kingdom (UK), we have investigated vector competence at two different temperatures. Culex pipiens f. pipiens were provided a bloodmeal containing JEV genotype I at 7.8 × 10⁸ PFU/mL. Mosquitoes were maintained for 14 days at 21 °C or 25 °C, and rates of infection, dissemination, and transmission potential were assessed. There was no evidence for virus infection, dissemination, or potential for transmission at 21 °C. However, at 25 °C, virus infection was detected in 5 of 36 mosquitoes (13.9%). Of these, JEV disseminated to legs and wings in three specimens (3/5) and viral RNA was detected in saliva from one specimen (1/3). These data indicate that at elevated temperatures of 25 °C, UK Cx. pipiens f. pipiens could transmit JEV genotype 1. Full article

Japanese encephalitis (JE), a mosquito-borne viral disease caused by the Japanese encephalitis virus (JEV), remains a significant public health threat in Asia. Although vaccination programs have successfully reduced the incidence of JE, challenges persist in the adult population, and the emergence of rare JEV genotypes poses additional risks. In this study, a phylogenetic analysis of the whole JEV genome sequence, along with a temporal–spatial analysis of isolates and a host–vector analysis, was used to examine the changes in JEV transmission dynamics before and after 2012. The results revealed persistent differences between the dominant G1 and G3 genotypes, as well as the re-emergence of G4 and G5 genotypes. Although JEV has been detected in non-traditional vectors and atypical mammalian hosts, Culex tritaeniorhynchus and pigs remain the primary vector and amplifying host, respectively. These findings underscore the need to enhance existing JEV genotype surveillance while addressing emerging threats from genotype diversity, host expansion, and geographic spread. Full article

The Japanese encephalitis virus is an arbovirus that causes severe damage to the central nervous system. At present, there are still 67,900 cases of Japanese encephalitis worldwide every year, which poses a global public health concern and causes great economic losses to animal husbandry. In this study, we analyzed the epidemiology, transmission, and evolution of JEV based on the NCBI database. E and NS1 were emphatically analyzed for amino acid variation and predicted protein structure. Gene recombination and the evolutionary rate of JEV were analyzed using RDP 4 and BEAST. The maximum clade credibility tree of E was reconstructed to estimate the time of the most recent common ancestor. Chinese genotype Ⅰ (GI) strain recombination events occurred in the C, M/PrM, E, NS2A, NS4B, and NS5 proteins, and genotype III (GIII) strains occurred in the E, NS1, NS3, NS4A, and NS5 proteins. The average evolutionary rates of JEV were comparable (3.3830 × 10⁻⁴, 2.0481 × 10⁻⁴, 3.5650 × 10⁻⁴, 2.2423 × 10⁻⁴, 3.0844 × 10⁻⁴, and 1.9757 × 10⁻⁴ substitutions/site/year for the JEV-I whole genome, JEV-III whole genome, JEV-I E gene, JEV-III E gene, JEV-I NS1 gene, and JEV-III NS1 gene, respectively). The MCC tree revealed the evolutionary order was GⅢ, GⅠ, GⅤ, GⅡ, and GⅣ. This study was expected to provide theoretical support for vaccine development and comprehensive prevention and treatment of JEV. Full article

