Search

Japanese encephalitis virus is mainly prevalent in the tropical and subtropical regions of Asia and Oceania. Through immunoprecipitation-mass spectrometry analysis using monoclonal antibodies targeting JEV E protein, we found that mosquito Histone 2A protein could bind to JEV particles. The binding of H2A and JEV was detected in the salivary gland and supernatant of mosquito cells. Furthermore, RNA interference experiments in vitro and in vivo confirmed that H2A protein promotes JEV infection in mosquitoes. In summary, we found that mosquito H2A is a factor that supports JEV infection and can potentially facilitate cross-species transmission of JEV. [ABSTRACT FROM AUTHOR]

Nonstructural protein 2A (NS2A) of the Japanese encephalitis virus (JEV) contributes to viral replication and pathogenesis; however, a lack of NS2A-specific antibodies restricts studies on the underlying mechanisms. In this study, we constructed a recombinant JEV with a hemagglutinin (HA)-tagged NS2A (JEV-HA/NS2A/∆NS1') to overcome this challenge. An HA-tag was fused to the N-terminus of NS2A (HA-NS2A) at the intergenic junction between NS1 and NS2A. A peptide linker, "FNG", was added to the N-terminus of HA-tag to ensure correct cleavage between the C-terminus of NS1 and the N-terminus of HA-NS2A. To avoid the side effects of an unwanted NS1' tagged with HA (HA-NS1'), an alanine-to-proline (A30P) substitution was introduced at residue 30 of NS2A to abolish HA-NS1' production. The HA-tag insertion and A30P substitution were stably present in JEV-HA/NS2A/∆NS1' after six passages and did not exhibit any significant effects on viral replication and plaque morphology. Taking advantage of HA-NS2A, we examined the activities of NS2A during JEV infection in vitro using anti-HA antibodies. NS2A was observed to be localized to the endoplasmic reticulum and interact with viral NS2B and NS3 during virus infection. These data suggest that JEV-HA/NS2A/∆NS1' can serve as a model for the analysis of the biological characteristics and functions of NS2A in vitro during JEV infection. [ABSTRACT FROM AUTHOR]

A total of 548 mosquitoes were collected from different animal farms located near to highly populated cities in Xinjiang and were subjected to metagenomic next‐generation sequencing (mNGS). The mNGS data demonstrated that 18,842 (XJ1 strain) and 1,077 (XJ2 strain) of Japanese encephalitis virus (JEV)‐related reads were detected in XJ1 and XJ2 mosquito samples collected from Wushi and Wensu counties of Aksu area, which accounted for 0.032% and 0.006% of the total clean reads generated from XJ1 and XJ2 samples, respectively. The Bayesian molecular phylogenetic analysis suggested that XJ1 and XJ2 strains belonged to JEV genotype III and were clustered with JEV strains isolated in China. Notably, Bayesian molecular time line phylogeny revealed that XJ1 strain shared its MRCA with JEV GSS strain about 67 YA, suggesting that XJ1 strain likely originated from linages closely related to GSS strain and spread to Xinjiang later. Overall, these findings suggest that Xinjiang was probably not free from JEV, and thus, a further surveillance of JEV is required in Xinjiang. [ABSTRACT FROM AUTHOR]

Japanese encephalitis virus (JEV) genotype dominance has shifted to genotype I (GI) from genotype III (GIII) in China as demonstrated by molecular epidemiological surveillance. In this study, we performed a serological survey in JEV-non-vaccinated pigs to confirm JEV genotype shift at the sero-epidemiological level. The average ratio of GI/GIII infection was 1.87, suggesting co-circulation of GI and GIII infections with GI infection being more prevalent in pigs in China. To gain an insight into the reasons for this JEV genotype shift, the replication kinetics of seven recently-isolated JEV isolates including three GI strains and four GIII strains were compared in mosquito C6/36 cells, chicken fibroblast cells (DF-1) and porcine iliac artery endothelial cells (PIEC). We observed that GI strains replicated more efficiently than GIII strains in DF-1 and PIEC cells, particularly in DF-1 cells with titers reaching 22.9–225.3 fold higher than GIII strains. This shows an enhanced replication efficiency of GI viruses in avian cells. To examine this enhanced replication efficiency in vivo, young domestic ducklings were used as the animal model and inoculated with GI and GIII strains at day 2 post-hatching. We observed that GI-inoculated ducklings developed higher viremia titers and displayed a comparatively longer viremic duration than GIII-inoculated ducklings. These results conform to the hypothesis of an enhanced replication efficiency for GI viruses in birds. There are 36 amino acid differences between GI and GIII viruses, some of which may be responsible for the enhanced replication efficiency of GI viruses in birds. Based on these findings, we speculated that the enhanced replication of GI viruses in birds would have resulted in higher exposure and therefore infection in mosquitoes, which could result in an increased transmission efficiency of GI viruses in the birds-mosquitoes-birds enzootic transmission cycle, thereby contributing to JEV genotype shift. [ABSTRACT FROM AUTHOR]

