ABSTRACT Japanese encephalitis virus (JEV) is an arthropod-borne, plus-strand flavivirus causing viral encephalitis in humans with a high case fatality rate. The JEV non-structural protein 5 (NS5) with the RNA-dependent RNA polymerase activity interacts with the viral and host proteins to constitute the replication complex. We have identified the multifunctional protein Nucleolin (NCL) as one of the several NS5-interacting host proteins. We demonstrate the interaction and colocalization of JEV NS5 with NCL in the virus-infected HeLa cells. The siRNA-mediated knockdown of NCL indicated that it was required for efficient viral replication. Importantly, JEV grew to higher titers in cells over-expressing exogenous NCL, demonstrating its pro-viral role. We demonstrated that NS5 interacted with the RRM and GAR domains of NCL. We show that the NCL-binding aptamer AS1411 containing the G-quadruplex (GQ) structure and the GQ ligand BRACO-19 caused significant inhibition of JEV replication. The antiviral effect of AS1411 and BRACO-19 could be overcome in HeLa cells by the overexpression of exogenous NCL. We demonstrated that the synthetic RNAs derived from the 3′-NCR of JEV genomic RNA containing the GQ sequence could bind NCL in vitro . The replication complex binding to the 3′-NCR is required for the viral RNA synthesis. It is likely that NCL present in the replication complex destabilizes the GQ structures in the genomic RNA, thus facilitating the movement of the replication complex resulting in efficient virus replication. IMPORTANCEJapanese encephalitis virus (JEV) is endemic in most parts of South-East Asia and the Western Pacific region, causing epidemics of encephalitis with a high case fatality rate. While a tissue culture-derived JEV vaccine is available, no antiviral therapy exists. The JEV NS5 protein has RNA-dependent RNA polymerase activity. Together with several host and viral proteins, it constitutes the replication complex necessary for virus replication. Understanding the interaction of NS5 with the host proteins could help design novel antivirals. We identified Nucleolin (NCL) as a crucial host protein interactor of JEV NS5 having a pro-viral role in virus replication. The NS5-interacting NCL binds to the G-quadruplex (GQ) structure sequence in the 3′-NCR of JEV RNA. This may smoothen the movement of the replication complex along the genomic RNA, thereby facilitating the virus replication. This study is the first report on how NCL, a host protein, helps in JEV replication through GQ-binding.

2024-07-01·Heliyon

Generation of rescued Japanese encephalitis virus genotype 1 from infectious full-size clone using reverse genetics

Article

作者: Nowakowska, Aleksandra ; Kim, Sehyun ; Bang, Hee Won ; Choi, Heejae ; Kim, Young Bong ; Kim, Min Sun ; Lee, Hee-Jung

Japanese encephalitis virus (JEV) is a pathogen responsible for high mortality and morbidity rates among children with encephalitis. Since JEV genotype 1 (GI) is the most prevalent strain in South Korea these days, corresponding research and vaccine development is urgently required. Molecular genetic studies on JEV vaccines can be boosted by obtaining genetically stable full-length infectious JEV complementary DNA (cDNA) clones. Furthermore, the significance of the reverse genetics system in facilitating molecular biological analyses of JEV properties has been demonstrated. This study constructed a recombinant JEV-GI strain using a reverse genetics system based on a Korean wild-type GI isolate (K05GS). RNA extracted from JEV-GI was used to synthesize cDNA, a recombinant full-length JEV clone, pTRE-JEVGI, was generated from the DNA fragment, and the virus was rescued. We performed in vitro and in vivo experiments to analyze the rescued JEV-GI virus. The rescued JEV-GI exhibited similar characteristics to wild-type JEV. These results suggest that our reverse genetics system can generate full-length infectious clones that can be used to analyze molecular biological factors that influence viral properties and immunogenicity. Additionally, it may be useful as a heterologous gene expression vector and help develop new strains for JEV vaccines.

2020-05-01·Transfusion3区 · 医学

A case series of inactivated Japanese encephalitis virus vaccination associated with positive West Nile virus blood donor screening nucleic acid tests

3区 · 医学

Article

作者: Makowski, Kai ; Bigham, Mark ; Hawes, Gordon ; Yan, Matthew T.S. ; Skeate, Robert ; Dimitrova, Kristina ; Young, Dale ; Ng, Michiko ; Fearon, Margaret ; Wood, Heidi ; Drews, Steven J.

ABSTRACTBACKGROUNDWest Nile Virus (WNV) is a member of the Japanese Encephalitis (JE) serocomplex within the Flaviviridae family. We report four whole blood donors and one plasma donor with WNV nucleic acid test (NAT)‐reactive donations between September 2018 and November 2019, following recent Japanese Encephalitis virus (JEV) vaccination.CASE SERIESCases 1 and 4 had reactive WNV NAT donations 1 day after receiving the JEV vaccine. Case 2 had a reactive WNV donation 3 days after receiving the JEV vaccine. Case 3 had a reactive WNV NAT donation 3 days after returning from Arizona and 1 day after receiving the JEV vaccine. Case 5 had a reactive WNV donation the same day as receiving the JEV vaccine.STUDY DESIGN AND METHODSWNV screening used the Roche cobas WNV nucleic acid test (NAT) (Roche Molecular Systems). Reference testing on WNV‐reactive donations was carried out by the National Microbiology Laboratory (NML). JEV vaccine dilutions were also analyzed.RESULTSSupplemental NAT was negative for WNV and JEV for Cases 1, 3, and 5. Case 2 had a weak amplification curve for one of two JEV NAT targets. Case 4 was JEV NAT‐positive, WNV NAT‐negative. Serologic testing on donation specimens for Cases 2, 4, and 5 did not support recent or remote WNV infection. JEV vaccine dilutions were detected by both cobas and supplemental NAT.CONCLUSIONSWe recommend implementing a temporary blood donor deferral following a JEV vaccination, if screening utilizes a WNV assay with the capability of detecting other members of the JE serocomplex.

100 项与 JEV vaccine (Vaxine) 相关的药物交易

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