Japanese Encephalitis Virus Genome Research Articles

One-Step Dual-Color Labeling of Viral Envelope and Intraviral Genome with Quantum Dots Harnessing Virus Infection.

Labeling the genome and envelope of a virus with multicolor quantum dots (QDs) simultaneously enables real-time monitoring of viral uncoating and genome release, contributing to our understanding of virus infection mechanisms. However, current labeling techniques require genetic modification, which alters the virus's composition and infectivity. To address this, we utilized the CRISPR/Cas13 system and a bioorthogonal metabolic method to label the Japanese encephalitis virus (JEV) genome and envelopes with different-colored QDs in situ. This technique allows one-step two-color labeling of the viral envelope and intraviral genome with QDs harnessing virus infection. In combination with single-virus tracking, we visualized JEV uncoating and genome release in real time near the endoplasmic reticulum of live cells. This labeling strategy allows for real-time visualization of uncoating and genome release at the single-virus level, and it is expected to advance the study of other viral infection mechanisms.

A Recombinant Genotype I Japanese Encephalitis Virus Expressing a Gaussia Luciferase Gene for Antiviral Drug Screening Assay and Neutralizing Antibodies Detection

Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in humans throughout Asia. In the past twenty years, the emergence of the genotype I (GI) JEV as the dominant genotype in Asian countries has raised a significant threat to public health security. However, no clinically approved drug is available for the specific treatment of JEV infection, and the commercial vaccines derived from the genotype III JEV strains merely provided partial protection against the GI JEV. Thus, an easy-to-perform platform in high-throughput is urgently needed for the antiviral drug screening and assessment of neutralizing antibodies specific against the GI JEV. In this study, we established a reverse genetics system for the GI JEV strain (YZ-1) using a homologous recombination strategy. Using this reverse genetic system, a gaussia luciferase (Gluc) expression cassette was inserted into the JEV genome to generate a reporter virus (rGI-Gluc). The reporter virus exhibited similar growth kinetics to the parental virus and remained genetically stable for at least ten passages in vitro. Of note, the bioluminescence signal strength of Gluc in the culture supernatants was well correlated with the viral progenies determined by viral titration. Taking advantage of this reporter virus, we established Gluc readout-based assays for antiviral drug screening and neutralizing antibody detection against the GI JEV. These Gluc readout-based assays exhibited comparable performance to the assays using an actual virus and are less time consuming and are applicable for a high-throughput format. Taken together, we generated a GI JEV reporter virus expressing a Gluc gene that could be a valuable tool for an antiviral drug screening assay and neutralization assay.

Molecular detection and characterisation of the first Japanese encephalitis virus belonging to genotype IV acquired in Australia.

A fatal case of Japanese encephalitis (JE) occurred in a resident of the Tiwi Islands, in the Northern Territory of Australia in February 2021, preceding the large JE outbreak in south-eastern Australia in 2022. This study reports the detection, whole genome sequencing and analysis of the virus responsible (designated JEV/Australia/NT_Tiwi Islands/2021). Reverse transcription quantitative PCR (RT-qPCR) testing was performed on post-mortem brain specimens using a range of JE virus (JEV)-specific assays. Virus isolation from brain specimens was attempted by inoculation of mosquito and mammalian cells or embryonated chicken eggs. Whole genome sequencing was undertaken using a combination of Illumina next generation sequencing methodologies, including a tiling amplicon approach. Phylogenetic and selection analyses were performed using alignments of the Tiwi Islands JEV genome and envelope (E) protein gene sequences and publicly available JEV sequences. Virus isolation was unsuccessful and JEV RNA was detected only by RT-qPCR assays capable of detecting all JEV genotypes. Phylogenetic analysis revealed that the Tiwi Islands strain is a divergent member of genotype IV (GIV) and is closely related to the 2022 Australian outbreak virus (99.8% nucleotide identity). The Australian strains share highest levels of nucleotide identity with Indonesian viruses from 2017 and 2019 (96.7-96.8%). The most recent common ancestor of this Australian-Indonesian clade was estimated to have emerged in 2007 (95% HPD range: 1998-2014). Positive selection was detected using two methods (MEME and FEL) at several sites in the E and non-structural protein genes, including a single site in the E protein (S194N) unique to the Australian GIV strains. This case represents the first detection of GIV JEV acquired in Australia, and only the second confirmed fatal human infection with a GIV JEV strain. The close phylogenetic relationship between the Tiwi Islands strain and recent Indonesian viruses is indicative of the origin of this novel GIV lineage, which we estimate has circulated in the region for several years prior to the Tiwi Islands case.

