The Highly Conserved Stem-Loop II Motif Is Dispensable for SARS-CoV-2.

The stem-loop II motif (s2m) is an RNA structural element that is found in the 3' untranslated region (UTR) of many RNA viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Though the motif was discovered over 25 years ago, its functional significance is unknown. In order to understand the importance of s2m, we created viruses with deletions or mutations of the s2m by reverse genetics and also evaluated a clinical isolate harboring a unique s2m deletion. Deletion or mutation of the s2m had no effect on growth in vitro or on growth and viral fitness in Syrian hamsters in vivo. We also compared the secondary structure of the 3' UTR of wild-type and s2m deletion viruses using selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) and dimethyl sulfate mutational profiling and sequencing (DMS-MaPseq). These experiments demonstrate that the s2m forms an independent structure and that its deletion does not alter the overall remaining 3'-UTR RNA structure. Together, these findings suggest that s2m is dispensable for SARS-CoV-2. IMPORTANCE RNA viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), contain functional structures to support virus replication, translation, and evasion of the host antiviral immune response. The 3' untranslated region of early isolates of SARS-CoV-2 contained a stem-loop II motif (s2m), which is an RNA structural element that is found in many RNA viruses. This motif was discovered over 25 years ago, but its functional significance is unknown. We created SARS-CoV-2 with deletions or mutations of the s2m and determined the effect of these changes on viral growth in tissue culture and in rodent models of infection. Deletion or mutation of the s2m element had no effect on growth in vitro or on growth and viral fitness in Syrian hamsters in vivo. We also observed no impact of the deletion on other known RNA structures in the same region of the genome. These experiments demonstrate that s2m is dispensable for SARS-CoV-2.

The highly conserved stem-loop II RNA element (s2m) is critical for the lifecycle of astrovirus VA1 but is dispensable for SARS-CoV-2

Background RNA viruses often contain important elements for the viral lifecycle in the untranslated regions (UTR) of the genome. The stem-loop II (s2m) element is a RNA structural motif in the 3' UTR that is 39-43 nucleotides in length and is present in members of the Astroviridae, Caliciviridae, Picornaviridae, Reoviridae, and Coronaviridae viral families, including SARS coronavirus 1 and 2 (SARS-CoV-1 and -2). Despite the first description of this element twenty-five years ago, the functional significance of the s2m sequence to the viral lifecycle remains poorly understood. The conservation of the s2m in many viral genomes suggests some fitness benefit to viruses that maintain it. Method To test the significance of the s2m element, we developed reverse genetic systems for astrovirus VA1 (VA1) and SARS-CoV-2. For both viruses, we introduced deletions and mutations predicted to significantly disrupt the secondary structure of the s2m. Recombinant viruses were passaged and the infectious viral titer was measured. To evaluate the in vivo significance of the s2m, we used a hamster model of SARS-CoV-2 infection. Recombinant viruses were intranasally inoculated and viral RNA and infectious viral titers measured from the nasal washes and the lungs three and six days after infection. A clinical SARS-CoV-2 isolate containing a deletion of the s2m was also propagated and the viral titer measured in a multi-step growth curve. Results The s2m in VA1 was critical to the viral lifecycle as deletion of the s2m resulted in recombinant virus that could not be rescued and propagated. Introduction of one or two nucleotide mutations that disrupt predicted G-C binding pairs in the s2m element also resulted in recombinant virus that was not viable. Compensatory mutations that restored G-C base pairings in the s2m element supported recovery and propagation of the virus with similar viral titers compared to wild-type (WT) VA-1. In contrast, the s2m in SARS-CoV-2 was dispensable. SARS-CoV-2 genomes containing a deletion or mutation of the s2m could be rescued and propagated in cell culture and had similar viral titers compared to the WT SARS-CoV-2 in multi-step growth curves. In the hamster model, there was also no difference in the amount of viral RNA and infectious virus in the lung and nasal washes of infected animals. We also identified a clinical SARS-CoV-2 isolate that contained a deletion of the s2m and it showed no differences in the growth in a multi-step growth curve compared to a SARS-CoV-2 virus with an intact s2m. Conclusion The s2m is essential for the lifecycle of astrovirus VA1 but dispensable for SARS-CoV-2, suggesting differing functions of the s2m that is virus dependent. Future experiments will identify the mechanistic basis for the divergent role of the s2m in each viral lifecycle.

