SARS-CoV-2 emerged in 2019 and has become a major global pathogen in an astonishingly short period of time. The emergence of SARS-CoV-2 also has been notable due to its impacts on individuals residing within skilled nursing facilities (SNFs) such as rehabilitation centers and nursing homes. SNF residents tend to possess several risk factors for the most severe outcomes of SARS-CoV-2 infection, including advanced age and the presence of multiple comorbidities. Indeed, residents of long-term care facilities represent approximately 40 percent of US SARS-CoV-2 deaths. To assess the prevalence and incidence of SARS-CoV-2 among SNF workers, determine the extent of asymptomatic infection by SARS-CoV-2, and provide information on the genomic epidemiology of the virus within these unique care settings, we sampled workers weekly at five SNFs in Colorado using nasopharyngeal swabs, determined the presence of viral RNA and infectious virus among these workers, and sequenced 48 nearly complete genomes. This manuscript reports results from the first five to six weeks of observation. Our data reveal a strikingly high degree of asymptomatic infection, a strong correlation between RNA detection and the presence of infectious virus in NP swabs, persistent RNA in a subset of individuals, and declining incidence over time. Our data suggests that asymptomatic individuals infected by SARS-CoV-2 may contribute to virus transmission within the workplace.

Arthropod-borne viruses (arboviruses) of vertebrates including dengue, zika, chikungunya, Rift Valley fever, and blue tongue viruses cause extensive morbidity and mortality in humans, agricultural animals, and wildlife across the globe. As obligate intercellular pathogens, arboviruses must be well adapted to the cellular and molecular environment of both their arthropod (invertebrate) and vertebrate hosts, which are vastly different due to hundreds of millions of years of separate evolution. Here we discuss the comparative pressures on arbovirus RNA genomes as a result of a dual host life cycle, focusing on pressures that do not alter amino acids. We summarize what is currently known about arboviral genetic composition, such as dinucleotide and codon usage, and how cyclical infection of vertebrate and invertebrate hosts results in different genetic profiles compared with single-host viruses. To serve as a comparison, we compile what is known about arthropod tRNA, dinucleotide, and codon usages and compare this with vertebrates. Additionally, we discuss the potential roles of genetic robustness in arboviral evolution and how it may vary from other viruses. Overall, both arthropod and vertebrate hosts influence the resulting genetic composition of arboviruses, but a great deal remains to be investigated.

The unwinding of double-stranded RNA intermediates is critical for replication and packaging of flavivirus RNA genomes. This unwinding activity is achieved by the ATP-dependent nonstructural protein 3 (NS3) helicase. In previous studies, we investigated the mechanism of energy transduction between the ATP and RNA binding pockets using molecular dynamics simulations and enzymatic characterization. Our data corroborated the hypothesis that Motif V is a communication hub for this energy transduction. More specifically, mutations T407A and S411A in Motif V exhibit a hyperactive helicase phenotype leading to the regulation of translocation and unwinding during replication. However, the effect of these mutations on viral infection in cell culture and in vivo is not well understood. Here, we investigated the role of Motif V in viral replication using T407A and S411A West Nile virus (Kunjin subtype) mutants in cell culture and in vivo. We were able to recover S411A Kunjin but unable to recover T407A Kunjin. Our results indicated that S411A Kunjin decreased viral infection, and increased cytopathogenicity in cell culture as compared to WT Kunjin. Similarly, decreased infection rates in surviving S411A-infected Culex quinquefasciatus mosquitoes were observed, but S411A Kunjin infection resulted in increased mortality compared to WT Kunjin. Additionally, S411A Kunjin increased viral dissemination and saliva positivity rates in surviving mosquitoes compared to WT Kunjin. These data suggest that S411A Kunjin increases pathogenesis in mosquitoes. Overall, these data indicate that NS3 Motif V may play a role in the pathogenesis, dissemination, and transmission efficiency of Kunjin virus. IMPORTANCE: Kunjin and West Nile viruses belong to the arthropod-borne flaviviruses, which can result in severe symptoms including encephalitis, meningitis, and death. Flaviviruses have expanded into new populations and emerged as novel pathogens repeatedly in recent years demonstrating they remain a global threat. Currently, there are no approved anti-viral therapeutics against either Kunjin or West Nile viruses. Thus, there is a pressing need for understanding the pathogenesis of these viruses in humans. In this study, we investigate the role of the Kunjin virus helicase on infection in cell culture and in vivo . This work provides new insight into how flaviviruses control pathogenesis and mosquito transmission through the nonstructural protein 3 helicase.