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Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection can cause neurological disease in humans, but little is known about the pathogenesis of SARS-CoV-2 infection in the central nervous system (CNS). Herein, using K18-hACE2 mice, we demonstrate that SARS-CoV-2 neuroinvasion and encephalitis is associated with mortality in these mice. Intranasal infection of K18-hACE2 mice with 105 plaque-forming units of SARS-CoV-2 resulted in 100% mortality by day 6 after infection. The highest virus titers in the lungs were observed on day 3 and declined on days 5 and 6 after infection. By contrast, very high levels of infectious virus were uniformly detected in the brains of all the animals on days 5 and 6. Onset of severe disease in infected mice correlated with peak viral levels in the brain. SARS-CoV-2-infected mice exhibited encephalitis hallmarks characterized by production of cytokines and chemokines, leukocyte infiltration, hemorrhage and neuronal cell death. SARS-CoV-2 was also found to productively infect cells within the nasal turbinate, eye and olfactory bulb, suggesting SARS-CoV-2 entry into the brain by this route after intranasal infection. Our data indicate that direct infection of CNS cells together with the induced inflammatory response in the brain resulted in the severe disease observed in SARS-CoV-2-infected K18-hACE2 mice.

Cas13a has been used to target RNA viruses in cell culture, but efficacy has not been demonstrated in animal models. In this study, we used messenger RNA (mRNA)-encoded Cas13a for mitigating influenza virus A and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in mice and hamsters, respectively. We designed CRISPR RNAs (crRNAs) specific for PB1 and highly conserved regions of PB2 of influenza virus, and against the replicase and nucleocapsid genes of SARS-CoV-2, and selected the crRNAs that reduced viral RNA levels most efficiently in cell culture. We delivered polymer-formulated Cas13a mRNA and the validated guides to the respiratory tract using a nebulizer. In mice, Cas13a degraded influenza RNA in lung tissue efficiently when delivered after infection, whereas in hamsters, Cas13a delivery reduced SARS-CoV-2 replication and reduced symptoms. Our findings suggest that Cas13a-mediated targeting of pathogenic viruses can mitigate respiratory infections. Cas13a delivered to the lung of mice and hamsters inhibits replication of influenza virus and SARS-CoV-2.

Oxidative stress activates the cellular kinase HRI, which then phosphorylates eIF2α, resulting in stalled translation initiation and the formation of stress granules (SGs). SG assembly redirects cellular translation to stress response mRNAs and inhibits cap-dependent viral RNA translation. Flavivirus infections were previously reported to induce oxidative stress in infected cells but flavivirus-infected cells paradoxically develop resistance to arsenite (Ars)-induced SG formation with time after infection. This resistance was previously postulated to be due to sequestration of the SG protein Caprin1 by Japanese encephalitis virus capsid protein. However, Caprin1 did not co-localize with West Nile virus (WNV) capsid protein in infected cells. Other stressors induced SGs with equal efficiency in mock- and WNV-infected cells indicating the intrinsic ability of cells to assemble SGs was not disabled. Induction of both reactive oxygen species (ROS) and the antioxidant response was detected at early times after WNV-infection. The transcription factors, Nrf2 and ATF4, which activate antioxidant genes, were upregulated and translocated to the nucleus. Knockdown of Nrf2, ATF4 or apoptosis-inducing factor (AIF), a mitochondrial protein involved in regenerating intracellular reduced glutathione (GSH) levels, with siRNA or treatment of cells with buthionine sulphoximine, which induces oxidative stress by inhibiting GSH synthesis, decreased intracellular GSH levels and increased the number of SG-positive, infected cells. Mitochondria were protected from Ars-induced damage by WNV infection until late times in the infection cycle. The results indicate that the increase in virus-induced ROS levels is counterbalanced by a virus-induced antioxidant response that is sufficient to also overcome the increase in ROS induced by Ars treatment and prevent Ars-induced SG assembly and mitochondrial damage. The virus-induced alterations in the cellular redox status appear to provide benefits for the virus during its lifecycle.