Virus-like particles (VLPs) are non-replicative vectors for the delivery of heterologous epitopes and are considered one of the most potent inducers of cellular and humoral immune responses in mice and guinea pigs. In the present study, VLP-JEVe was constructed by the insertion of six Japanese encephalitis virus (JEV) envelope protein epitopes into different surface loop regions of PPV VP2 by the substitution of specific amino acid sequences without altering the assembly of the virus; subsequently, the protective efficacy of this VLP-JEVe was evaluated against JEV challenge in mice and guinea pigs. Mice immunized with the VLP-JEVe antigen developed high titers of neutralizing antibodies and 100% protection against lethal JEV challenge. The neutralizing and hemagglutination inhibition (HI) antibody responses were also induced in guinea pigs vaccinated with VLP-JEVe. In addition, immunization with VLP-JEVe in mice induced effective neutralizing antibodies and protective immunity against PPV (porcine parvovirus) challenge in guinea pigs. These studies suggest that VLP-JEVe produced as described here could be a potential candidate for vaccine development. [ABSTRACT FROM AUTHOR]

Understanding the proteolytic processing of polyprotein mediated by NS2B-NS3 protease contributes to the exploration of the mechanisms underlying infection of Japanese encephalitis virus (JEV), a zoonotic flavivirus. In this study, eukaryotic and prokaryotic cell models were employed to identify the cleavage sites mediated by viral NS2B-NS3 protease in JEV polyprotein. Artificial green fluorescent protein (GFP) substrates that contained the predicted cleavage site sequences of JEV polyprotein were expressed in swine testicle (ST) cells in the presence and absence of JEV infection, or co-expressed in E. coli with the recombinant NS2B-NS3 protease that was generated by fusing the N-terminal protease domain of NS3 to the central hydrophilic domain of NS2B. The cleavage of GFP substrates was examined by western blot. Among twelve artificial GFP substrates containing the cleavage site sequences predictively processed by host cell and/or NS2B-NS3 proteases, all sites were found to be cleaved by host cell proteases with different efficiencies. The sites at internal C, NS2A/NS2B, NS2B/NS3 and NS3/NS4A junctions, but not the sites at internal NS3, internal NS4A and NS4B/NS5 junctions were identified to be cleaved by JEV NS2B-NS3 protease. These data provide insight into the proteolytic processing of polyprotein, which is useful for understanding JEV replication and pathogenesis. [ABSTRACT FROM AUTHOR]

Japanese encephalitis virus (JEV) is a zoonotic pathogen that is maintained by mosquito vectors and vertebrate hosts including birds in a natural transmission cycle. Domestic ducklings are sensitive to JEV infection, but the clinical responses of domestic ducklings to natural JEV infection are unknown. In this study, we simulated the natural JEV infection of domestic ducklings via JEV-infected mosquito bites to evaluate the pathogenicity of JEV in domestic ducklings. Specific pathogen-free domestic ducklings were infected at day 2 post-hatching with JEV-infected Culex pipiens mosquito bites and monitored for clinical responses. Among 20 ducklings exposed to JEV-infected mosquitoes, six showed mild and non-characteristic clinical signs starting at two days post-infection, then died suddenly with neurological signs of opisthotonos (a condition of spasm of the back muscles causing the head and limbs to bend backward and the trunk to arch forward) between two and three days post-infection. The mortality of the affected ducklings was 30% (6/20). Multifocal lymphohistiocytic perivascular cuffs and lymphohistiocytic meningitis were macroscopically observed in the affected duckling brains. JEV was detected in the cytoplasm of neuronal cells in the affected duckling brains by immunohistochemical assays and was recovered from the affected duckling brains by viral isolation. These observations indicated that JEV infection via mosquito bites causes mortality associated with viral encephalitis in newly hatched domestic ducklings, thus demonstrating the potential pathogenicity of JEV in domestic ducklings under natural conditions. [ABSTRACT FROM AUTHOR]

