An RBM10 and NF-κB interacting host lncRNA promotes JEV replication and neuronal cell death.

IMPORTANCE: Central nervous system infection by flaviviruses such as Japanese encephalitis virus, Dengue virus, and West Nile virus results in neuroinflammation and neuronal damage. However, little is known about the role of long non-coding RNAs (lncRNAs) in flavivirus-induced neuroinflammation and neuronal cell death. Here, we characterized the role of a flavivirus-induced lncRNA named JINR1 during the infection of neuronal cells. Depletion of JINR1 during virus infection reduces viral replication and cell death. An increase in GRP78 expression by JINR1 is responsible for promoting virus replication. Flavivirus infection induces the expression of a cellular protein RBM10, which interacts with JINR1. RBM10 and JINR1 promote the proinflammatory transcription factor NF-κB activity, which is detrimental to cell survival.

The RNA binding protein Quaking represses splicing of the Fibronectin EDA exon and downregulates the interferon response.

Quaking (QKI) controls RNA metabolism in many biological processes including innate immunity, where its roles remain incompletely understood. To illuminate these roles, we performed genome scale transcriptome profiling in QKI knockout cells with or without poly(I:C) transfection, a double-stranded RNA analog that mimics viral infection. Analysis of RNA-sequencing data shows that QKI knockout upregulates genes induced by interferons, suggesting that QKI is an immune suppressor. Furthermore, differential splicing analysis shows that QKI primarily controls cassette exons, and among these events, we noted that QKI silences splicing of the extra domain A (EDA) exon in fibronectin (FN1) transcripts. QKI knockout results in elevated production and secretion of FN1-EDA protein, which is a known activator of interferons. Consistent with an upregulation of the interferon response in QKI knockout cells, our results show reduced production of dengue virus-2 and Japanese encephalitis virus in these cells. In conclusion, we demonstrate that QKI downregulates the interferon system and attenuates the antiviral state.

Endoplasmic Reticulum-Associated Degradation Controls Virus Protein Homeostasis, Which Is Required for Flavivirus Propagation.

Many positive-stranded RNA viruses encode polyproteins from which viral proteins are generated by processing the polyproteins. This system produces an equal amount of each viral protein, though the required amounts for each protein are not the same. In this study, we found the extra membrane-anchored nonstructural (NS) proteins of Japanese encephalitis virus and dengue virus are rapidly and selectively degraded by the endoplasmic reticulum-associated degradation (ERAD) pathway. Our gene targeting study revealed that ERAD involving Derlin2 and SEL1L, but not Derlin1, is required for the viral genome replication. Derlin2 is predominantly localized in the convoluted membrane (CM) of the viral replication organelle, and viral NS proteins are degraded in the CM. Hence, these results suggest that viral protein homeostasis is regulated by Derlin2-mediated ERAD in the CM, and this process is critical for the propagation of these viruses. IMPORTANCE The results of this study reveal the cellular ERAD system controls the amount of each viral protein in virus-infected cells and that this "viral protein homeostasis" is critical for viral propagation. Furthermore, we clarified that the "convoluted membrane (CM)," which was previously considered a structure with unknown function, serves as a kind of waste dump where viral protein degradation occurs. We also found that the Derlin2/SEL1L/HRD1-specific pathway is involved in this process, whereas the Derlin1-mediated pathway is not. This novel ERAD-mediated fine-tuning system for the stoichiometries of polyprotein-derived viral proteins may represent a common feature among polyprotein-encoding viruses.

Mechanism through Which Retrocyclin Targets Flavivirus Multiplication.