The West Nile virus minus-strand 3' terminal stem loop (SL) RNA was previously shown to bind specifically to cellular stress granule (SG) components, T cell intracellular antigen-1 (TIA-1) and the related protein TIAR. In vitro TIAR binding was 10 times more efficient than TIA-1. The 3'(-)SL functions as the promoter for genomic RNA synthesis. Colocalization of TIAR and TIA-1 with the viral replication complex components dsRNA and NS3 was observed in the perinuclear regions of West Nile virus- and dengue virus-infected cells. The kinetics of accumulation of TIAR in the perinuclear region was similar to those of genomic RNA synthesis. In contrast, relocation of TIA-1 to the perinuclear region began only after maximal levels of RNA synthesis had been achieved, except when TIAR was absent. Virus infection did not induce SGs and progressive resistance to SG induction by arsenite developed coincident with TIAR relocation. A progressive decrease in the number of processing bodies was secondarily observed in infected cells. These data suggest that the interaction of TIAR with viral components facilitates flavivirus genome RNA synthesis and inhibits SG formation, which prevents the shutoff of host translation.

Potential G-quadruplex sites have been identified in the genomes of DNA and RNA viruses and proposed as regulatory elements. The genus Orthoflavivirus contains arthropod-transmitted, positive-sense, single-stranded RNA viruses that cause significant human disease globally. Computational studies have identified multiple potential G-quadruplex sites that are conserved across members of this genus. Subsequent biophysical studies established that some G-quadruplexes predicted in Zika and tickborne encephalitis virus genomes can form and known quadruplex binders reduced viral yields from cells infected with these viruses. The susceptibility of RNA to degradation and the variability of loop regions have made structure determination challenging. Despite these difficulties, we report a high-resolution structure of the NS5-B quadruplex from the West Nile virus genome. Analysis reveals two stacked tetrads that are further stabilized by a stacked triad and transient noncanonical base pairing. This structure expands the landscape of solved RNA quadruplex structures and demonstrates the diversity and complexity of biological quadruplexes. We anticipate that the availability of this structure will assist in solving further viral RNA quadruplexes and provides a model for a conserved antiviral target in Orthoflavivirus genomes. G-quadruplex are higher-order nucleotide structures within G-rich sequences with regulatory roles. Here, the authors provide the structure of a viral genomic RNA G-quadruplex using X-ray crystallography. They report unusual features of the West Nile virus NS5B quadruplex which expands our knowledge of quadruplex complexity and will aid in designing molecules to target them.

The human OAS1 (hOAS1) gene produces multiple possible isoforms due to alternative splicing events and sequence variation among individuals, some of which affect splicing. The unique C-terminal sequences of the hOAS1 isoforms could differentially affect synthetase activity, protein stability, protein partner interactions and/or cellular localization. Recombinant p41, p42, p44, p46, p48, p49 and p52 hOAS1 isoform proteins expressed in bacteria were each able to synthesize trimer and higher order 2′-5′ linked oligoadenylates in vitro in response to poly(I:C). The p42, p44, p46, p48 and p52 isoform proteins were each able to induce RNase-mediated rRNA cleavage in response to poly(I:C) when overexpressed in HEK293 cells. The expressed levels of the p42 and p46 isoform proteins were higher than those of the other isoforms, suggesting increased stability in mammalian cells. In a yeast two-hybrid screen, Fibrillin1 (FBN1) was identified as a binding partner for hOAS1 p42 isoform, and Supervillin (SVIL) as a binding partner for the p44 isoform. The p44-SVIL interaction was supported by co-immunoprecipitation data from mammalian cells. The data suggest that the unique C-terminal regions of hOAS1 isoforms may mediate the recruitment of different partners, alternative functional capacities and/or different cellular localization.