Japanese encephalitis virus (JEV) is regarded as an emerging and reemerging pathogen that is a primary cause of viral encephalitis in humans. However, there is a scarcity of research on the prevalence of JEV genotypes across the different mosquito species in Shanghai. In this study, the diversity of mosquito species and prevalence of JEV in pig and sheep farms in Shanghai were surveyed in 2022. A total of 24,073 mosquitoes, belonging to four genera and seven species, were collected from pig and sheep enclosures in the Chongming, Jiading, Pudong, Fengxian, and Jinshan Districts of Shanghai. Culex tritaeniorhynchus was identified as the predominant species (87.09%, 20,965/24,073) with the highest JEV detection rate. Six strains of JEV were isolated in this study, and genetic analysis revealed that five strains (SH22-M5, SH22-M9, SH22-M14, SH22-M41, and SH22-M52) belonged to genotype I (GI), while one strain (SH22-M1) was classified as genotype III (GIII). The sequence homology was highest between SH22-M9 and SD-1 (99.87%) and between SH22-M14 and SD12 (99.53%). SH22-M5, SH22-M41, and SH22-M52 shared the highest sequence homology with the HEN07011 strain (99.73–99.93%). SH22-M1 was most closely related to SH18, with a sequence homology of 99.8%. Additionally, for the first time, the GI JEV strain was isolated from mosquitoes in sheep sties in this study. The findings highlight the necessity of enhancing the surveillance of JEV in pigs and other livestock farms, including sheep, as well as monitoring the mosquitoes present in these environments. It is recommended that livestock farming areas be kept separate from human habitation to reduce the risk of JEV infections in humans. Full article

Japanese encephalitis virus (JEV) NS2B-NS3 is a protein complex composed of NS3 proteases and an NS2B co-factor. The N-terminal protease domain (180 residues) of NS3 (NS3(pro)) interacts directly with a central 40-amino acid hydrophilic domain of NS2B (NS2B(H)) to form an active serine protease. In this study, the recombinant NS2B(H)-NS3(pro) proteases were prepared in E. coli and used to compare the enzymatic activity between genotype I (GI) and III (GIII) NS2B-NS3 proteases. The GI NS2B(H)-NS3(pro) was able to cleave the sites at the internal C, NS2A/NS2B, NS2B/NS3, and NS3/NS4A junctions that were identical to the sites proteolytically processed by GIII NS2B(H)-NS3(pro). Analysis of the enzymatic activity of recombinant NS2B(H)-NS3(pro) proteases using a model of fluorogenic peptide substrate revealed that the proteolytical processing activity of GIII NS2B(H)-NS3(pro) was significantly higher than that of GI NS2B(H)-NS3(pro). There were eight amino acid variations between GI and GIII NS2B(H)-NS3(pro), which may be responsible for the difference in enzymatic activities between GI and GIII proteases. Therefore, recombinant mutants were generated by exchanging the NS2B(H) and NS3(pro) domains between GI and GIII NS2B(H)-NS3(pro) and subjected to protease activity analysis. Substitution of NS2B(H) significantly altered the protease activities, as compared to the parental NS2B(H)-NS3(pro), suggesting that NS2B(H) played an essential role in the regulation of NS3(pro) protease activity. To further identify the amino acids responsible for the difference in protease activities, multiple substitution mutants including the individual and combined mutations at the variant residues 55 and 65 of NS2B(H) were generated and subjected to protease activity analysis. Replacement of NS2B-55 and NS2B-65 of GI to GIII significantly increased the enzymatic activity of GI NS2B(H)-NS3(pro) protease, whereas mutation of NS2B-55 and NS2B-65 of GIII to GI remarkably reduced the enzymatic activity of GIII NS2B(H)-NS3(pro) protease. Overall, these data demonstrated that NS2B-55 and NS2B-65 variations in the hydrophilic domain of NS2B co-contributed to the difference in NS2B(H)-NS3(pro) protease activities between GI and GIII. However, it will be crucial to explore these mutations in other in vivo and/or in vitro models. Collectively, these observations will be useful for understanding the replication of JEV GI and GIII viruses. Full article