The phenotypic and genotypic characteristics of a live-attenuated genotype I (GI) strain (SD12-F120) of Japanese encephalitis virus (JEV) were compared with its virulent parental SD12 strain to gain an insight into the genetic changes acquired during the attenuation process. SD12-F120 formed smaller plaque on BHK-21 cells and showed reduced replication in mouse brains compared with SD12. Mice inoculated with SD12-F120 via either intraperitoneal or intracerebral route showed no clinical symptoms, indicating a highly attenuated phenotype in terms of both neuroinvasiveness and neurovirulence. SD12-F120 harbored 29 nucleotide variations compared with SD12, of which 20 were considered silent nucleotide mutations, while nine resulted in eight amino acid substitutions. Comparison of the amino acid variations of SD12-F120 vs. SD12 pair with those from other four isogenic pairs of the attenuated and their virulent parental strains revealed that the variations at E138 and E176 positions of E protein were identified in four and three pairs, respectively, while the remaining amino acid variations were almost unique to their respective strain pairs. These observations suggest that the genetic changes acquired during the attenuation process were likely to be strain-specific and that the mechanisms associated with JEV attenuation/virulence are complicated. [ABSTRACT FROM AUTHOR]

Highlights • Some JEV strains caused notable mortality in newly hatched domestic ducklings. • JEV infection resulted in stunted growth in newly hatched domestic ducklings. • Response and susceptibility of ducklings to JEV infection differed among JEV strains. Abstract Japanese encephalitis virus (JEV) is a zoonotic flavivirus that is transmitted by mosquitoes and vertebrate-amplifying hosts, including birds. Domestic ducks are susceptible to JEV infection and develop various levels of viremia. We tested the pathogenicities of seven JEV strains in newly hatched domestic ducklings. All inoculated ducklings showed stunted growth. Two JEV strains caused notable mortalities of 12.7% and 31.7%, respectively, highlighting that some emerged JEV strains may thus be pathogenic in newly hatched domestic ducklings. [ABSTRACT FROM AUTHOR]

Japanese encephalitis (JE) is a viral encephalitis disease caused by infection with the Japanese encephalitis virus (JEV). The virus can cross the blood-brain barrier and cause death or long-term sequela in infected humans or animals. In this study, we first investigated the distribution of JEV infection in brain and further analyzed the dynamic change in inflammation related genes, chemokines, as well as pathological characteristics. Results demonstrated that CCR2 and CCR5 antagonist could significantly inhibit the inflammation. The mice treated with CCR2 and CCR5 antagonists had a higher survival rate between 60% and 70%, respectively. In summary, our study thoroughly illustrated the characteristics of the dynamic change in inflammation related genes and chemokines induced by JEV infection. We further indicated that CCR5 and CCR2 are potential targets for treatment of JE. [ABSTRACT FROM AUTHOR]

• Mosquito defensin facilitates JEV infection within mosquito. • Mosquito defensin down-regulates cell surface antiviral protein HSC70B. • Mosquito defensin-HSC70B axis impacts JEV infection in in vivo and in vitro. Japanese encephalitis virus (JEV) is a viral zoonosis that can cause viral encephalitis, death and disability whose primary vector is the Culex mosquito. Viral infection induces a series of antimicrobial peptide responses in mosquitoes, and the effector defensin enhances JEV replication in mosquitoes. However, the underlying mechanisms by which defensin enhances JEV are not fully understood. Here, we found that mosquito defensin could downregulate the antiviral protein HSC70B and enhance virus infection in mosquitoes. The cell-surface protein HSC70B was significantly downregulated by JEV infection and defensin treatment. Low levels of HSC70B were beneficial to JEV infection in mosquitoes. Taken together, these findings show that defensin and HSC70B axis facilitates JEV infection in the mosquito. [ABSTRACT FROM AUTHOR]

• SD12-F120VC has higher titer and adaptation to growth in Vero cells • SD12-F120VC has high attenuated phenotype in suckling mice and has small plaque morphology • SD12-F120VC provided the complete protection against challenge with homologous strain, but not for the heterologous strain. • The neutralizing antibodies titer was high against homologous strains but low for heterologous strain. • Some mutations in the pr domain of prM may be involved in Vero cell adaptation The SD12-F120 is a live-attenuated genotype I strain of Japanese encephalitis virus (JEV) and was obtained by serial passage of wild-type strain SD12 on BHK-21 cells combined with multiple plaque purification and virulence selection in mice. The large scale production and vast clinical trials always demand ideal safety and efficacy profile of live-attenuated vaccines. In the present study, SD12-F120VC has undergone serial passaging of P1-P30 in WHO qualified Vero cells to assess the potential effect of adaptation to growth on Vero cells. The series of experiments showed that vaccine SD12-F120VC (Vero cell adapted) variants have consistently increased in peak virus titer compared to early passages and have good adaptation to growth in Vero cells. The animal experiments showed that Vero cell adapted SD12-F120VC variants have attenuation phenotype in suckling mice and the plaque morphology for all SD12-F120VC variants was small. Vaccination of mice with SD12-F120VC vaccine produced complete protection for homologous SD12 genotype I strain, but failed to give the complete protection of vaccinated mice against the challenge of heterologous N28 genotype III strain. In response to immunization of SD12-F120VC in mice, the neutralizing antibodies titer against homologous SD12-F120VC and SD12 (GI) was higher than heterologous N28 (GIII) strain. The prM protein has 6 amino acid substitutions, of which 5 amino acid changes were confined at the start of the pr domain in the ∼40 amino acids, and some mutations in the pr domain of prM might contribute to Vero cell adaptation. Our findings in this study are important for validation, evaluation and quality control study of live attenuated flaviviruses vaccines and show that Vero cells are a suitable substrate for the production of a safe and stable live-attenuated JEV vaccine. [ABSTRACT FROM AUTHOR]

