Despite entering an endemic phase, SARS-CoV-2 remains a significant burden to public health across the global community. Wastewater sampling has consistently proven utility to understanding SARS-CoV-2 prevalence trends and genetic variation as it represents a less biased assessment of the corresponding communities. Here, we report that ongoing monitoring of SARS-CoV-2 genetic variation in samples obtained from the wastewatersheds of the city of Louisville in Jefferson county Kentucky has revealed the periodic reemergence of the Delta strain in the presence of the presumed dominant Omicron strain. Unlike previous SARS-CoV-2 waves/emergence events, the Delta reemergence events were geographically restricted in the community and failed to spread into other areas as determined by wastewater analyses. Moreover, the reemergence of the Delta strain did not correlate with vaccination rates as communities with lower relative vaccination have been, to date, not affected. Importantly, Delta reemergence events correlate with increased public health burdens, as indicated by increased daily case rates and mortality relative to non-Delta wastewatershed communities. While the underlying reasons for the reemergence of the Delta variant remain unclear, these data reaffirm the ongoing importance of wastewater genomic analyses towards understanding SARS-CoV-2 as it enters the endemic phase.

Alphaviruses are positive-sense RNA viruses that utilize a 5′ cap structure to facilitate translation of viral proteins and to protect the viral RNA genome. Nonetheless, significant quantities of viral genomic RNAs that lack a canonical 5′ cap structure are produced during alphaviral replication and packaged into viral particles. However, the role/impact of the noncapped genomic RNA (ncgRNA) during alphaviral infection in vivo has yet to be characterized. To determine the importance of the ncgRNA in vivo, the previously described D355A and N376A nsP1 mutations, which increase or decrease nsP1 capping activity, respectively, were incorporated into the neurovirulent AR86 strain of Sindbis virus to enable characterization of the impact of altered capping efficiency in a murine model of infection. Mice infected with the N376A nsP1 mutant exhibited slightly decreased rates of mortality and delayed weight loss and neurological symptoms, although levels of inflammation in the brain were similar to those of wild-type infection. Although the D355A mutation resulted in decreased antiviral gene expression and increased resistance to interferon in vitro, mice infected with the D355A mutant showed significantly reduced mortality and morbidity compared to mice infected with wild-type virus. Interestingly, expression of proinflammatory cytokines was found to be significantly decreased in mice infected with the D355A mutant, suggesting that capping efficiency and the production of ncgRNA are vital to eliciting pathogenic levels of inflammation. Collectively, these data indicate that the ncgRNA have important roles during alphaviral infection and suggest a novel mechanism by which noncapped viral RNAs aid in viral pathogenesis. IMPORTANCE Mosquito-transmitted alphaviruses have been the cause of widespread outbreaks of disease that can range from mild illness to lethal encephalitis or severe polyarthritis. There are currently no safe and effective vaccines or therapeutics with which to prevent or treat alphaviral disease, highlighting the need to better understand alphaviral pathogenesis to develop novel antiviral strategies. This report reveals production of noncapped genomic RNAs (ncgRNAs) to be a novel determinant of alphaviral virulence and offers insight into the importance of inflammation to pathogenesis. Taken together, the findings reported here suggest that the ncgRNAs contribute to alphaviral pathogenesis through the sensing of the ncgRNAs during alphaviral infection and are necessary for the development of severe disease.

Alphaviruses cause significant outbreaks of febrile illness and debilitating multi-joint arthritis for prolonged periods after initial infection. We have previously reported that several host hnRNP proteins bind to the Sindbis virus (SINV) RNAs, and disrupting the sites of these RNA–protein interactions results in decreased viral titers in tissue culture models of infection. Intriguingly, the primary molecular defect associated with the disruption of the hnRNP interactions is enhanced viral structural protein expression; however, the precise underlying mechanisms spurring the enhanced gene expression remain unknown. Moreover, our previous efforts were unable to functionally dissect whether the observed phenotypes were due to the loss of hnRNP binding or the incorporation of polymorphisms into the primary nucleotide sequence of SINV. To determine if the loss of hnRNP binding was the primary cause of attenuation or if the disruption of the RNA sequence itself was responsible for the observed phenotypes, we utilized an innovative protein tethering approach to restore the binding of the hnRNP proteins in the absence of the native interaction site. Specifically, we reconstituted the hnRNP I interaction by incorporating the 20nt bovine immunodeficiency virus transactivation RNA response (BIV-TAR) at the site of the native hnRNP I interaction sequence, which will bind with high specificity to proteins tagged with a TAT peptide. The reestablishment of the hnRNP I–vRNA interaction via the BIV-TAR/TAT tethering approach restored the phenotype back to wild-type levels. This included an apparent decrease in structural protein expression in the absence of the native primary nucleotide sequences corresponding to the hnRNP I interaction site. Collectively, the characterization of the hnRNP I interaction site elucidated the role of hnRNPs during viral infection.