The emergence of Japanese encephalitis virus (JEV) in eastern Australia in 2022 caused extensive reproductive disease in pigs and is a threat to public health. Groups of weaned piglets were experimentally infected with the Australian outbreak strain of JEV (genotype 4). All pigs challenged at 5 weeks of age were infected after an intradermal injection of 1 × 10^5.5 (n = 4) or 1 × 10^4.5 TCID₅₀/pig (n = 5). Intranasal instillation was less effective at this age, infecting 3/4 pigs with the same higher dose and 1/5 with the lower dose. Intradermal injection using 1 × 10^5.0 TCID₅₀/pig also infected 9/9 pigs at 11 weeks of age. Infection in all cases was confirmed by qRT-PCR of blood samples, which identified a viremia peak at 3–4 days and detected JEV-specific antibodies as early as 5 days after the challenge. The detection of JEV in oral and nasal swabs and in saliva from chew ropes was less consistent. JEV was detected in the tonsils of 21/22 infected pigs and was isolated from the tonsils of 9/9 pigs sampled 19 days after the challenge at 11 weeks of age. The infected pigs showed no clinical signs other than pyrexia on Days 4–6. Histopathology consistent with JEV infection was evident in the nervous tissues of all but two pigs sampled 28 days after the challenge and was characterized by meningitis, encephalitis and gliosis throughout the brain. Serological studies showed extensive cross-reactivity between JEV and Murray Valley encephalitis virus using blocking ELISAs. However, the determination of limiting-dilution titres allowed for the identification of the infecting virus. This in vivo infection model will be useful in evaluating JEV vaccines and for comparative pathogenesis studies with other JEV genotypes. Full article

Japanese encephalitis is a disease caused by the Japanese encephalitis virus (JEV) and is a concern for U.S. military personnel stationed in the Republic of Korea (ROK). The recent literature reports a potential shift from GI to GV as the dominant genotype circulating in east Asia. In the ROK, GV has been reported in a few Culex spp., but not in the main JEV vector, Cx. tritaeniorhynchus. The goal of this surveillance was to shed light on the current knowledge of the epidemiology of JEV in the ROK by analyzing mosquito collection data from three consecutive years, 2021–2023, and molecularly detecting and genotyping JEV in all Culex spp. collected in several military locations across the ROK. In this study, we detected only JEV GI in Cx. tritaeniorhynchus in 2021 samples. In contrast, all 2022 and 2023 positive samples were GV and detected in Cx. bitaeniorhynchus, Cx. orientalis, and Cx. pipiens. Results support a shift in JEV genotype in the ROK and suggest that for GV, Culex spp. other than Cx. tritaeniorhynchus may be playing an important role. Full article

Pigs are the most common amplifying hosts of the Japanese encephalitis virus (JEV). In 2016, four residents on Tsushima Island who did not own pig farms were diagnosed with JE. Therefore, a serosurvey was conducted to estimate the risk and seroprevalence of JEV after the outbreak. Sera collected from 560 Tsushima Island residents between January and September 2017 were tested for neutralizing antibodies against JEV strains JaGAr01 (genotype 3) and Muar (genotype 5). Sera collected from six wild boars between June and July 2022 were tested. The seroprevalence rates of neutralizing antibodies against JaGAr01 and Muar were 38.8% and 24.6%, respectively. High anti-JEV neutralizing antibody titers of ≥320 were identified in 16 residents, including 3 younger than 6 years with prior JEV vaccination, 2 in their 40s, and 11 older than 70. However, no anti-JEV-specific IgM was detected. Residents who engaged in outdoor activities had higher anti-JEV antibody titers. Sera from wild boars were negative for JEV RNA, but four of six samples contained neutralizing antibodies against JEV. Therefore, JEV transmission continues on Tsushima Island, even in the absence of pig farms, and wild boars might serve as the amplifying hosts. Full article

Japanese encephalitis virus (JEV), a flavivirus transmitted by mosquitoes, has caused epidemics and severe neurological diseases in Asian countries. In this study, we developed a cDNA infectious clone, pBAC JYJEV3, of the JEV genotype 3 strain (EF571853.1) using a bacterial artificial chromosome (BAC) vector. The constructed infectious clone was transfected into Vero cells, where it exhibited infectivity and induced cytopathic effects akin to those of the parent virus. Confocal microscopy confirmed the expression of the JEV envelope protein. Comparative analysis of growth kinetics revealed similar replication dynamics between the parental and recombinant viruses, with peak titers observed 72 h post-infection (hpi). Furthermore, plaque assays demonstrated comparable plaque sizes and morphologies between the viruses. Cryo-electron microscopy confirmed the production of recombinant virus particles with a morphology identical to that of the parent virus. Immunization studies in mice using inactivated parental and recombinant viruses revealed robust IgG responses, with neutralizing antibody production increasing over time. These results showcase the successful generation and characterization of a recombinant JEV3 virus and provide a platform for further investigations into JEV pathogenesis and vaccine development. Full article

