The RNA helicase DDX39B activates FOXP3 RNA splicing to control T regulatory cell fate.

Genes associated with increased susceptibility to multiple sclerosis (MS) have been identified, but their functions are incompletely understood. One of these genes codes for the RNA helicase DExD/H-Box Polypeptide 39B (DDX39B), which shows genetic and functional epistasis with interleukin-7 receptor-α gene (IL7R) in MS-risk. Based on evolutionary and functional arguments, we postulated that DDX39B enhances immune tolerance thereby decreasing MS risk. Consistent with such a role we show that DDX39B controls the expression of many MS susceptibility genes and important immune-related genes. Among these we identified Forkhead Box P3 (FOXP3), which codes for the master transcriptional factor in CD4+/CD25+ T regulatory cells. DDX39B knockdown led to loss of immune-regulatory and gain of immune-effector expression signatures. Splicing of FOXP3 introns, which belong to a previously unrecognized type of introns with C-rich polypyrimidine tracts, was exquisitely sensitive to DDX39B levels. Given the importance of FOXP3 in autoimmunity, this work cements DDX39B as an important guardian of immune tolerance.

Identification of novel orthonairoviruses from rodents and shrews in Gabon, Central Africa.

In Africa, several emerging zoonotic viruses have been transmitted from small mammals such as rodents and shrews to humans. Although no clinical cases of small mammal-borne viral diseases have been reported in Central Africa, potential zoonotic viruses have been identified in rodents in the region. Therefore, we hypothesized that there may be unrecognized zoonotic viruses circulating in small mammals in Central Africa. Here, we investigated viruses that have been maintained among wild small mammals in Gabon to understand their potential risks to humans. We identified novel orthonairoviruses in 24.6 % of captured rodents and shrews from their kidney total RNA samples. Phylogenetic analysis revealed that the novel viruses, Lamusara virus (LMSV) and Lamgora virus, were closely related to Erve virus, which was previously identified in shrews of the genus Crocidura and has been suspected to cause neuropathogenic diseases in humans. Moreover, we show that the LMSV ovarian tumour domain protease, one of the virulence determination factors of orthonairoviruses, suppressed interferon signalling in human cells, suggesting the possible human pathogenicity of this virus. Taken together, our study demonstrates the presence of novel orthonairoviruses that may pose unrecognized risks of viral disease transmission in Gabon.

A screen of FDA-approved drugs with minigenome identified tigecycline as an antiviral targeting nucleoprotein of Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) belongs to the genus Orthonairovirus and is the causative agent of a viral hemorrhagic disease with a case fatality rate of 30%. However, limited studies have been conducted to explore antiviral compounds specific to CCHFV. In this study, we developed a minigenome system of orthonairoviruses, CCHFV and Hazara virus to analyze viral replication and screened an FDA-approved compound library. The transfection of the minigenome components induced marked increase in luciferase expression, indicating the sufficient replication and translation of reporter RNA. Compound library screening identified 14 candidate compounds that significantly decreased luciferase activity. Some of the compounds also inhibited the replication of the infectious Hazara virus. The mechanism of inhibition by tigecycline was further analyzed, and a decrease in the interaction between the viral N protein and RNA by tigecycline was observed. This work provides a basis for validation using animal models and the design of chemical derivatives with stronger activity in future studies on the development of an antiviral against CCHFV.

Y-shaped RNA secondary structure of a noncoding region in the genomic RNA of tick-borne encephalitis virus affects pathogenicity.

Tick-borne encephalitis virus (TBEV) is a zoonotic virus that causes encephalitis in humans. Various deletions have been reported in a variable region of the 3' untranslated region of the TBEV genome. This study analyzed the role of a Y-shaped secondary structure in the pathogenicity of TBEV by using reverse genetics. Deletion of the structure increased the mortality rate of virus-infected mice but did not affect the virus multiplication in cultured cells and organs. The results indicate that the secondary structure is involved in the regulation of TBEV pathogenesis.

Topoisomerase III-ß is required for efficient replication of positive-sense RNA viruses.

Based on genome-scale loss-of-function screens we discovered that Topoisomerase III-ß (TOP3B), a human topoisomerase that acts on DNA and RNA, is required for yellow fever virus and dengue virus-2 replication. Remarkably, we found that TOP3B is required for efficient replication of all positive-sense-single stranded RNA viruses tested, including SARS-CoV-2. While there are no drugs that specifically inhibit this topoisomerase, we posit that TOP3B is an attractive anti-viral target.