Perceptions of risk of SARS-CoV-2 transmission in social and educational activities by infectious diseases and general pediatric healthcare providers, a pre-vaccine risk perception cross-sectional survey.

BACKGROUND: The perception of the transmission risks of SARS-CoV-2 in social and educational settings by US healthcare providers have not been previously quantified. METHODS: Respondents completed an online survey between September and October 2020 to estimate the risk of SARS-CoV-2 transmission on a scale of 0-10 for different social and educational activities prior to the availability of the SARS-CoV-2 vaccines. Demographic information and experiences during the pandemic were also collected. The risk assessment was emailed to three listservs of healthcare providers, including national listservs of pediatric (PID) and adult infectious diseases (AID) providers, and a listserv of general pediatric practitioners in the St Louis, USA metropolitan area. RESULTS: Respondents identified the highest risk of SARS-CoV-2 transmission in spending time in a bar, eating at a restaurant, and attending an indoor sporting event. In the school setting, lower risk was identified in elementary and daycare students compared to high school or university-level students. Comparatively, the risk of transmission to students and teachers was lower than the identified high-risk social activities. Factors increasing risk perception in social activities included the absence of children in the respondent's household and female gender. For the school setting, AID providers perceived greater risk compared to PID providers or pediatric practitioners. CONCLUSIONS: Respondents identified high risk activities that were associated with a high density of participants in an indoor space where masks are removed for eating and drinking. Differences were apparent in the school setting where pediatric providers perceived lower risks when compared to adult providers.

Progression of SARS-CoV-2 Seroprevalence in St. Louis, Missouri, through January 2021.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seropositivity was assessed for 3,066 individuals visiting hospitals in St. Louis, Missouri, during July 2020, November 2020, or January 2021. Seropositivity in children increased from 5.22% in July to 21.16% in January. In the same time frame, seropositivity among adults increased from 4.52% to 19.03%, prior to initiation of mass vaccination. IMPORTANCE This study determined the percentage of children and adult samples from the St. Louis metropolitan area in Missouri with SARS-CoV-2 antibodies during three collection periods spanning July 2020 to January 2021. By January 2021, 20.68% of the tested individuals had antibodies. These results show the evolution of the SARS-CoV-2 pandemic in St. Louis, Missouri, and provide a snapshot of the extent of infection just prior to the start of mass vaccination.

Seroprevalence of SARS-CoV-2 Antibodies in Children and Adults in St. Louis, Missouri, USA.

Reported coronavirus disease 2019 (COVID-19) case counts likely underestimate the true prevalence because mild or asymptomatic cases often go untested. Here, we use a sero-survey to estimate the seroprevalence of IgG antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the St. Louis, MO, metropolitan area in a symptom-independent manner. Five hundred three adult and 555 pediatric serum/plasma samples were collected from patients presenting to Barnes-Jewish Hospital or St. Louis Children's Hospital between 14 April 2020 and 12 May 2020. We developed protocols for in-house enzyme-linked immunosorbent assays (ELISAs) using spike and nucleoprotein and used the assays to estimate a seroprevalence rate based on our samples. Overall IgG seropositivity was estimated to be 1.71% (95% credible interval [CI], 0.04% to 3.38%) in pediatric samples and 3.11% (95% CI, 0.92% to 5.32%) in adult samples. Seropositivity was significantly lower in children under 5 years of age than in adults, but rates between adults and children aged 5 or older were similar. Of the 176 samples tested from children under 4 years of age, none were positive.IMPORTANCE This study determined the percentages of both children and adult samples from the greater St. Louis metropolitan area who had antibodies to SARS-CoV-2 in late April to early May 2020. Approximately 1.7 to 3.1% of the tested individuals had antibodies, indicating that they had previously been infected by SARS-CoV-2. These results demonstrate that the extent of infection was about 10 times greater than the number of confirmed cases at that time. Furthermore, it demonstrated that by 5 years of age, children were infected to an extent similar to that of adults.