Currently, there are no approved drugs for the treatment of flavivirus infection. Accordingly, we tested the inhibitory effects of the novel θ-defensin retrocyclin-101 (RC-101) against flavivirus infection and investigated the mechanism underlying the potential inhibitory effects. First, RC-101 robustly inhibited both Japanese encephalitis virus (JEV) and Zika virus (ZIKV) infections. RC-101 exerted inhibitory effects on the entry and replication stages. Results also indicated that the nonstructural protein NS2B-NS3 serine protease might serve as a potential viral target. Furthermore, RC-101 inhibited protease activity at the micromolar level. We also demonstrated that with respect to the glycoprotein E protein of flavivirus, the DE loop of domain III (DIII), which is the receptor-binding domain of the E protein, might serve as another viral target of RC-101. Moreover, a JEV DE mutant exhibited resistance to RC-101, which was associated with deceased binding affinity of RC-101 to DIII. These findings provide a basis for the development of RC-101 as a potential candidate for the treatment of flavivirus infection. IMPORTANCE Retrocyclin is an artificially humanized circular θ-defensin peptide, containing 18 residues, previously reported to possess broad antimicrobial activity. In this study, we found that retrocyclin-101 inhibited flavivirus (ZIKV and JEV) infections. Retrocyclin-101 inhibited NS2B-NS3 serine protease activity, suggesting that the catalytic triad of the protease is the target. Moreover, retrocyclin-101 bound to the DE loop of the E protein of flavivirus, which prevented its entry.

Evaluating the competence of the primary vector, Culex tritaeniorhynchus, and the invasive mosquito species, Aedes japonicus japonicus, in transmitting three Japanese encephalitis virus genotypes.

Japanese encephalitis virus (JEV) is maintained in an enzootic cycle between swine, water birds, and mosquitoes. JEV has circulated indigenously in Asia, with Culex tritaeniorhynchus as the primary vector. In some areas where the primary vector is scarce or absent, sporadic cases of Japanese encephalitis have been reported, with Aedes japonicus japonicus presumed to have the potential as a secondary vector. As one of the world's most invasive culicid species, Ae. j. japonicus carries a considerable health risk for spreading diseases to wider areas, including Europe and North America. Thus, evaluation of its competency as a JEV vector, particularly in a native population, will be essential in preventing potential disease spread. In this study, the two mosquito species' vector competence in transmitting three JEV genotypes (I, III, and V) was assessed, with Cx. tritaeniorhynchus serving as a point of reference. The mosquitoes were virus-fed and the infection rate (IR), dissemination rate (DR), and transmission rate (TR) evaluated individually by either RT-qPCR or focus forming assay. Results showed striking differences between the two species, with IR of 95% (261/274) and 9% (16/177) in Cx. tritaeniorhynchus and Ae. j. japonicus, respectively. Both mosquitoes were susceptible to all three JEV genotypes with significant differences in IR and mean viral titer. Results confirm the primary vector's competence, but the fact that JEV was able to establish in Ae. j. japonicus is of public health significance, and with 2%-16% transmission rate it has the potential to successfully transmit JEV to the next host. This may explain the human cases and infrequent detection in primary vector-free areas. Importantly, Ae. j. japonicus could be a relevant vector spreading the disease into new areas, indicating the need for security measures in areas where the mosquito is distributed or where it may be introduced.

Identifying optimal capsid duplication length for the stability of reporter flaviviruses.

ABSTRACT Mosquito-transmitted flaviviruses cause widespread disease across the world. To provide better molecular tools for drug screens and pathogenesis studies, we report a new approach to produce stable NanoLuc-tagged flaviviruses, including dengue virus serotypes 1-4, Japanese encephalitis virus, yellow fever virus, West Nile virus, and Zika virus. Since the reporter gene is often engineered at the capsid gene region, the capsid sequence must be duplicated to flank the reporter gene; such capsid duplication is essential for viral replication. The conventional approach for stabilizing reporter flaviviruses has been to shorten or modify the duplicated capsid sequence to minimize homologous recombination. No study has examined the effects of capsid duplication length on reporter virus stability. Here we report an optimal length to stabilize reporter flaviviruses. These viruses were stable after ten rounds of cell culture passaging, and in the case of stable NanoLuc-tagged Zika virus (ZIKV C38), the virus replicated to 107 FFU/ml in cell culture and produced robust luciferase signal after inoculation in mosquitoes. Mechanistically, the optimal length of capsid duplication may contain all the cis-acting RNA elements required for viral RNA replication, thus reducing the selection pressure for recombination. Together, these data describe an improved method of constructing optimal reporter flaviviruses.

Inactivation of Japanese encephalitis virus in plasma by methylene blue combined with visible light and in platelet concentrates by ultraviolet C light.