Topoisomerase III-ß is required for efficient replication of positive-sense RNA viruses.

Based on genome-scale loss-of-function screens we discovered that Topoisomerase III-ß (TOP3B), a human topoisomerase that acts on DNA and RNA, is required for yellow fever virus and dengue virus-2 replication. Remarkably, we found that TOP3B is required for efficient replication of all positive-sense-single stranded RNA viruses tested, including SARS-CoV-2. While there are no drugs that specifically inhibit this topoisomerase, we posit that TOP3B is an attractive anti-viral target.

Characterization of tick-borne encephalitis virus isolated from a tick in central Hokkaido in 2017.

Tick-borne encephalitis virus (TBEV) is a zoonotic virus in the genus Flavivirus, family Flaviviridae. TBEV is widely distributed in northern regions of the Eurasian continent, including Japan, and causes severe encephalitis in humans. Tick-borne encephalitis (TBE) was recently reported in central Hokkaido, and wild animals with anti-TBEV antibodies were detected over a wide area of Hokkaido, although TBEV was only isolated in southern Hokkaido. In this study, we conducted a survey of ticks to isolate TBEV in central Hokkaido. One strain, designated Sapporo-17-Io1, was isolated from ticks (Ixodes ovatus) collected in Sapporo city. Sequence analysis revealed that the isolated strain belonged to the Far Eastern subtype of TBEV and was classified in a different subcluster from Oshima 5-10, which had previously been isolated in southern Hokkaido. Sapporo-17-Io1 showed similar growth properties to those of Oshima 5-10 in cultured cells and mouse brains. The mortality rate of mice infected intracerebrally with each virus was similar, but the survival time of mice inoculated with Sapporo-17-Io1 was significantly longer than that of mice inoculated with Oshima 5-10. These results indicate that the neurovirulence of Sapporo-17-Io1 was lower than that of Oshima 5-10. Using an infectious cDNA clone, the replacement of genes encoding non-structural genes from Oshima 5-10 with those from Sapporo-17-Io1 attenuated the neuropathogenicity of the cloned viruses. This result indicated that the non-structural proteins determine the neurovirulence of these two strains. Our results provide important insights for evaluating epidemiological risk in TBE-endemic areas of Hokkaido.

West Nile virus capsid protein inhibits autophagy by AMP-activated protein kinase degradation in neurological disease development.

West Nile virus (WNV) belongs to the Flaviviridae family and has emerged as a significant cause of viral encephalitis in birds and animals including humans. WNV replication directly induces neuronal injury, followed by neuronal cell death. We previously showed that accumulation of ubiquitinated protein aggregates was involved in neuronal cell death in the WNV-infected mouse brain. In this study, we attempted to elucidate the mechanisms of the accumulation of protein aggregates in the WNV-infected cells. To identify the viral factor inducing the accumulation of ubiquitinated proteins, intracellular accumulation of ubiquitinated proteins was examined in the cells expressing the viral protein. Expression of capsid (C) protein induced the accumulation, while mutations at residues L51 and A52 in C protein abrogated the accumulation. Wild-type (WT) or mutant WNV in which mutations were introduced into the residues was inoculated into human neuroblastoma cells. The expression levels of LC3-II, an autophagy-related protein, and AMP-activated protein kinase (AMPK), an autophagy inducer, were reduced in the cells infected with WT WNV, while the reduction was not observed in the cells infected with WNV with the mutations in C protein. Similarly, ubiquitination and degradation of AMPK were only observed in the cells infected with WT WNV. In the cells expressing C protein, AMPK was co-precipitated with C protein and mutations in L51 and A52 reduced the interaction. Although the viral replication was not affected, the accumulation of ubiquitinated proteins in brain and neurological symptoms were attenuated in the mouse inoculated with WNV with the mutations in C protein as compared with that with WT WNV. Taken together, ubiquitination and degradation of AMPK by C protein resulted in the inhibition of autophagy and the accumulation of protein aggregates, which contributes to the development of neurological disease.

Development of a serodiagnostic IgM-ELISA for tick-borne encephalitis virus using subviral particles with strep-tag.