Japanese encephalitis virus (JEV) has a positive-sense single-stranded RNA genome and belongs to the genus Flavivirus of the family Flaviviridae. Persistent JEV infection was previously shown in pig blood cells, which act as a natural reservoir of this virus. We aimed to determine the pathogenicity factors involved in persistent JEV infection by analyzing the pathogenicity and genome sequences of a virus isolated from a persistent infection model. We established persistent JEV infections in cells by inoculating mouse fetus primary cell cultures with the Beijing-1 strain of JEV and then performing repeated infected cell passages, harvesting viruses after each passage while monitoring the plaque size over 100 generations. The virus growth rate was compared among Vero, C6/36, and Neuro-2a cells. The pathogenicity was examined in female ICR mice at several ages. Additionally, we determined the whole-genome sequences. The 134th Beijing-1-derived persistent virus (ME134) grew in Vero cells at a similar rate to the parent strain but did not grow well in C6/36 or Neuro-2a cells. No differences were observed in pathogenicity after intracerebral inoculation in mice of different ages, but the survival time was extended in older mice. Mutations in the persistent virus genomes were found across all regions but were mainly focused in the NS3, NS4b, and 3′NCR regions, with a 34-base-pair deletion found in the variable region. The short deletion in the 3′NCR region appeared to be responsible for the reduced pathogenicity and growth efficiency. Full article

The Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, has a wide host range, extending from pigs and ardeid birds to opportunistic dead-end hosts, such as humans and horses. However, JEV encephalitis infections in aquatic mammals are rare, with only two cases in seals reported to date. Here, we report a lethal case of JEV and Schizophyllum commune co-infection in an aquarium-housed harbor seal in Japan. We isolated JEV from the brain of the dead seal and characterized its phylogeny and pathogenicity in mice. The virus isolate from the seal was classified as genotype GIb, which aligns with recent Japanese human and mosquito isolates as well as other seal viruses detected in China and Korea, and does not exhibit a unique sequence trait distinct from that of human and mosquito strains. We demonstrated that the seal isolate is pathogenic to mice and causes neuronal symptoms. These data suggest that seals should be considered a susceptible dead-end host for circulating JEV in natural settings. Full article

Japanese encephalitis (JE) is a very severe disease characterized by high fatality rates and the development of permanent behavioral, psychiatric, and neurological sequelae among survivors. Japanese encephalitis virus (JEV), a flavivirus, is responsible for JE. In Asia, Genotype I (GI) has emerged as the dominant strain, replacing Genotype III (GIII). However, no clinically approved drug is available to treat JEV infection, and currently available commercial vaccines derived from JEV GIII strains provide only partial protection against GI. Utilizing a reverse genetics system, this study attempted to produce a novel chimeric JEV strain with high efficacy against JEV GI. Accordingly, a GI/GIII intertypic recombinant strain, namely SA14-GI env, was generated by substituting the E region of the GIII SA14-14-2 strain with that of the GI strain, K05GS. The neurovirulence of the mutant virus was significantly reduced in mice. Analysis of the immunogenicity of the chimeric virus revealed that it induced neutralizing antibodies against JEV GI in mice, and the protective efficacy of SA14-GI env was higher than that of SA14-14-2. These findings suggest that SA14-GI env may be a safe and effective live-attenuated vaccine candidate against JEV GI. Full article