West Nile virus (WNV) is a mosquito-borne flavivirus that primarily infects birds but occasionally also infects humans and horses. In recent years, the frequency of WNV outbreaks in humans has increased, and these outbreaks have been associated with a higher incidence of severe disease. In 1999, the geographical distribution of WNV expanded to the Western hemisphere. WNV has a positive strand RNA genome of about 11 kb that encodes a single polyprotein. WNV replicates in the cytoplasm of infected cells. Although there are still many questions to be answered, a large body of data on the molecular biology of WNV and other flaviviruses has already been obtained. Aspects of virion structure, the viral replication cycle, viral protein function, genome structure, conserved viral elements, host factors, virus-host interactions, and vaccines are discussed in this review.

West Nile virus (WNV), a category B pathogen endemic in parts of Africa, Asia and Europe, emerged in North America in 1999, and spread rapidly across the continental U.S. Outcomes of infection with WNV range from asymptomatic to severe neuroinvasive disease manifested as encephalitis, paralysis, and/or death. Neuroinvasive WNV disease occurs in less than one percent of cases, and although host genetic factors are thought to influence risk for symptomatic disease, the identity of these factors remains largely unknown. We tested 360 common haplotype tagging and/or functional SNPs in 86 genes that encode key regulators of immune function in 753 individuals infected with WNV including: 422 symptomatic WNV cases and 331 cases with asymptomatic infections. After applying a Bonferroni correction for multiple tests and controlling for population stratification, SNPs in IRF3 (OR 0.54, p = 0.035) and MX1, (OR 0.19, p = 0.014) were associated with symptomatic WNV infection and a single SNP in OAS1 (OR 9.79, p = 0.003) was associated with increased risk for West Nile encephalitis and paralysis (WNE/P). Together, these results suggest that genetic variation in the interferon response pathway is associated with both risk for symptomatic WNV infection and WNV disease progression.

The 2′-5′-oligoadenylate synthetase (2′-5′-OAS) family members are interferon-induced antiviral proteins. Twenty-three single nucleotide polymorphisms located within the OAS1, OAS2, OAS3, and OASL genes were analyzed in 142 patients with Russian tick-borne encephalitis. Statistically significant differences in genotype, allele, and haplotype frequencies for 3 OAS2 single nucleotide polymorphisms (rs1293762, rs15895, and rs1732778) and 2 OAS3 single nucleotide polymorphisms (rs2285932 and rs2072136) were detected between patients with central nervous system disease and both those with fever and/or meningitis and the control group. The data suggest a possible association between these 5 OAS single nucleotide polymorphisms and the outcome of tick-borne encephalitis virus infection in a Russian population.

Background. The live attenuated yellow fever vaccine 17D (YF-17D) is one of the most effective vaccines. Despite its excellent safety record, some cases of viscerotropic adverse events develop, which are sometimes fatal. The mechanisms underlying such events remain a mystery. Here, we present an analysis of the immunologic and genetic factors driving disease in a 64-year-old male who developed viscerotropic symptoms. Methods. We obtained clinical, serologic, virologic, immunologic and genetic data on this case patient. Results. Viral RNA was detected in the blood 33 days after vaccination, in contrast to the expected clearance of virus by day 7 after vaccination in healthy vaccinees. Vaccination induced robust antigen-specific T and B cell responses, which suggested that persistent virus was not due to adaptive immunity of suboptimal magnitude. The genes encoding OAS1, OAS2, TLR3, and DC-SIGN, which mediate antiviral innate immunity, were wild type. However, there were heterozygous genetic polymorphisms in chemokine receptor CCR5, and its ligand RANTES, which influence the migration of effector T cells and CD14+CD16bright monocytes to tissues. Consistent with this, there was a 200-fold increase in the number of CD14+CD16bright monocytes in the blood during viremia and even several months after virus clearance. Conclusion. In this patient, viscerotropic disease was not due to the impaired magnitude of adaptive immunity but instead to anomalies in the innate immune system and a possible disruption of the CCR5-RANTES axis.