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Background Early in the pandemic, transmission risk from asymptomatic infection was unclear, making it imperative to monitor infection in workplace settings. Further, data on SARS-CoV-2 seroprevalence within university populations has been limited. Methods We performed a longitudinal study of University research employees on campus July-December 2020. We conducted questionnaires on COVID-19 risk factors, RT-PCR testing, and SARS-CoV-2 serology using an in-house spike RBD assay, laboratory-based Spike NTD assay, and standard nucleocapsid platform assay. We estimated prevalence and cumulative incidence of seroconversion with 95% confidence intervals using the inverse of the Kaplan-Meier estimator. Results 910 individuals were included in this analysis. At baseline, 6.2% (95% CI 4.29–8.19) were seropositive using the spike RBD assay; four (0.4%) were seropositive using the nucleocapsid assay, and 44 (4.8%) using the Spike NTD assay. Cumulative incidence was 3.61% (95% CI: 2.04–5.16). Six asymptomatic individuals had positive RT-PCR results. Conclusions Prevalence and incidence of SARS-CoV-2 infections were low; however, differences in target antigens of serological tests provided different estimates. Future research on appropriate methods of serological testing in unvaccinated and vaccinated populations is needed. Frequent RT-PCR testing of asymptomatic individuals is required to detect acute infections, and repeated serosurveys are beneficial for monitoring subclinical infection.

SARS-CoV-2 is constantly evolving. Prior studies have focused on high case-density locations, such as the Northern and Western metropolitan areas in the U.S. This study demonstrates continued SARS-CoV-2 evolution in a suburban Southern U.S. region by high-density amplicon sequencing of symptomatic cases. 57% of strains carried the spike D614G variant. The presence of D614G was associated with a higher genome copy number and its prevalence expanded with time. Four strains carried a deletion in a predicted stem loop of the 3’ untranslated region. The data are consistent with community spread within the local population and the larger continental U.S. No strain had mutations in the target sites used in common diagnostic assays. The data instill confidence in the sensitivity of current tests and validate “testing by sequencing” as a new option to uncover cases, particularly those not conforming to the standard clinical presentation of COVID-19. This study contributes to the understanding of COVID-19 by providing an extensive set of genomes from a non-urban setting and further informs vaccine design by defining D614G as a dominant and emergent SARS-CoV-2 isolate in the U.S.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is constantly evolving. Prior studies focused on high-case-density locations, such as the northern and western metropolitan areas of the United States. This study demonstrates continued SARS-CoV-2 evolution in a suburban southern region of the United States by high-density amplicon sequencing of symptomatic cases. 57% of strains carry the spike D614G variant, which is associated with higher genome copy numbers, and its prevalence expands with time. Four strains carry a deletion in a predicted stem loop of the 3′ UTR. The data are consistent with community spread within local populations and the larger continental United States. The data instill confidence in current testing sensitivity and validate “testing by sequencing” as an option to uncover cases, particularly nonstandard coronavirus disease 2019 (COVID-19) clinical presentations. This study contributes to the understanding of COVID-19 through an extensive set of genomes from a non-urban setting and informs vaccine design by defining D614G as a dominant and emergent SARS-CoV-2 isolate in the United States., Graphical Abstract, Highlights • NGS of SARS-CoV-2 from a rural/suburban area shows local spread as an epidemic driver • The D614G spike mutant is observed in >50% of cases • Deletion in the 3′ UTR of SARS-CoV-2 is identified • Targeted NGS has 100% specificity and is as sensitive as qPCR, McNamara et al. use next-generation sequencing (NGS) with a high-density tiling array across SARS-CoV-2 to find a deletion and document how the D614G spike protein mutation rapidly swept through a rural/suburban population. D614G is associated with slightly higher viral loads.

The primary function of toll-like receptor 8 (TLR-8) is the detection of viruses and other microbial pathogens. Recent evidence suggests that TLR-8 also senses host microRNAs (miRNAs) and implicate TLR-8 in autoimmune disorders. This study examined the interaction between miR-1294 and TLR-8. We first performed a BLAST search to identify miRNAs with the same sequences as two core motifs of miR-1294. Next, we examined NFḱB activation induced by the binding of miR-1294 mimic to endosomal TLR-8. HEK-Blue™ hTLR-8 cells (Invivogen), a HEK293 cell line co-transfected with human TLR-8 gene, were incubated with miR-1294 mimic. A TLR-8 agonist ssRNA40, was used as a positive control. Using the same experimental set up, we also examined the effects of miR-1294 and its two core motifs (Integrated DNA Technologies) on IL-8, IL-1β, and TNFα. Data were analyzed using t-test or one-way ANOVA and Dunnets post-hoc test. Using miRCarta we identified 29 other mature human miRNAs or their precursors which contain the same core motifs as miR-1294. Our data show that miR-1294 activates NFḱB in cells expressing TLR-8 (p < 0.05). miR-1294, and its core motifs induce expression of IL-8, IL-1β, and TNFα via TLR8 activation (p < 0.05). This constitutes a novel mechanism by which endosomal TLR-8 senses host miRNAs resulting in the release of pro-inflammatory cytokines and thus potentially contributing to autoimmune disorders.