Alphaviruses are arthropod-borne, single-stranded positive sense RNA viruses that rely on the engagement of host RNA-binding proteins to efficiently complete the viral lifecycle. Because of this reliance on host proteins, the identification of host/pathogen interactions and the subsequent characterization of their importance to viral infection has been an intensive area of study for several decades. Many of these host protein interaction studies have evaluated the Protein:Protein interactions of viral proteins during infection and a significant number of host proteins identified by these discovery efforts have been RNA Binding Proteins (RBPs). Considering this recognition, the field has shifted towards discovery efforts involving the direct identification of host factors that engage viral RNAs during infection using innovative discovery approaches. Collectively, these efforts have led to significant advancements in the understanding of alphaviral molecular biology; however, the precise extent and means by which many RBPs influence viral infection is unclear as their specific contributions to infection, as per any RNA:Protein interaction, have often been overlooked. The purpose of this review is to summarize the discovery of host/pathogen interactions during alphaviral infection with a specific emphasis on RBPs, to use new ontological analyses to reveal potential functional commonalities across alphaviral RBP interactants, and to identify host RBPs that have, and have yet to be, evaluated in their native context as RNA:Protein interactors.

Alphaviruses are positive-sense RNA viruses that utilize a 5’ cap structure to facilitate translation of viral proteins and to protect the viral RNA genome. Nonetheless, significant quantities of viral genomic RNAs that lack a canonical 5’ cap structure are produced during alphaviral replication and packaged into viral particles. However, the role/impact of the noncapped genomic RNA (ncgRNA) during alphaviral infection in vivo has yet to be characterized. To determine the importance of the ncgRNA in vivo, the previously described D355A and N376A nsP1 mutations, which increase or decrease nsP1 capping activity respectively, were incorporated into the neurovirulent AR86 strain of Sindbis virus to enable characterization of the impact of altered capping efficiency in a murine model of infection. Mice infected with the N376A nsP1 mutant exhibited slightly decreased rates of mortality and delayed weight loss and neurological symptoms, although levels of inflammation in the brain were similar to wild type infection. The mice infected with the D355A nsP1 mutant showed significantly reduced mortality and morbidity compared to mice infected with wild type virus. Interestingly, both capping mutants had roughly equivalent viral titer in the brain compared to wild type virus, illustrating that the changes in mortality were not due to deficits in viral replication or dissemination. Examination of the brain tissue revealed that mice infected with the D355A capping mutant had significantly reduced cell death and immune cell infiltration compared to the N376A mutant and wild type virus. Finally, expression of proinflammatory cytokines was found to be significantly decreased in mice infected with the D355A mutant, suggesting that capping efficiency and the production of ncgRNA are vital to eliciting pathogenic levels of inflammation. Collectively, these data indicate that the ncgRNA have important roles during alphaviral infection and suggest a novel mechanism by which noncapped viral RNA aid in viral pathogenesis.AUTHOR SUMMARYMosquito transmitted alphaviruses have been the cause of widespread outbreaks of disease which can range from mild illness to lethal encephalitis or severe polyarthritis. In order to successfully replicate, the alphavirus RNA genome needs a 5’ cap structure so that the genome can be translated and produce the viral replication machinery. Despite this, a large number of viral genomes produced during infection do not have a 5’ cap structure, and their role during infection is unknown. Using mouse models of infection and point mutations in the nsP1 protein of Sindbis virus which alter the amount of noncapped genomic RNA (ncgRNA) produced, we found the decreasing the production of ncgRNA greatly reduced morbidity and mortality as well as proinflammatory cytokine expression, resulting in less tissue-damaging inflammation in the brain. These studies suggest that the ncgRNAs contribute to pathogenesis through the sensing of the ncgRNAs during alphaviral infection and are necessary for the development of severe disease.