Japanese encephalitis virus (JEV) is endemic to tropical areas in Asia and the Western Pacific. It can cause fatal encephalitis, although most infected individuals are asymptomatic. JEV is mainly transmitted to humans through the bite of an infected mosquito, but can also be transmitted through blood transfusion. To manage the potential risk of transfusion transmission, pathogen inactivation (PI) technologies, such as THERAFLEX MB-Plasma and THERAFLEX UV-Platelets systems, have been developed. We examined the efficacy of these two PI systems to inactivate JEV. STUDY DESIGN AND METHODS: Japanese encephalitis virus-spiked plasma units were treated using the THERAFLEX MB-Plasma system (visible light doses, 20, 40, 60, and 120 [standard] J/cm2) in the presence of methylene blue at approximately 0.8 µmol/L and spiked platelet concentrates (PCs) were treated using the THERAFLEX UV-Platelets system (UVC doses, 0.05, 0.10, 0.15, and 0.20 [standard] J/cm2). Samples were taken before the first and after each illumination dose and tested for infectivity using an immunoplaque assay. RESULTS: Treatment of plasma with the THERAFLEX MB-Plasma system resulted in an average of 6.59 log reduction in JEV infectivity at one-sixth of the standard visible light dose (20 J/cm2). For PCs, treatment with the THERAFLEX UV-Platelet system resulted in an average of 7.02 log reduction in JEV infectivity at the standard UVC dose (0.20 J/cm2). CONCLUSIONS: The THERAFLEX MB-Plasma and THERAFLEX UV-Platelets systems effectively inactivated JEV in plasma or PCs, and thus these PI technologies could be an effective option to reduce the risk of JEV transfusion transmission.

Amino Acid at Position 166 of NS2A in Japanese Encephalitis Virus (JEV) is Associated with In Vitro Growth Characteristics of JEV.

We previously showed that the growth ability of the Japanese encephalitis virus (JEV) genotype V (GV) strain Muar is clearly lower than that of the genotype I (GI) JEV strain Mie/41/2002 in murine neuroblastoma cells. Here, we sought to identify the region in GV JEV that is involved in its low growth potential in cultured cells. An intertypic virus containing the NS1-3 region of Muar in the Mie/41/2002 backbone (NS1-3Muar) exhibited a markedly diminished growth ability in murine neuroblastoma cells. Moreover, the growth rate of a Muar NS2A-bearing intertypic virus (NS2AMuar) was also similar to that of Muar in these cells, indicating that NS2A of Muar is one of the regions responsible for the Muar-specific growth ability in murine neuroblastoma cells. Sequencing analysis of murine neuroblastoma Neuro-2a cell-adapted NS1-3Muar virus clones revealed that His-to-Tyr mutation at position 166 of NS2A (NS2A166) could rescue the low replication ability of NS1-3Muar in Neuro-2a cells. Notably, a virus harboring a Tyr-to-His substitution at NS2A166 (NS2AY166H) showed a decreased growth ability relative to that of the parental virus Mie/41/2002, whereas an NS2AMuar-based mutant virus, NS2AMuar-H166Y, showed a higher growth ability than NS2AMuar in Neuro-2a cells. Thus, these results indicate that the NS2A166 amino acid in JEV is critical for the growth and tissue tropism of JEV in vitro.

Establishment and characterization of the pig tonsil epithelial (PT) cell line as a new model for persist infection of Japanese Encephalitis Virus.

Japanese encephalitis virus (JEV) causes a serious zoonotic disease worldwide, pig is the reservoir and amplifying host of JEV. JEV can persist infect tonsil in pig, but the relation between persist infection in tonsil and reservoir are not clear until now. A stable pig tonsil cell line is necessary for JEV persist infection research. In this study, we established a continuous epithelial cell line, named PT cell, from the pig tonsil. This cell is susceptible to JEV. We determined the growth characteristics, molecular properties, microstructure profiles of PT cell. JEV is easy to enter PT cell which may partly explain the reason of persist infection. We further determined that LMAN2L, a mannose lectin proteins, is the primary viral receptors for JEV entry in PT cell. IFITM3, an cellular surface antiviral factor, is underexpression in PT cell after JEV infection. All these results provide solid evidence that PT cell will promote additional research on JEV persist infection in pig tonsil.

The emerged genotype I of Japanese encephalitis virus shows an infectivity similar to genotype III in Culex pipiens mosquitoes from China.