Molecular pathogenesis of Japanese encephalitis and possible therapeutic strategies.

Japanese encephalitis virus (JEV), a single-stranded, enveloped RNA virus, is a health concern across Asian countries, associated with severe neurological disorders, especially in children. Primarily, pigs, bats, and birds are the natural hosts for JEV, but humans are infected incidentally. JEV requires a few host proteins for its entry and replication inside the mammalian host cell. The endoplasmic reticulum (ER) plays a significant role in JEV genome replication and assembly. During this process, the ER undergoes stress due to its remodelling and accumulation of viral particles and unfolded proteins, leading to an unfolded protein response (UPR). Here, we review the overall strategy used by JEV to infect the host cell and various cytopathic effects caused by JEV infection. We also highlight the role of JEV structural proteins (SPs) and non-structural proteins (NSPs) at various stages of the JEV life cycle that are involved in up- and downregulation of different host proteins and are potentially relevant for developing efficient therapeutic drugs.Graphical abstract

Cellular Interleukin Enhancer-Binding Factor 2, ILF2, Inhibits Japanese Encephalitis Virus Replication In Vitro.

Japanese encephalitis virus (JEV) is a zoonotic mosquito-borne flavivirus which is the leading causative agent of viral encephalitis in endemic regions. JEV NS3 is a component of the viral replicase complex and is a multifunctional protein. In this study, interleukin enhancer-binding factor 2 (ILF2) is identified as a novel cellular protein interacting with NS3 through co-immunoprecipitation assay and LC-MS/MS. The expression of ILF2 is decreased in JEV-infected human embryonic kidney (293T) cells. The knockdown of endogenous ILF2 by special short hairpin RNA (shRNA) positively regulates JEV propagation, whereas the overexpression of ILF2 results in a significantly reduced JEV genome synthesis. Further analysis revealed that the knockdown of ILF2 positively regulates viral replication by JEV replicon system studies. These results suggest that ILF2 may act as a potential antiviral agent against JEV infection.

Towards Obtaining a Nanoscale Structure of Terminal Regions of japanese Encephalitis Virus Genome

Japanese encephalitis virus (JEV) belongs to the Flaviviridae family of RNA viruses - a family that includes other pathogenic viruses such as Dengue, West Nile, and Zika. These viruses have a conserved 3′ terminal region that plays a significant role in viral replication by mediating long-range interactions with the 5′ terminal region of the genome. The 3′ terminal region also interacts with host proteins (e.g. DEAD-box family of RNA helicases), aiding viral replication. Despite the significance these terminal regions play in human infection, their structural details are largely unknown. Plasmids were designed that encode for the entire ∼600 kb long 3′ terminal region of JEV, as well as numerous fragments therein. The resulting RNA was purified using size-exclusion chromatography (SEC). Structural studies of the constructs were performed using small angle X-ray scattering (SAXS) to generate low-resolution models of the RNA and selective 2' hydroxyl acetylation analyzed by primer extension (SHAPE). Using in vitro transcription, full-length and fragments of the JEV 3′ terminal region RNA have been generated. Preliminary SAXS data indicate that one of the segments of the terminal region adopts multiple conformations in solution, whereas other regions have elongated shapes. The data is being combined with that of SHAPE in an iterative process to obtain high-resolution models of these constructs. Future studies will focus on the structural analysis of the 3′ terminal region in complex with the 5′ terminal region as well as various host proteins, including DDX5, to give further insight into viral replication and thus give rise to potential new antiviral targets.

