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New variants of SARS-CoV-2 are continually emerging with mutations in spike associated with increased transmissibility and immune escape. Phenotypic maps can inform the prediction of concerning mutations from genomic surveillance, however most of these maps currently derive from studies using monomeric RBD, while spike is trimeric, and contains additional domains. These maps may fail to reflect interdomain interactions in the prediction of phenotypes. To try to improve on this, we developed a platform for deep mutational scanning using whole trimeric spike. We confirmed a previously reported epistatic effect within the RBD affecting ACE2 binding, that highlights the importance of updating the base spike sequence for future mutational scanning studies. Using post vaccine sera, we found that the immune response of vaccinated individuals was highly focused on one or two epitopes in the RBD and that single point mutations at these positions can account for most of the immune escape mediated by the Omicron BA.1 RBD. However, unexpectedly we found that the BA.1 RBD alone does not account for the high level of antigenic escape by BA.1 spike. We show that the BA.1 NTD amplifies the immune evasion of its associated RBD. BA.1 NTD reduces neutralistion by RBD directed monoclonal antibodies, and impacts ACE2 interaction. NTD variation is thus an important mechanism of immune evasion by SARS-CoV-2. Such effects are not seen when pre-stabilized spike proteins are used, suggesting the interdomain effects require protein mobility to express their phenotype., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Kugathasan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

SARS-CoV-2 has a broad mammalian species tropism infecting humans, cats, dogs, and farmed mink. Since the start of the 2019 pandemic, several reverse zoonotic outbreaks of SARS-CoV-2 have occurred in mink, one of which reinfected humans and caused a cluster of infections in Denmark. Here we investigate the molecular basis of mink and ferret adaptation and demonstrate the spike mutations Y453F, F486L, and N501T all specifically adapt SARS-CoV-2 to use mustelid ACE2. Furthermore, we risk assess these mutations and conclude mink-adapted viruses are unlikely to pose an increased threat to humans, as Y453F attenuates the virus replication in human cells and all three mink adaptations have minimal antigenic impact. Finally, we show that certain SARS-CoV-2 variants emerging from circulation in humans may naturally have a greater propensity to infect mustelid hosts and therefore these species should continue to be surveyed for reverse zoonotic infections., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: To establish the presence of live virus and its association with polymerase chain reaction (PCR) positivity and antibody status in patients with COVID-19 undergoing tracheotomy., Study Design: Prospective observational study., Setting: Single institution across 3 hospital sites during the first wave of the COVID-19 pandemic., Methods: Patients who were intubated for respiratory wean tracheotomy underwent SARS-CoV-2 PCR nasal, throat, and endotracheal tube swabs at the time of the procedure. These were assessed via quantitative real-time reverse transcription PCR. The tracheal tissue excised during the tracheotomy was cultured for SARS-CoV-2 with Vero E6 and Caco2 cells. Serum was assessed for antibody titers against SARS-CoV-2 via neutralization assays., Results: Thirty-seven patients were included in this study. The mean number of days intubated prior to undergoing surgical tracheotomy was 27.8. At the time of the surgical tracheotomy, PCR swab testing yielded 8 positive results, but none of the 35 individuals who underwent tissue culture were positive for SARS-CoV-2. All 18 patients who had serum sampling demonstrated neutralization antibodies, with a minimum titer of 1:80., Conclusion: In our series, irrespective of positive PCR swab, the likelihood of infectivity during tracheotomy remains low given negative tracheal tissue cultures. While our results do not undermine national and international guidance on tracheotomy after day 10 of intubation, given the length of time to procedure in our data, infectivity at 10 days cannot be excluded. We do however suggest that a preoperative negative PCR swab not be a prerequisite and that antibody titer levels may serve as a useful adjunct for assessment of infectivity.

SARS-CoV-2 entry requires sequential cleavage of the spike glycoprotein at the S1/S2 and the S2' cleavage sites to mediate membrane fusion. SARS-CoV-2 has a polybasic insertion (PRRAR) at the S1/S2 cleavage site that can be cleaved by furin. Using lentiviral pseudotypes and a cell-culture-adapted SARS-CoV-2 virus with an S1/S2 deletion, we show that the polybasic insertion endows SARS-CoV-2 with a selective advantage in lung cells and primary human airway epithelial cells, but impairs replication in Vero E6, a cell line used for passaging SARS-CoV-2. Using engineered spike variants and live virus competition assays and by measuring growth kinetics, we find that the selective advantage in lung and primary human airway epithelial cells depends on the expression of the cell surface protease TMPRSS2, which enables endosome-independent virus entry by a route that avoids antiviral IFITM proteins. SARS-CoV-2 virus lacking the S1/S2 furin cleavage site was shed to lower titres from infected ferrets and was not transmitted to cohoused sentinel animals, unlike wild-type virus. Analysis of 100,000 SARS-CoV-2 sequences derived from patients and 24 human postmortem tissues showed low frequencies of naturally occurring mutants that harbour deletions at the polybasic site. Taken together, our findings reveal that the furin cleavage site is an important determinant of SARS-CoV-2 transmission.