RNase H-dependent gapmer antisense oligonucleotides (ASOs) are a promising therapeutic approach via sequence-specific binding to and degrading target RNAs. However, the efficacy and mechanism of antiviral gapmer ASOs have remained unclear. Here, we investigated the inhibitory effects of gapmer ASOs containing locked nucleic acids (LNA gapmers) on proliferating a mosquito-borne flavivirus, Japanese encephalitis virus (JEV), with high mortality. We designed several LNA gapmers targeting the 3′ untranslated region of JEV genomic RNAs. In vitro screening by plaque assay using Vero cells revealed that LNA gapmers targeting a stem-loop region effectively inhibit JEV proliferation. Cell-based and RNA cleavage assays using mismatched LNA gapmers exhibited an underlying mechanism where the inhibition of viral production results from JEV RNA degradation by LNA gapmers in a sequence- and modification-dependent manner. Encouragingly, LNA gapmers potently inhibited the proliferation of five JEV strains of predominant genotypes I and III in human neuroblastoma cells without apparent cytotoxicity. Database searching showed a low possibility of off-target binding of our LNA gapmers to human RNAs. The target viral RNA sequence conservation observed here highlighted their broad-spectrum antiviral potential against different JEV genotypes/strains. This work will facilitate the development of an antiviral LNA gapmer therapy for JEV and other flavivirus infections. Full article

Japanese encephalitis (JE), found in pigs, is a serious mosquito-borne zoonotic infectious disease caused by the Japanese encephalitis virus (JEV). JEV is maintained in an enzootic cycle between mosquitoes and amplifying vertebrate hosts, mainly pigs and wading birds. It is transmitted to humans through the bite of an infected mosquito, allowing the pathogen to spread and cause disease epidemics. However, there is little research on JEV genotype variation in mosquitoes and pigs in Fujian province. Previous studies have shown that the main epidemic strain of JEV in Fujian Province is genotype III. In this study, a survey of mosquito species diversity in pig farms and molecular evolutionary analyses of JEV were conducted in Fujian, China, in the summer of 2019. A total of 19,177 mosquitoes were collected at four sites by UV trap. Four genera were identified, of which the Culex tritaeniorhynchus was the most common mosquito species, accounting for 76.4% of the total (14,651/19,177). Anopheles sinensi (19.25%, 3691/19,177) was the second largest species. High mosquito infection rateswere an important factor in the outbreak. The captured mosquito samples were milled and screened with JEV-specific primers. Five viruses were isolated, FJ1901, FJ1902, FJ1903, FJ1904, and FJ1905. Genetic affinity was determined by analyzing the envelope (E) gene variants. The results showed that they are JEV gene type I and most closely related to the strains SH-53 and SD0810. In this study, it was found through genetic evolution analysis that the main epidemic strain of JE in pig farms changed from gene type III to gene type I. Compared with the SH-53 and SD0810 strains, we found no change in key sites related to antigenic activity and neurovirulence of JEV in Fujian JEV and pig mosquito strains, respectively. The results of the study provide basic data for analyzing the genotypic shift of JEV in Fujian Province and support the prevention and control of JEV. Full article

The Japanese encephalitis virus (JEV) is prevalent in Asian countries, including Korea, Japan, China, Vietnam, and India. JEV is transmitted to humans by Culex mosquitoes. Despite extensive research efforts, no approved antiviral agents are currently available, although JE can be prevented by vaccination. DNA endonuclease-targeted CRISPR trans reporter (DETECTR) is a newly emerging CRISPR-Cas12a-based molecular diagnostic method combined with isothermal nucleic acid amplification. In this study, DETECTR with reverse transcription–recombinase polymerase amplification (RT-RPA) was effectively utilized for JEV diagnosis and detected down to 10 RNA copies for JEV genotype I (GI) and 1 × 10² copies for both GIII and GV, achieving similar sensitivity to RT-PCR while displaying no cross-reaction with other viruses. A one-tube, one-temperature format of DETECTR was further developed, and its efficiency compared with that of conventional DETECTR. Full article