The conserved stem-loop II structure at the 3' untranslated region of Japanese encephalitis virus genome is required for the formation of subgenomic flaviviral RNA.

Flaviviruses accumulate abundant subgenomic RNA (sfRNA) in infected cells. It has been reported that sfRNA results from stalling of host 5’-to-3’ exoribonuclease XRN1 at the highly structured RNA of the 3’ untranslated region (UTR). Although XRN1 digestion of a 3’-terminal 800-nt RNA could stall at a position to generate the sfRNA in vitro, we found that knocking out XRN1 had no effect on the accumulation of sfRNA in Japanese encephalitis virus (JEV) infected cells. Mutagenesis studies revealed that the stemloop II (SLII) at the 3’ UTR is required for the accumulation of sfRNA. According to the results of an in vitro RNA-dependent RNA polymerase (RdRp) assay, the (-)10431-10566 RNA fragment, containing the putative promoter on the antigenome for the sfRNA transcription, binds to RdRp protein and exhibits a strong promoter activity. Taken together, our results indicate that the JEV sfRNA could be transcribed initially and then be trimmed by XRN1 or other unidentified exoribonucleases.

Full-Length Genome Sequence of Japanese Encephalitis Virus Strain FC792, Isolated from Guangxi, China.

ABSTRACTWe report here the complete genomic sequence of Japanese encephalitis virus (JEV) strain FC792, isolated from aborted fetuses of sows which were unimmunized with JEV vaccines in Guangxi Province, southern China. The complete JEV genome of strain FC792 had the highest nucleotide homology (99.7%) and amino acid identity (99.4%) with the sequence of JEV strain SA14-14-2 (GenBank accession number AF315119). Phylogenetic analysis showed that strain FC792 had the closest phylogenetic relationship to the sequence of strain YUNNAN0901 (GenBank accession number JQ086762). This study will help us understand the molecular pathogenesis and genetic diversity of genotype III Japanese encephalitis virus in pigs.

Single-Round Infectious Particle Antiviral Screening Assays for the Japanese Encephalitis Virus.

Japanese Encephalitis virus (JEV) is a mosquito-borne flavivirus with a positive-sense single-stranded RNA genome that contains a big open reading frame (ORF) flanked by 5′- and 3′- untranslated regions (UTRs). Nearly 30,000 JE cases with 10,000 deaths are still annually reported in East Asia. Although the JEV genotype III vaccine has been licensed, it elicits a lower protection against other genotypes. Moreover, no effective treatment for a JE case is developed. This study constructed a pBR322-based and cytomegaloviruses (CMV) promoter-driven JEV replicon for the production of JEV single-round infectious particles (SRIPs) in a packaging cell line expressing viral structural proteins. Genetic instability of JEV genome cDNA in the pBR322 plasmid was associated with the prokaryotic promoter at 5′ end of the JEV genome that triggers the expression of the structural proteins in E. coli. JEV structural proteins were toxic E. coli, thus the encoding region for structural proteins was replaced by a reporter gene (enhanced green fluorescent protein, EGFP) that was in-frame fused with the first eight amino acids of the C protein at N-terminus and the foot-and-mouth disease virus (FMDV) 2A peptide at C-terminus in a pBR322-based JEV-EGFP replicon. JEV-EGFP SRIPs generated from JEV-EGFP replicon-transfected packaging cells displayed the infectivity with cytopathic effect induction, self-replication of viral genomes, and the expression of EGFP and viral proteins. Moreover, the combination of JEV-EGFP SRIP plus flow cytometry was used to determine the half maximal inhibitory concentration (IC50) values of antiviral agents according to fluorescent intensity and positivity of SRIP-infected packaging cells post treatment. MJ-47, a quinazolinone derivative, significantly inhibited JEV-induced cytopathic effect, reducing the replication and expression of JEV-EGFP replicon in vitro. The IC50 value of 6.28 µM for MJ-47 against JEV was determined by the assay of JEV-EGFP SRIP infection in packaging cells plus flow cytometry that was more sensitive, effective, and efficient compared to the traditional plaque assay. Therefore, the system of JEV-EGFP SRIPs plus flow cytometry was a rapid and reliable platform for screening antiviral agents and evaluating antiviral potency.