The ongoing SARS-CoV-2 epidemic was marked by the repeated emergence and replacement of "variants" with genetic and phenotypic distance from the ancestral strains, the most recent examples being viruses of the Omicron lineage. Here, we describe a hamster direct contact exposure challenge model to assess protection against reinfection conferred by either vaccination or prior infection. We found that two doses of self-amplifying RNA vaccine based on the ancestral Spike ameliorated weight loss following Delta infection and decreased viral loads but had minimal effect on Omicron BA.1 infection. Prior vaccination followed by Delta or BA.1 breakthrough infections led to a high degree of cross-reactivity to all tested variants, suggesting that repeated exposure to antigenically distinct Spikes, via infection and/or vaccination drives a cross-reactive immune response. Prior infection with ancestral or Alpha variant was partially protective against BA.1 infection, whereas all animals previously infected with Delta and exposed to BA.1 became reinfected, although they shed less virus than BA.1-infected naive hamsters. Hamsters reinfected with BA.1 after prior Delta infection emitted infectious virus into the air, indicating that they could be responsible for onwards airborne transmission. We further tested whether prior infection with BA.1 protected from reinfection with Delta or later Omicron sublineages BA.2, BA.4, or BA.5. BA.1 was protective against BA.2 but not against Delta, BA.4, or BA.5 reinfection. These findings suggest that cohorts whose only immune experience of COVID-19 is Omicron BA.1 infection may be vulnerable to future circulation of reemerged Delta-like derivatives, as well as emerging Omicron sublineages. [ABSTRACT FROM AUTHOR]

Background: Accurate antibody tests are essential to monitor the SARS-CoV-2 pandemic. Lateral flow immunoassays (LFIAs) can deliver testing at scale. However, reported performance varies, and sensitivity analyses have generally been conducted on serum from hospitalised patients. For use in community testing, evaluation of finger-prick self-tests, in non-hospitalised individuals, is required.Methods: Sensitivity analysis was conducted on 276 non-hospitalised participants. All had tested positive for SARS-CoV-2 by reverse transcription PCR and were ≥21 days from symptom onset. In phase I, we evaluated five LFIAs in clinic (with finger prick) and laboratory (with blood and sera) in comparison to (1) PCR-confirmed infection and (2) presence of SARS-CoV-2 antibodies on two 'in-house' ELISAs. Specificity analysis was performed on 500 prepandemic sera. In phase II, six additional LFIAs were assessed with serum.Findings: 95% (95% CI 92.2% to 97.3%) of the infected cohort had detectable antibodies on at least one ELISA. LFIA sensitivity was variable, but significantly inferior to ELISA in 8 out of 11 assessed. Of LFIAs assessed in both clinic and laboratory, finger-prick self-test sensitivity varied from 21% to 92% versus PCR-confirmed cases and from 22% to 96% versus composite ELISA positives. Concordance between finger-prick and serum testing was at best moderate (kappa 0.56) and, at worst, slight (kappa 0.13). All LFIAs had high specificity (97.2%-99.8%).Interpretation: LFIA sensitivity and sample concordance is variable, highlighting the importance of evaluations in setting of intended use. This rigorous approach to LFIA evaluation identified a test with high specificity (98.6% (95%CI 97.1% to 99.4%)), moderate sensitivity (84.4% with finger prick (95% CI 70.5% to 93.5%)) and moderate concordance, suitable for seroprevalence surveys. [ABSTRACT FROM AUTHOR]

Vaccines for SARS-CoV-2 have been hugely successful in alleviating hospitalization and deaths caused by the newly emerged coronavirus that is the cause of COVID. However, although the parentally administered vaccines are very effective at reducing severe disease, they do not induce sterilizing immunity. As the virus continues to circulate around the globe, it is still not clear how long protection will last, nor whether variants will emerge that escape vaccine immunity. Animal models can be useful to complement studies of antigenicity of novel variants and inform decision making about the need for vaccine updates. The Syrian golden hamster is the preferred small animal model for SARS-CoV-2 infection. Since virus is efficiently transmitted between hamsters, we developed a transmission challenge model that presents a more natural dose and route of infection than the intranasal challenge usually employed. Our studies demonstrate that an saRNA vaccine based on the earliest Wuhan-like virus spike sequence induced neutralizing antibodies in sera of immunized hamsters at similar titres to those in human convalescent sera or vaccine recipients. The saRNA vaccine was equally effective at abrogating clinical signs in animals who acquired through exposure to cagemates infected either with a virus isolated in summer 2020 or with a representative Alpha (B.1.1.7) variant isolated in December 2020. The vaccine also reduced shedding of infectious virus from the nose, further reinforcing its likely effectiveness at reducing onwards transmission. This model can be extended to test the effectiveness of vaccination in blocking infections with and transmission of novel variants as they emerge. [ABSTRACT FROM AUTHOR]