Tick-borne encephalitis virus (TBEV) is a zoonotic agent causing severe encephalitis in humans. IgM antibody detection is useful for the serological diagnosis of TBEV infection, because IgM has high specificity for each flavivirus and indicates a recent infection. Commercial IgM-ELISA kits are somewhat expensive and difficulties in their sensitivity have been suggested due to their format and formalin-inactivated antigens. Therefore, the development of an inexpensive IgM-ELISA with high specificity and sensitivity is needed. In this study, a µ-capture ELISA was developed to detect TBEV-specific IgM antibodies using subviral particles (SPs) with strep-tag (strep-SP-IgM-ELISA). The results of our strep-SP-IgM-ELISA were highly correlated with diagnoses made by the neutralization test (sensitivity: 94.1%), and our strep-SP-IgM-ELISA could detect anti-TBEV IgM antibodies in patients who could not be diagnosed with the neutralization test. Besides, 51 of 52 positive samples by a commercial IgM-ELISA were also diagnosed as positive by our strep-SP-IgM-ELISA (98.1%), and our strep-SP-IgM-ELISA could detect anti-TBEV IgM antibodies in all samples that were inconclusive based on the commercial IgM-ELISA. Our strep-SP-IgM-ELISA will be useful for diagnoses in TBE-endemic areas.

Identification and analysis of host proteins that interact with the 3'-untranslated region of tick-borne encephalitis virus genomic RNA.

Tick-borne encephalitis virus (TBEV) causes severe neurological disease, but the pathogenetic mechanism is unclear. The conformational structure of the 3'-untranslated region (UTR) of TBEV is associated with its virulence. We tried to identify host proteins interacting with the 3'-UTR of TBEV. Cellular proteins of HEK293T cells were co-precipitated with biotinylated RNAs of the 3'-UTR of low- and high-virulence TBEV strains and subjected to mass spectrometry analysis. Fifteen host proteins were found to bind to the 3'-UTR of TBEV, four of which-cold shock domain containing-E1 (CSDE1), spermatid perinuclear RNA binding protein (STRBP), fragile X mental retardation protein (FMRP), and interleukin enhancer binding factor 3 (ILF3)-bound specifically to that of the low-virulence strain. An RNA immunoprecipitation and pull-down assay confirmed the interactions of the complete 3'-UTRs of TBEV genomic RNA with CSDE1, FMRP, and ILF3. Partial deletion of the stem loop (SL) 3 to SL 5 structure of the variable region of the 3'-UTR did not affect interactions with the host proteins, but the interactions were markedly suppressed by deletion of the complete SL 3, 4, and 5 structures, as in the high-virulence TBEV strain. Further analysis of the roles of host proteins in the neurologic pathogenicity of TBEV is warranted.

Dendritic transport of tick-borne flavivirus RNA by neuronal granules affects development of neurological disease.

Neurological diseases caused by encephalitic flaviviruses are severe and associated with high levels of mortality. However, little is known about the detailed mechanisms of viral replication and pathogenicity in the brain. Previously, we reported that the genomic RNA of tick-borne encephalitis virus (TBEV), a member of the genus Flavivirus, is transported and replicated in the dendrites of neurons. In the present study, we analyzed the transport mechanism of the viral genome to dendrites. We identified specific sequences of the 5' untranslated region of TBEV genomic RNA that act as a cis-acting element for RNA transport. Mutated TBEV with impaired RNA transport in dendrites caused a reduction in neurological symptoms in infected mice. We show that neuronal granules, which regulate the transport and local translation of dendritic mRNAs, are involved in TBEV genomic RNA transport. TBEV genomic RNA bound an RNA-binding protein of neuronal granules and disturbed the transport of dendritic mRNAs. These results demonstrated a neuropathogenic virus hijacking the neuronal granule system for the transport of viral genomic RNA in dendrites, resulting in severe neurological disease.

Escape of Tick-Borne Flavivirus from 2'-C-Methylated Nucleoside Antivirals Is Mediated by a Single Conservative Mutation in NS5 That Has a Dramatic Effect on Viral Fitness.