Complete Genome Sequence of a Genotype III Japanese Encephalitis Virus, Isolated from Pigs in Sichuan, China.

A complete genomic sequence of Japanese encephalitis virus (JEV) was detected by viral metagenome analysis on aborted piglets. A phylogenetic analysis of this genome reveals that it is highly similar to previously reported India JEV genomes. The complete JEV sequence is 10,718 nucleotides long.

Genetic instability of Japanese encephalitis virus cDNA clones propagated in Escherichia coli.

The genetic instability of Flavivirus cDNA clones in transformed bacteria is a common phenomenon. Herein, a cDNA fragment of the nucleotide (nt) 1-2913 of the genome of a flavivirus, Japanese encephalitis virus (JEV), was used to investigate factors that caused the instability of cDNA clones. Several cDNA fragments with different 5'- or 3'-termini of the 2913-nt cDNA were obtained by PCR amplification or restriction enzyme digestion and cloned into a pCR-Blunt II-TOPO vector. All the cDNA fragments were stably propagated at 25 °C. However, the 5'-untranslated region and half of the 3'-E gene could cause the instability of the 2913-nt cDNA at 37 °C. The 5'-terminus sequences of the 2913-nt fragment were subjected to testing of the prokaryotic promoter activity by luciferase assay and Western blot. The sequences of 54-120 nt of the JEV genome exhibited high prokaryotic promoter activity at 37 °C, and the activity declined markedly at 25 °C. These findings revealed that the high prokaryotic promoter activity of the 54-120 nt sequences of the JEV genome together with expression of JEV structural genes determined the instability of a JEV cDNA clone. Growth at room temperature may reduce the prokaryotic promoter activity of 5'-sequences of the JEV genome and could represent an effective way to improve the stability of flavivirus cDNA clones in host bacteria.

An in vitro recombination-based reverse genetic system for rapid mutagenesis of structural genes of the Japanese encephalitis virus.

Japanese encephalitis virus (JEV) is one of the most common pathogens of severe viral encephalitis, which is a severe threat to human health. Despite instability of the JEV genome in bacteria, many strategies have been developed to establish molecular clone systems of JEV, providing convenient tools for studying the virus life cycle and virus-host interactions. In this study, we adapted an In-Fusion enzyme-based in vitro recombination method to construct a reverse genetic system of JEV, thereby providing a rapid approach to introduce mutations into the structural genes. A truncated genome without the structural genes was constructed as the backbone, and the complementary segment containing the structural genes was recombined in vitro, which was then transfected directly into virus-permissive cells. The progeny of the infectious virus was successfully detected in the supernatant of the transfected cells, and showed an identical phenotype to its parental virus. To provide a proof-of-principle, the 12 conserved cysteine residues in the envelope (E) protein of JEV were respectively mutated using this approach, and all mutations resulted in a complete failure to generate infectious virus. However, a leucine-tophenylanine mutation at amino acid 107 of the E protein did not interfere with the production of the infectious virus. These results suggested that all 12 cysteines in the E protein are essential for the JEV life cycle. In summary, a novel reverse genetic system of JEV was established for rapidly introducing mutations into structural genes, which will serve as a useful tool for functional studies.