• Rapid differention of Genotype I and III Japanese Encephalitis Virus by a novel duplex TaqMan probe-based RT-qPCR assay. • Rapid detection of Japanese Encephalitis Virus in clinical samples from pigs and mosquitoes. • Co-circulation of GI and GIII infections with GI infection being more prevalent in pigs or mosquitoes in eastern China. Japanese Encephalitis (JE) is an acute infectious disease that threatens both human and pig populations throughout Asia. JE is caused by the Japanese Encephalitis Virus (JEV), of which genotype III (GIII) had been the most prevalent strain throughout Asia, but recent studies have shown that genotype I (GI) has replaced GIII as the predominant version. Pigs and mosquitoes play a primary role in JEV transmission. However, a method for the rapid differentiation between JEV G I and G III remains unavailable. This study aimed to establish a rapid JEV genotyping method using novel duplex TaqMan RT-qPCR assay.specific primer and probes located in the PrM/M gene that were able to specifically differentiate GI and GIII JEV, was selected as the duplex TaqMan RT-qPCR target.The specificity, sensitivity and reproducibility test of this assay were validated. The sensitivity of the assay was 10 genomic RNA copies for both GI and GIII JEV in field mosquito and pig samples,and more sensitive than the current methods. In addition, the novel assay can be completed in less than 1 h. Therefore, This duplex TaqMan RT-qPCR assay is a promising tool for rapid differential detection and epidemiology of GI and GIII JEV strains in China. The results showed that co-circulation of GI and GIII infections with GI infection being more prevalent in pigs or mosquitoes in eastern China. [ABSTRACT FROM AUTHOR]

Japanese Encephalitis virus (JEV) is a zoonotic flavivirus that is the most significant etiological agent of childhood viral neurological infections. However, no specific antiviral drug is currently available to treat JEV infections. The JEV envelope (E) protein is a class II viral fusion protein that mediates host cell entry, making interference with the interaction between the E protein of JEV and its cognate receptors an attractive strategy for anti-JEV drug development. In this study, we identified a peptide derived from a phage display peptide library against the E protein of JEV, designated P1, that potentially inhibits in vitro and in vivo JEV infections. P1 inhibits JEV infection in BHK-21 cells with 50% inhibitory capacity at a concentration of 35.9 μM. The time-of-addition assay indicates that JEV replication is significantly inhibited during pre-infection and co-infection of P1 with JEV while post-infection treatments with P1 have very little impact on JEV proliferation, showing that P1 inhibits JEV infection at early stages and indicating the potential prophylactic effect of P1. We adapted an in vitro BiFC assay system and demonstrated that P1 interacts with JEV E proteins and blocks their entry into cells. We also evaluated the therapeutic efficacy of P1 in a lethal JEV mouse model exhibiting systemic and brain infections. Interestingly, P1 treatment protected C57BL/6 mice against mortality, markedly reduced the viral loads in blood and brain, and diminished the histopathological lesions in the brain cells. In addition to controlling systemic infection, P1 has a very low level of cytotoxicity and acts in a sequence-specific manner, as scrambled peptide sP1 does not show any antiviral activity. In conclusion, our in vitro and in vivo experimental findings show that P1 possesses antiviral activity against JEV infections, is safe to use, and has potential for further development as an antiviral treatment against JEV infections. • Peptide P1 target to E protein by phage display screening inhibits JEV infection by blocking JEV attachment to host cells. • P1 decreases histopathological damages and viral load in mouse brain. • P1 increases survival rate of mice in lethal JEV challenge after preincubation with JEV. [ABSTRACT FROM AUTHOR]