Japanese Encephalitis virus (JEV) is a zoonotic flavivirus that represents the most significant etiology of childhood viral neurological infections throughout the Asia. During the last 20 years, JEV genotype dominance has shifted from genotype III (GIII) to genotype I (GI). To date, the exact mechanism of this displacement is still not known. Culex (Cx.) mosquitoes are the most common species in China and play an essential role in maintaining JEV enzootic transmission cycle. In this study, we used Cx. pipiens mosquitoes from China as an in vivo mosquito model to explore if mosquitoes played a potential role in JEV genotype shift. We exposed female Cx. pipiens mosquitoes orally to either GI or GIII JEV strains. Midgut, whole mosquitoes, secondary organs, and salivary glands of JEV-infected mosquitoes were collected at 7 and 14 days of post infection (dpi) and subjected to measure the infection rate, replication kinetics, dissemination rate and transmission potential of the infected JEV strains in Cx. pipiens mosquitoes by 50% tissue culture infective dose assay. We found that Cx. pipiens mosquito was competent vector for both GI and GIII JEV infection, with similar infection rates and growth kinetics. After the establishment of infection, Cx. pipiens mosquitoes disseminated both JEV genotypes to secondary organs at similar rates of dissemination. A few GI-infected mosquito salivary glands (16.2%) were positive for GI virus, whereas GIII virus was undetectable in GIII-infected mosquito salivary glands at 7 dpi. However, 29.4% (5/17) and 36.3% (8/22) were positive for GI- and GIII-infected mosquito salivary glands at 14 dpi, respectively, showing an increase in JEV positive rate. No statistical difference in the transmission rate between GI- and GIII-infected mosquitoes was detected. Our experiment data demonstrated that GI and GIII viruses have similar infectivity in Cx. pipiens mosquitoes, suggesting that Cx. pipiens mosquitoes from China may not play a critical role in JEV genotype shift. Although the current data were obtained solely from Cx. pipiens mosquitoes, it is likely that the conclusion drawn could be extrapolated to the role of mosquitoes in JEV genotype shift.

Functional Correlation between Subcellular Localizations of Japanese Encephalitis Virus Capsid Protein and Virus Production.

The flavivirus capsid protein is considered to be essential for the formation of nucleocapsid complexes with viral genomic RNA at the viral replication organelle that appears on the endoplasmic reticulum (ER), as well as for incorporation into virus particles. However, this protein is also detected at the lipid droplet (LD) and nucleolus, and physiological roles of these off-site localizations are still unclear. In this study, we made a series of alanine substitution mutants of Japanese encephalitis virus (JEV) capsid protein that cover all polar and hydrophobic amino acid residues to identify the molecular surfaces required for virus particle formation and for localization at the LD and nucleolus. Five mutants exhibited a defect in the formation of infectious particles, and two of these mutants failed to be incorporated into the subviral particles (SVP). Three mutants lost the ability to localize to the nucleolus, and only a single mutant, with mutations at α2, was unable to localize to the LD. Unlike the cytoplasmic capsid protein, the nucleolar capsid protein was resistant to detergent treatment, and the α2 mutant was hypersensitive to detergent treatment. To scrutinize the relationship between these localizations and viral particle formation, we made eight additional alanine substitution mutants and found that all the mutants that did not localize at the LD or nucleolus failed to form normal viral particles. These results support the functional correlation between LD or nucleolus localization of the flaviviral capsid protein and the formation of infectious viral particles.IMPORTANCE This study is the first to report the comprehensive mutagenesis of a flavivirus capsid protein. We assessed the requirement of each molecular surface for infectious viral particle formation as well as for LD and nucleolar localization and found functional relationships between the subcellular localization of the virus capsid protein and infectious virus particle formation. We developed a system to independently assess the packaging of viral RNA and that of the capsid protein and found a molecular surface of the capsid protein that is crucial for packaging of viral RNA but not for packaging of the capsid protein itself. We also characterized the biochemical properties of capsid protein mutants and found that the capsid protein localizes at the nucleolus in a different manner than for its localization to the LD. Our comprehensive alanine-scanning mutagenesis study will aid in the development of antiflavivirus small molecules that can target the flavivirus capsid protein.

Upregulated expression of the antioxidant sestrin 2 identified by transcriptomic analysis of Japanese encephalitis virus-infected SH-SY5Y neuroblastoma cells.