Profiling of viral proteins expressed from the genomic RNA of Japanese encephalitis virus using a panel of 15 region-specific polyclonal rabbit antisera: implications for viral gene expression.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is closely related to West Nile (WN), yellow fever (YF), and dengue (DEN) viruses. Its plus-strand genomic RNA carries a single open reading frame encoding a polyprotein that is cleaved into three structural (C, prM/M, and E) and at least seven nonstructural (NS1/NS1', NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins, based on previous work with WNV, YFV, and DENV. Here, we aimed to profile experimentally all the viral proteins found in JEV-infected cells. We generated a collection of 15 JEV-specific polyclonal antisera covering all parts of the viral protein-coding regions, by immunizing rabbits with 14 bacterially expressed glutathione-S-transferase fusion proteins (for all nine viral proteins except NS2B) or with a chemically synthesized oligopeptide (for NS2B). In total lysates of JEV-infected BHK-21 cells, immunoblotting with these antisera revealed: (i) three mature structural proteins (~12-kDa C, ~8-kDa M, and ~53-kDa E), a precursor of M (~24-kDa prM) and three other M-related proteins (~10-14 kDa); (ii) the predicted ~45-kDa NS1 and its frameshift product, ~58-kDa NS1', with no evidence of the predicted ~25-kDa NS2A; (iii) the predicted but hardly detectable ~14-kDa NS2B and an unexpected but predominant ~12-kDa NS2B-related protein; (iv) the predicted ~69-kDa NS3 plus two major cleavage products (~34-kDa NS3N-term and ~35-kDa NS3C-term), together with at least nine minor proteins of ~16-52 kDa; (v) the predicted ~14-kDa NS4A; (vi) two NS4B-related proteins (~27-kDa NS4B and ~25-kDa NS4B'); and (vii) the predicted ~103-kDa NS5 plus at least three other NS5-related proteins (~15 kDa, ~27 kDa, and ~90 kDa). Combining these data with confocal microscopic imaging of the proteins’ intracellular localization, our study is the first to provide a solid foundation for the study of JEV gene expression, which is crucial for elucidating the regulatory mechanisms of JEV genome replication and pathobiology.

Phylogeny and Homologous Recombination in Japanese Encephalitis Viruses

Japanese encephalitis virus (JEV) is a significant causative agent of arthropod-borne encephalitis and what is less clear that the factors cause the virus wide spread. The objective was to confirm whether the homologous recombination imposed on JEV. The phylogenetic and homologous recombination analyses were performed based on 163 complete JEV genomes which were recently isolated. They were still separated into five genotypes (GI-GV) and the most of recently isolated JEVs were GI rather than GIII in Asian areas including mainland China. Two recombinant events were identified in JEV and the evidence of the recombination was observed between China and Japan isolates that partitioned into two distinct subclades, but still the same genotype (GIII). Our data further suggested that most of the nucleotides in JEV genome were under negative selection; however, changes within codon 2 316 (amino acid NS4b-44) showed an evidence of the positive selection.

Molecular Characterization of Full-length Genome of Japanese Encephalitis Virus Genotype V Isolated from Tibet, China

ObjectiveTo determine the molecular characterization of full-length genome of Japanese encephalitis virus (JEV) genotype V. MethodsThe full-length nucleotide sequences of JEV strains isolated from different locations and sources were used in sequence and phylogenetic analysis. ResultsThe full-length genome of genotypes V JEV, XZ0934, and Muar strain were composed of 10 983 and 10 988 nucleotides respectively and shared a lower level of identity with JEV genotypes I-IV, ranging from 78.4% (G I, KV1899) to 79.7% (G III, JaGAr01), for the nucleotide sequences, and from 90.0% (G I, KV1899) to 91.8% (G III, JaGAr01) for the amino acid sequences. The open reading frame (ORF) of JEV genotype V spanned nucleotides 96 to 10 397 and encoded 3 433 amino acids. Interestingly, a comparison with JEV genotype I-IV revealed that 3 nucleotides (encoded with a serine residue) were inserted in the NS4A gene of JEV genotype V, and the insertion of nucleotides was also found in downstream of the ORF stop codon in 3’-untranslated region. Moreover, numerous amino acid mutations were observed in 3 functional domains of the E gene of JEV genotype V. ConclusionsThe molecular characterization of JEV genotype V is significantly different from that of the known genotypes I-IV. The mutations located in the coding region and the non-coding region may be molecular markers of JEV genotype V and warrant further studies to determine their effects on biology and immunogenicity of genotype V strains.