Tick-borne encephalitis virus (TBEV) causes a severe and potentially fatal neuroinfection in humans. Despite its high medical relevance, no specific antiviral therapy is currently available. Here we demonstrate that treatment with a nucleoside analog, 7-deaza-2'-C-methyladenosine (7-deaza-2'-CMA), substantially improved disease outcomes, increased survival, and reduced signs of neuroinfection and viral titers in the brains of mice infected with a lethal dose of TBEV. To investigate the mechanism of action of 7-deaza-2'-CMA, two drug-resistant TBEV clones were generated and characterized. The two clones shared a signature amino acid substitution, S603T, in the viral NS5 RNA-dependent RNA polymerase (RdRp) domain. This mutation conferred resistance to various 2'-C-methylated nucleoside derivatives, but no cross-resistance was seen with other nucleoside analogs, such as 4'-C-azidocytidine and 2'-deoxy-2'-beta-hydroxy-4'-azidocytidine (RO-9187). All-atom molecular dynamics simulations revealed that the S603T RdRp mutant repels a water molecule that coordinates the position of a metal ion cofactor as 2'-C-methylated nucleoside analogs approach the active site. To investigate its phenotype, the S603T mutation was introduced into a recombinant TBEV strain (Oshima-IC) generated from an infectious cDNA clone and into a TBEV replicon that expresses a reporter luciferase gene (Oshima-REP-luc2A). The mutants were replication impaired, showing reduced growth and a small plaque size in mammalian cell culture and reduced levels of neuroinvasiveness and neurovirulence in rodent models. These results indicate that TBEV resistance to 2'-C-methylated nucleoside inhibitors is conferred by a single conservative mutation that causes a subtle atomic effect within the active site of the viral NS5 RdRp and is associated with strong attenuation of the virus.IMPORTANCE This study found that the nucleoside analog 7-deaza-2'-C-methyladenosine (7-deaza-2'-CMA) has high antiviral activity against tick-borne encephalitis virus (TBEV), a pathogen that causes severe human neuroinfections in large areas of Europe and Asia and for which there is currently no specific therapy. Treating mice infected with a lethal dose of TBEV with 7-deaza-2'-CMA resulted in significantly higher survival rates and reduced the severity of neurological signs of the disease. Thus, this compound shows promise for further development as an anti-TBEV drug. It is important to generate drug-resistant mutants to understand how the drug works and to develop guidelines for patient treatment. We generated TBEV mutants that were resistant not only to 7-deaza-2'-CMA but also to a broad range of other 2'-C-methylated antiviral medications. Our findings suggest that combination therapy may be used to improve treatment and reduce the emergence of drug-resistant viruses during nucleoside analog therapy for TBEV infection.

A novel reverse genetics system for production of infectious West Nile virus using homologous recombination in mammalian cells.

Reverse genetics systems facilitate investigation of many aspects of the life cycle and pathogenesis of viruses. However, genetic instability in Escherichia coli has hampered development of a reverse genetics system for West Nile virus (WNV). In this study, we developed a novel reverse genetics system for WNV based on homologous recombination in mammalian cells. Introduction of the DNA fragment coding for the WNV structural protein together with a DNA-based replicon resulted in the release of infectious WNV. The growth rate and plaque size of the recombinant virus were almost identical to those of the parent WNV. Furthermore, chimeric WNV was produced by introducing the DNA fragment coding for the structural protein and replicon plasmid derived from various strains. Here, we report development of a novel system that will facilitate research into WNV infection.

Development of a serodiagnostic multi-species ELISA against tick-borne encephalitis virus using subviral particles.

Tick-borne encephalitis virus (TBEV) is a zoonotic agent causing severe encephalitis in humans. A wide range of animal species could be infected with TBEV in endemic areas. A serological survey of wild animals is effective in identifying TBEV-endemic areas. Safe, simple, and reliable TBEV serodiagnostic tools are needed to test animals. In this study, ELISA was developed to detect anti-TBEV specific antibodies in multi-species of animals, using recombinant subviral particles (SPs) with an affinity tag and protein A/G. A Strep-tag was fused at the N terminus of the E protein of the plasmid coding TBEV prME. The E proteins with Strep-tag were secreted as SPs, of which Strep-tag was exposed on the surface. The tagged E proteins were associated with prM. The SPs with Strep-tag were applied as the antigen of ELISA, and TBEV-specific antibodies were detected by the protein A/G. Compared to neutralization test results, the ELISA showed 96.8% sensitivity and 97.7% specificity in rodents and 95.1% sensitivity and 96.0% specificity in humans, without cross-reactivity with antibodies to Japanese encephalitis virus. These results indicate that our ELISA would be useful to detect TBE-specific antibodies in a wide range of animal species.