Japanese encephalitis virus (JEV) exerts a profound burden of viral encephalitis. We have investigated the differentially expressed transcripts in the neuronal transcriptome during JEV infection by RNA sequencing (RNA-Seq) of virus-infected SH-SY5Y human neuroblastoma cells. Gene ontology analysis revealed significant enrichment from two main pathways: endoplasmic reticulum (ER)-nucleus signaling (P value: 5.75E-18; false discovery rate [FDR] 3.11E-15) and the ER unfolded protein response (P value: 7.58E-18; FDR 3.11E-15). qPCR validation showed significant upregulation and differential expression (P < 0.01) of ER stress-signaling transcripts (SESN2, TRIB3, DDIT3, DDIT4, XBP1, and ATF4) at 24 h post-infection for both low (LN) and high (HN) neurovirulence JEV strains. Immunoblot analysis following JEV infection of SH-SY5Y cells showed an increase in levels of SESN2 protein following JEV infection. Similarly, Zika virus (MR766) infection of SH-SY5Y showed a titer-dependent increase in ER stress-signaling transcripts; however, this was absent or diminished for DDIT4 and ATF4, respectively, suggestive of differences in the induction of stress-response transcripts between flaviviruses. Interestingly, SLC7A11 and SLC3A2 mRNA were also both deregulated in JEV-infected SH-SY5Y cells and encode the two constituent subunits of the plasma membrane xCT amino acid antiporter that relieves oxidative stress by export of glutamate and import of cystine. Infection of SH-SY5Y and HEK293T cells by the JEV HN strain Sw/Mie/40/2004 lead to significant upregulation of the SLC7A11 mRNA to levels comparable to DDIT3. Our findings suggest upregulation of antioxidants including SESN2 and, also, the xCT antiporter occurs to counteract the oxidative stress elicited by JEV infection.

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.

Differential replication efficiencies between Japanese encephalitis virus genotype I and III in avian cultured cells and young domestic ducklings.

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.

Genetic and neuroattenuation phenotypic characteristics and their stabilities of SA14-14-2 vaccine seed virus.

Japanese encephalitis (JE) live attenuated vaccine SA14-14-2 is the most widely used JE vaccine in the world. Large-scale clinical trials have demonstrated satisfactory safety and efficacy profiles. The establishment of genetic and attenuated neurovirulence characteristics and their stabilities of SA14-14-2 virus are important in relation to vaccine safety in humans. Therefore, several researchers have studied and analyzed the full-length gene sequences of the SA14-14-2 virus strain. However, sequencing results have shown a significant difference. Here, we further studied the full-length sequence of three class seed virus banks of the vaccine as well as two vaccine viruses with different passages in primary hamster kidney cells, and compared them with our original stored SA14 parent virus (low passage in mouse brain). The full-length gene sequence determined in this study indicates there were 57 nucleotide and 25 amino acid substitutions of the SA14-14-2 strain compared to its parental SA14 virus strain. The full-length sequences of the three class seed bank viruses and the vaccine virus PHKC8 were completely identical among them, but the working seed virus passaged in primary hamster kidney cells for 17 generations (PHKC17) had a single nucleotide change at the 5' NCR. Both KM and ICR mice tested by intracerebral (i.c.) or subcutaneous (s.c.) routes with the three class seed viruses and vaccine viruses with ≥5.7 lgpfu/mL remained healthy, but all the mice inoculated with the SA14 parental virus strain died as early as day 5 post-inoculation. The present study provided new information on the full-length gene sequence and attenuated neurovirulence of SA14-14-2. They can be used as a reference sequence for vaccine quality control and surveillance of neurovirulence reversion following vaccination. Moreover, the present results further demonstrated the high genetic and phenotypic stabilities of the SA14-14-2 virus, suggesting the neurovirulence reversion of the vaccine strain will be highly unlikely.

Host Factor SPCS1 Regulates the Replication of Japanese Encephalitis Virus through Interactions with Transmembrane Domains of NS2B.