A novel approach to propagate flavivirus infectious cDNA clones in bacteria by introducing tandem repeat sequences upstream of virus genome.

Despite tremendous efforts to improve the methodology for constructing flavivirus infectious cDNAs, the manipulation of flavivirus cDNAs remains a difficult task in bacteria. Here, we successfully propagated DNA-launched type 2 dengue virus (DENV2) and Japanese encephalitis virus (JEV) infectious cDNAs by introducing seven repeats of the tetracycline-response element (7×TRE) and a minimal cytomegalovirus (CMVmin) promoter upstream of the viral genome. Insertion of the 7×TRE-CMVmin sequence upstream of the DENV2 or JEV genome decreased the cryptic E. coli promoter (ECP) activity of the viral genome in bacteria, as measured using fusion constructs containing DENV2 or JEV segments and the reporter gene Renilla luciferase in an empty vector. The growth kinetics of recombinant viruses derived from DNA-launched DENV2 and JEV infectious cDNAs were similar to those of parental viruses. Similarly, RNA-launched DENV2 infectious cDNAs were generated by inserting 7×TRE-CMVmin, five repeats of the GAL4 upstream activating sequence, or five repeats of BamHI linkers upstream of the DENV2 genome. All three tandem repeat sequences decreased the ECP activity of the DENV2 genome in bacteria. Notably, 7×TRE-CMVmin stabilized RNA-launched JEV infectious cDNAs and reduced the ECP activity of the JEV genome in bacteria. The growth kinetics of recombinant viruses derived from RNA-launched DENV2 and JEV infectious cDNAs displayed patterns similar to those of the parental viruses. These results support a novel methodology for constructing flavivirus infectious cDNAs, which will facilitate research in virology, viral pathogenesis and vaccine development of flaviviruses and other RNA viruses.

Effective inhibition of Japanese encephalitis virus replication by shRNAs targeting various viral genes in vitro and in vivo

Japanese encephalitis virus (JEV) is a serious mosquito-borne flavivirus that causes acute encephalitis in humans and many animals, with a high fatality rate. RNA interference (RNAi) is an evolutionarily conserved mechanism for the specific suppression of gene expression, which can be used as a reasonable antiviral strategy. In this study, 10 shRNAs targeting different regions of the JEV genome were designed, and their inhibition of viral replication in vitro and in vivo was determined. Treatment with these shRNAs significantly inhibited JEV replication in BHK-21 and SK-N-SH cells. An immunohistochemical analysis of suckling mice showed that brain sections pretreated with pGP-JE-1, pGP-JE-2 or pGP-JE-3 lacked viral particles. The survival of BALB/c mice challenged with 20 LD50 of the JEV NJ2008 strain at 24h post-injection or simultaneously with pGP-JE-2 was 83.3% and 66.7%, respectively. The results demonstrated that the efficiency of gene silencing and virus inhibition varied between shRNAs to different target genes and sites. Meanwhile, the shRNA-mediated antiviral effect was dose- and time-dependent, including prophylactic and therapeutic effect on virus infection both in vitro and in vivo. The whole results indicate that these shRNAs can inhibit JEV infection sufficiently in vitro and in vivo and might be a potential new tool for controlling JEV infection.

