Variant mutation in SARS-CoV-2 nucleocapsid enhances viral infection via altered genomic encapsidation (preprint)

The evolution of SARS-CoV-2 variants and their respective phenotypes represents an important set of tools to understand basic coronavirus biology as well as the public health implications of individual mutations in variants of concern. While mutations outside of Spike are not well studied, the entire viral genome is undergoing evolutionary selection, particularly the central disordered linker region of the nucleocapsid (N) protein. Here, we identify a mutation (G215C), characteristic of the Delta variant, that introduces a novel cysteine into this linker domain, which results in the formation of a disulfide bond and a stable N-N dimer. Using reverse genetics, we determined that this cysteine residue is necessary and sufficient for stable dimer formation in a WA1 SARS-CoV-2 background, where it results in significantly increased viral growth both in vitro and in vivo. Finally, we demonstrate that the N:G215C virus packages more nucleocapsid per virion and that individual virions are larger, with elongated morphologies.

Surveillance of Vermont wildlife in 2021-2022 reveals no detected SARS-CoV-2 viral RNA (preprint)

Previous studies have documented natural infections of SARS-CoV-2 in various domestic and wild animals. More recently, studies have been published noting the susceptibility of members of the Cervidae family, and infections in both wild and captive cervid populations. In this study, we investigated the presence of SARS-CoV-2 in mammalian wildlife within the state of Vermont. 739 nasal or throat samples were collected from wildlife throughout the state during the 2021 and 2022 harvest season. Data was collected from red and gray foxes (Vulpes vulples and Urocyon cineroargentus, respectively), fishers (Martes pennati), river otters (Lutra canadensis), coyotes (Canis lantrans), bobcats (Lynx rufus rufus), black bears (Ursus americanus), and white-tailed deer (Odocoileus virginianus). Samples were tested for the presence of SARS-CoV-2 via quantitative RT-qPCR using the CDC N1/N2 primer set and/or the WHO-E gene primer set. Our results indicate that no sampled wildlife were positive for SARS-CoV-2. This finding is surprising, given that most published North America studies have found SARS-CoV-2 within their deer populations. The absence of SARS-CoV-2 RNA in populations sampled here may provide insights in to the various environmental and anthropogenic factors that reduce spillover and spread in North American's wildlife populations.

Viral infectivity in pediatric SARS-CoV-2 clinical samples does not vary by age (preprint)

During the early months of the SARS-CoV-2 pandemic, notable uncertainty emerged regarding the role of children in transmission dynamics. With time, it became more clear that children were susceptible to infection with SARS-CoV-2, but that the vast majority of children experienced mild symptoms with lower incidence of severe disease. This pattern remained consistent despite the later emergence of SARS-CoV-2 variants, including Delta and Omicron, even among children <5 ineligible for vaccination. The relative lack of severe disease in the pediatric population raised questions regarding viral kinetics and infectivity in children versus adults. We hypothesized that unique virologic features in children could explain this apparent decrease in symptoms and transmissibility early in the pandemic.

Quantitative measurement of infectious virus in SARS-CoV-2 Alpha, Delta and Epsilon variants reveals higher infectivity (viral titer:RNA ratio) in clinical samples containing the Delta and Epsilon variants. (preprint)

BackgroundNovel SARS-CoV-2 Variants of Concern (VoC) pose a challenge to controlling the COVID-19 pandemic. Previous studies indicate that clinical samples collected from individuals infected with the Delta variant may contain higher levels of RNA than previous variants, but the relationship between viral RNA and infectious virus for individual variants is unknown. MethodsWe measured infectious viral titer (using a micro-focus forming assay) as well as total and subgenomic viral RNA levels (using RT-PCR) in a set of 165 clinical samples containing SARS-CoV-2 Alpha, Delta and Epsilon variants that were processed within two days of collection from the patient. ResultsWe observed a high degree of variation in the relationship between viral titers and RNA levels. Despite the variability we observed for individual samples the overall infectivity differed among the three variants. Both Delta and Epsilon had significantly higher infectivity than Alpha, as measured by the number of infectious units per quantity of viral E gene RNA (6 and 4 times as much, p=0.0002 and 0.009 respectively) or subgenomic E RNA (11 and 7 times as much, p<0.0001 and 0.006 respectively). ConclusionIn addition to higher viral RNA levels reported for the Delta variant, the infectivity (amount of replication competent virus per viral genome copy) may also be increased compared to Alpha. Measuring the relationship between live virus and viral RNA is an important step in assessing the infectivity of novel SARS-CoV-2 variants. An increase in the infectivity of the Delta variant may further explain increased spread and suggests a need for increased measures to prevent viral transmission. SIGNIFICANCE STATEMENTCurrent and future SARS-CoV-2 variants threaten our ability to control the COVID-19 pandemic. Variants with increased transmission, higher viral loads, or greater immune evasion are of particular concern. Viral loads are currently measured by the amount of viral RNA in a clinical sample rather than the amount of infectious virus. We measured both RNA and infectious virus levels directly in a set of 165 clinical specimens from Alpha, Epsilon or Delta variants. Our data shows that Delta is more infectious compared to Alpha, with [~] six times as much infectious virus for the same amount of RNA. This increase in infectivity suggests increased measures (vaccination, masking, distancing, ventilation) are needed to control Delta compared to Alpha.

RT-qPCR DETECTION OF SARS-CoV-2 RNA FROM PATIENT NASOPHARYNGEAL SWAB USING QIAGEN RNEASY KITS OR DIRECTLY VIA OMISSION OF AN RNA EXTRACTION STEP (preprint)

The ongoing COVID-19 pandemic has caused an unprecedented need for rapid diagnostic testing. The Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) recommend a standard assay that includes an RNA extraction step from a nasopharyngeal (NP) swab followed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect the purified SARS-CoV-2 RNA. The current global shortage of RNA extraction kits has caused a severe bottleneck to COVID-19 testing. We hypothesized that SARS-CoV-2 RNA could be detected from NP samples via a direct RT-qPCR assay that omits the RNA extraction step altogether, and tested this hypothesis on a series of blinded clinical samples. The direct RT-qPCR approach correctly identified 92% of NP samples (n = 155) demonstrated to be positive for SARS-CoV-2 RNA by traditional clinical diagnostic RT-qPCR that included an RNA extraction. Thus, direct RT-qPCR could be a front-line approach to identify the substantial majority of COVID-19 patients, reserving a repeat test with RNA extraction for those individuals with high suspicion of infection but an initial negative result. This strategy would drastically ease supply chokepoints of COVID-19 testing and should be applicable throughout the world.