Signal peptidase complex subunit 1 (SPCS1) is a newly identified host factor that regulates flavivirus replication, but the molecular mechanism is not fully understood. Here, using Japanese encephalitis virus (JEV) as a model, we investigated the mechanism through which the host factor SPCS1 regulates the replication of flaviviruses. We first validated the regulatory function of SPCS1 in JEV propagation by knocking down and knocking out endogenous SPCS1. The loss of SPCS1 function markedly reduced intracellular virion assembly and the production of infectious JEV particles but did not affect cell entry, RNA replication, or translation of the virus. SPCS1 was found to interact with nonstructural protein 2B (NS2B), which is involved in posttranslational protein processing and virus assembly. Serial deletion mutation of the JEV NS2B protein revealed that two transmembrane domains, NS2B(1-49) and NS2B(84-131), interact with SPCS1. Further mutagenesis analysis of conserved flavivirus residues in two SPCS1 interaction domains of NS2B demonstrated that G12A, G37A, and G47A in NS2B(1-49) and P112A in NS2B(84-131) weakened the interaction with SPCS1. Deletion mutation of SPCS1 revealed that SPCS1(91-169), which contains two transmembrane domains, was involved in interactions with both NS2B(1-49) and NS2B(84-131). Taken together, these results demonstrate that SPCS1 affects viral replication by interacting with NS2B, thereby influencing the posttranslational processing of JEV proteins and the assembly of virions.IMPORTANCE Understanding virus-host interactions is important for elucidating the molecular mechanisms of virus propagation and identifying potential antiviral targets. Previous reports demonstrated that SPCS1 is involved in the flavivirus life cycle, but the mechanism remains unknown. In this study, we confirmed that SPCS1 participates in the posttranslational protein processing and viral assembly stages of the JEV life cycle but not in the cell entry, genome RNA replication, or translation stages. Furthermore, we found that SPCS1 interacts with two independent transmembrane domains of the flavivirus NS2B protein. NS2B also interacts with NS2A, which is proposed to mediate virus assembly. Therefore, we propose a protein-protein interaction model showing how SPCS1 participates in the assembly of JEV particles. These findings expand our understanding of how host factors participate in the flavivirus replication life cycle and identify potential antiviral targets for combating flavivirus infection.

Mutation of I176R in the E coding region weakens Japanese encephalitis virus neurovirulence, but not its growth rate in BHK-21 cells.

Previously, we isolated the Japanese encephalitis virus (JEV) strain SCYA201201. In this study, we passed the SCYA201201 strain in Syrian baby hamster kidney (BHK-21) cells 120 times to obtain the SCYA201201-0901 strain, which exhibited an attenuated phenotype in mice. Comparison of SCYA201201-0901 amino acid sequences with those of other JEV strains revealed a single mutation, I176R, in the E coding region. Using reverse genetic technology, we provide evidence that this single E-I176R mutation does not affect virus growth in BHK-21 cells but significantly decreases JEV neurovirulence in mice. This study provides critical information for understanding the molecular mechanism of JEV attenuation.

Identification of a novel porcine OASL variant exhibiting antiviral activity.

2', 5'-Oligoadenylate synthetase-lilke (OASL) protein is an atypical oligoadenylate synthetase (OAS) family member, which possesses antiviral activity but lacks 2', 5'-oligoadenylate synthetase activity. Here, a novel variant of porcine OASL (pOASL2) was identified through RT-PCR amplification. This gene is distinguishable from the previously described wild-type porcine OASL (pOASL1). The gene appears to be derived from a truncation of exon 4 plus 8 nucleotides of exon 5 with a premature termination, measuring only 633 bp in length, although its position corresponds to that of pOASL1. Given this novel gene appears to be a variant of pOASL, we assayed for antiviral activity of the protein. We demonstrated that pOASL2 could inhibit Japanese encephalitis virus (JEV) proliferation as well as pOASL1 in a transient overexpression assay of pOASL1 and pOASL2 in PK-15 and Vero cells. In addition to JEV, pOASL1 and pOASL2 also decreased the proliferations of Porcine reproductive and respiratory syndrome virus (PRRSV) and vesicular stomatitis virus (VSV), but did not exhibit antiviral activity against pseudorabies virus (PRV). Structural analysis showed that the pOASL2 gene retained only the first three exons at the 5'-. To investigate the role of the αN4 helix in pOASL in antiviral responses like that in hOASL, we mutated key residues in the anchor domain of the αN4 helix in pOASL2, based on the domain's location in hOASL. However, the antiviral activity of pOASL2 was not affected. Thus, the αN4 helix of pOASL likely does not play a significant role in its antiviral activity. In conclusion, pOASL2 acts as a new splice isoform of pOASL that plays a role in resistance to infection of several kinds of RNA viruses.