Recovery of a chemically synthesized Japanese encephalitis virus reveals two critical adaptive mutations in NS2B and NS4A

A full-length genome infectious clone is a powerful tool for functional assays in virology. In this study, using a chemical synthesized complete genome of Japanese encephalitis virus (JEV) strain SA14 (GenBank accession no. U14163), we constructed a full-length genomic cDNA clone of JEV. The recovered virus from the cDNA clone replicated poorly in baby hamster kidney (BHK-21) cells and in suckling mice brain. Following serial passage in BHK-21 cells, adaptive mutations within the NS2B and NS4A proteins were recovered in the passaged viruses leading to viruses with a large-plaque phenotype. Mutagenesis analysis, using a genome-length RNA and a replicon of JEV, demonstrated that the adaptive mutations restored replication to different degrees, and the restoration efficiencies were in the order: NS2B-T102M<NS4A-R79K<NS2B-T102M+NS4A-R79K. An in vivo virulence assay in mice showed that the recombinant virus containing double mutations showed similar virulence to the WT SA14 (GenBank accession no. M55506). This study reports the first chemically synthesized JEV. A reverse genetics assay demonstrated that substitutions of NS2B-T102M and NS4A-R79K altered JEV replication.

Heat Shock Protein 70 Is Associated with Replicase Complex of Japanese Encephalitis Virus and Positively Regulates Viral Genome Replication

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes the most prevalent viral encephalitis in Asia. The NS5 protein of JEV is a key component of the viral replicase complex, which plays a crucial role in viral pathogenesis. In this study, tandem affinity purification (TAP) followed by mass spectrometry analysis was performed to identify novel host proteins that interact with NS5. Heat shock protein 70 (Hsp70), eukaryotic elongation factor 1-alpha (eEF-1α) and ras-related nuclear protein (Ran) were demonstrated to interact with NS5. In addition to NS5, Hsp70 was also found to interact with NS3 which is another important member of the replicase complex. It was observed that the cytoplasmic Hsp70 partially colocalizes with the components of viral replicase complex including NS3, NS5 and viral dsRNA during JEV infection. Knockdown of Hsp70 resulted in a significantly reduced JEV genome replication. Further analysis reveals that Hsp70 enhances the stability of viral proteins in JEV replicase complex. These results suggest an important role for Hsp70 in regulating JEV replication, which provides a potential target for the development of anti-JEV therapies.

Molecular phylogenetic and positive selection analysis of Japanese encephalitis virus strains isolated from pigs in China

Japanese encephalitis virus (JEV) is one of the most important virus which causes encephalitis. This disease is most prevalent in the south, southeast and the east region of Asia. In this study, two JEV strains, named JEV/SW/GD/01/2009 and JEV/SW/GZ/09/2004, were isolated from aborted fetuses and seminal fluid of pigs in China. To determine the characteristic of these virus isolates, the virulence of two newly JEV isolates was investigated, the result evidenced that the JEV/SW/GD/01/2009 did not kill mice, while the JEV/SW/GZ/09/2004 displayed neurovirulence with 0.925log10 p.f.u./LD50. Additionally, the full genome sequences of JEV were determined and compared with other known JEV strains. Results demonstrated that the genome of two JEV isolates was 10,976 nucleotides (nt) in length. As compared to the Chinese vaccine strain SA14-14-2, the JEV/SW/GD/01/2009 and the JEV/SW/GZ/09/2004 showed 99.7% and 97.5% identity at the nucleotide level, 99.6% and 96.7% identity at the amino acid level, respectively. Phylogenetic analysis, based on the full-length genome revealed that two JEV isolates were all clustered into genotype III compared to the reference strains. Furthermore, selection analyses revealed that dominant selective pressure acting on the JEV genome was purifying selection. Four sites under positive selection were identified: codon 521 (amino acid E-227), 2296 (amino acid NS4b-24), 3048 (amino acid NS5-521) and 3055 (amino acid NS5-528). Amino acid E-227 was proved to be related to neurovirulence. Taken together, the molecular epidemiology and functional of positively selected amino acid sites of two newly JEV isolates were fully understood, which might be helpful to predict possible changes in virulence.