Virulence of tick-borne encephalitis virus is associated with intact conformational viral RNA structures in the variable region of the 3'-UTR.

Tick-borne encephalitis virus (TBEV) is maintained between ticks and mammals in nature and causes severe neurological disease in human. However, the mechanism of viral pathogenicity is unknown. Previously, we showed that the deletion in the variable region of the 3'-untranslated region (UTR) is involved in the pathogenicity of the strains from the Far-Eastern subtype of TBEV. To investigate the detailed function of the variable region, we constructed recombinant TBEV with partial deletions in the region. In a mouse model, the partial deletions drastically increased the virulence of the virus, with no effect on virus multiplication in mouse brain. Furthermore, the mutations did not affect the production of subgenomic flavivirus RNA from the 3'-UTR, and the induction of interferon (IFN) and IFN-stimulated genes. These data suggested that the conformational structure of the variable region is associated with the pathogenicity of the Far-Eastern subtype of TBEV. These findings provide a foundation for further research to identify the pathogenic mechanisms of TBEV.

Variable region of the 3' UTR is a critical virulence factor in the Far-Eastern subtype of tick-borne encephalitis virus in a mouse model.

Tick-borne encephalitis virus (TBEV) is a major arbovirus that causes thousands of cases of severe neurological illness in humans annually. However, virulence factors and pathological mechanisms of TBEV remain largely unknown. To identify the virulence factors, we constructed chimeric viruses between two TBEV strains of the Far-Eastern subtype, Sofjin-HO (highly pathogenic) and Oshima 5-10 (low pathogenic). The replacement of the coding region for the structural and non-structural proteins from Sofjin into Oshima showed a partial increase of the viral pathogenicity in a mouse model. Oshima-based chimeric viruses with the variable region of the 3' UTR of Sofjin, which had a deletion of 207 nt, killed 100 % of mice and showed almost the same virulence as Sofjin. Replacement of the variable region of the 3' UTR from Sofjin into Oshima did not increase viral multiplication in cultured cells and a mouse model at the early phase of viral entry into the brain. At the terminal phase of viral infection in mice, the virus titre of the Oshima-based chimeric virus with the variable region of the 3' UTR of Sofjin reached a level identical to that of Sofjin and showed a similar histopathological change in the brain tissue. This is the first report to show that the variable region of the 3' UTR is a critical virulence factor in mice. These findings encourage further study to understand the mechanisms of the pathogenicity of TBEV, and to develop preventative and therapeutic strategies for tick-borne encephalitis.

Tick-borne flaviviruses alter membrane structure and replicate in dendrites of primary mouse neuronal cultures.

Neurological diseases caused by encephalitic flaviviruses are severe and associated with high levels of mortality. However, detailed mechanisms of viral replication in the brain and features of viral pathogenesis remain poorly understood. We carried out a comparative analysis of replication of neurotropic flaviviruses: West Nile virus, Japanese encephalitis virus and tick-borne encephalitis virus (TBEV), in primary cultures of mouse brain neurons. All the flaviviruses multiplied well in primary neuronal cultures from the hippocampus, cerebral cortex and cerebellum. The distribution of viral-specific antigen in the neurons varied: TBEV infection induced accumulation of viral antigen in the neuronal dendrites to a greater extent than infection with other viruses. Viral structural proteins, non-structural proteins and dsRNA were detected in regions in which viral antigens accumulated in dendrites after TBEV replication. Replication of a TBEV replicon after infection with virus-like particles of TBEV also induced antigen accumulation, indicating that accumulated viral antigen was the result of viral RNA replication. Furthermore, electron microscopy confirmed that TBEV replication induced characteristic ultrastructural membrane alterations in the neurites: newly formed laminal membrane structures containing virion-like structures. This is the first report describing viral replication in and ultrastructural alterations of neuronal dendrites, which may cause neuronal dysfunction. These findings encourage further work aimed at understanding the molecular mechanisms of viral replication in the brain and the pathogenicity of neurotropic flaviviruses.