Identification and characterization of differentially expressed genes from human microglial cell samples infected with Japanese encephalitis virus.

BACKGROUND & OBJECTIVES: Limited studies have been reported on Japanese encephalitis (JE) with reference to microarray data analysis. The present study involved an in silico approach for identification and characterization of differentially expressed genes in human microglial cell (CHME3) samples, infected with P20778 strain of Japanese encephalitis virus (JEV). METHODS: Gene expression data (GSE57330) belonging to mRNA expression profile of CHME3 cells infected with JEV, was downloaded from the gene expression omnibus (GEO) database, processed and normalized by robust multichip averaging (RMA) method using affy packages of R. The Bayes method was used to correct multiple testing. The log fold change (logFC > 1) and p< 0.05 were used as cut-off to identify differentially expressed genes (DEGs). The newly identified hub genes were set at the centre for construction of protein-protein interaction network using search tool for the retrieval of interacting genes/proteins (STRING) database considering human genome as reference. Gene ontology and pathway enrichment analysis of the hub gene and its associated genes were performed using STRING and DAVID tool. RESULTS: Microarray data analysis revealed that STAT1 gene was down-regulated during JEV infection. STAT1 gene was found to interact with tyrosine protein kinase family members, and showed strong interaction with JAK1 and JAK2 genes. INTERPRETATION & CONCLUSION: The identified transcription factors and the binding sites in the promoter region of STAT1 gene might act as potential drug targets in near future.

Genome-Wide Screening Uncovers the Significance of N-Sulfation of Heparan Sulfate as a Host Cell Factor for Chikungunya Virus Infection.

The molecular mechanisms underlying chikungunya virus (CHIKV) infection are poorly characterized. In this study, we analyzed the host factors involved in CHIKV infection using genome-wide screening. Human haploid HAP1 cells, into which an exon-trapping vector was introduced, were challenged with a vesicular stomatitis virus pseudotype bearing the CHIKV E3 to E1 envelope proteins. Analysis of genes enriched in the cells resistant to the pseudotyped virus infection unveiled a critical role of N-sulfation of heparan sulfate (HS) for the infectivity of the clinically isolated CHIKV Thai#16856 strain to HAP1 cells. Knockout of NDST1 that catalyzes N-sulfation of HS greatly decreased the binding and infectivity of CHIKV Thai#16856 strain but not infectivity of Japanese encephalitis virus (JEV) and yellow fever virus (YFV). While glycosaminoglycans were commonly required for the efficient infectivity of CHIKV, JEV, and YFV, as shown by using B3GAT3 knockout cells, the tropism for N-sulfate was specific to CHIKV. Expression of chondroitin sulfate (CS) in NDST1-knockout HAP1 cells did not restore the binding of CHIKV Thai#16856 strain and the infectivity of its pseudotype but restored the infectivity of authentic CHIKV Thai#16856, suggesting that CS functions at later steps after CHIKV binding. Among the genes enriched in this screening, we found that TM9SF2 is critical for N-sulfation of HS and therefore for CHIKV infection because it is involved in the proper localization and stability of NDST1. Determination of the significance of and the relevant proteins to N-sulfation of HS may contribute to understanding mechanisms of CHIKV propagation, cell tropism, and pathogenesis.IMPORTANCE Recent outbreaks of chikungunya fever have increased its clinical importance. Chikungunya virus (CHIKV) utilizes host glycosaminoglycans to bind efficiently to its target cells. However, the substructure in glycosaminoglycans required for CHIKV infection have not been characterized. Here, we unveil that N-sulfate in heparan sulfate is essential for the efficient infection of a clinical CHIKV strain to HAP1 cells and that chondroitin sulfate does not help the CHIKV binding but does play roles at the later steps in HAP1 cells. We show, by comparing previous reports using Chinese hamster ovary cells, along with another observation that enhanced infectivity of CHIKV bearing Arg82 in envelope E2 does not depend on glycosaminoglycans in HAP1 cells, that the infection manner of CHIKV varies among host cells. We also show that TM9SF2 is required for CHIKV infection to HAP1 cells because it is involved in the N-sulfation of heparan sulfate through ensuring NDST1 activity.