SARS-CoV-2 serosurvey of healthy, privately owned cats presenting to a New York City animal hospital in the early phase of the COVID-19 pandemic (2020-2021) (preprint)

Both domestic and non-domestic cats are now established to be susceptible to infection by SARS-CoV-2, the cause of the ongoing COVID-19 pandemic. While serious disease in cats may occur in some instances, the majority of infections appear to be subclinical. Differing prevalence data for SARS-CoV-2 infection of cats have been reported, and are highly context-dependent. Here, we report a retrospective serological survey of cats presented to an animal practice in New York City, located in close proximity to a large medical center that treated the first wave of COVID-19 patients in the US in the Spring of 2020. We sampled 79, mostly indoor, cats between June 2020 to May 2021, the early part of which time the community was under a strict public health lock-down. Using a highly sensitive and specific fluorescent bead-based multiplex assay, we found an overall prevalence of 13/79 (16%) serologically-positive animals for the study period; however, cats sampled in the Fall of 2020 had a confirmed positive prevalence of 44%. For SARS-CoV-2 seropositive cats, we performed viral neutralization test with live SARS-CoV-2 to additionally confirm presence of SARS-CoV-2 specific antibodies. Of the thirteen seropositive cats, 7/13 (54%) were also positive by virus neutralization, and 2 of seropositive cats had previously documented respiratory signs, with high neutralization titers of 1:1024 and 1:4096; overall however, there was no statistically significant association of SARS-CoV-2 seropositivity with respiratory signs, or with breed, sex or age of the animals. Follow up sampling of cats, while limited in scope, showed that positive serological titers were maintained over time. In comparison, we found an overall confirmed positive prevalence of 51% for feline coronavirus (FCoV), an endemic virus of cats, with 30% confirmed negative for FCoV. We demonstrate the impact of SARS-CoV in a defined feline population during the first wave of SARS-CoV-2 infection of humans, and suggest that human-cat transmission was substantial in our study group. Our data provide a new context for SARS-CoV-2 transmission events across species.

Spike Protein Cleavage-Activation Mediated by the SARS-CoV-2 P681R Mutation: A Case-Study From Its First Appearance in Variant of Interest (VOI) A.23.1 Identified in Uganda (preprint)

The African continent like all other parts of the world with high infection/low vaccination rates can, and will, be a source of novel SARS-CoV-2 variants. The A.23 viral lineage, characterized by three spike mutations F157L, V367F and Q613H, was first identified in COVID-19 cases from a Ugandan prison in July 2020, and then was identified in the general population with the additional spike mutation P681R at the S1/S2 cleavage site to comprise lineage A.23.1 by September 2020 with subsequent spread to 26 other countries. The P681R spike substitution of A.23.1 is of note as it increases the number of basic residues in the sub-optimal SARS-CoV-2 spike protein furin cleavage site; as such, this substitution may affect viral replication, transmissibility, or pathogenic properties. The same P681R substitution has also subsequently appeared in B.1.617 variants, including B.1.617.2 (Delta). Here, we performed assays using fluorogenic peptides mimicking the S1/S2 from A.23.1 and B.1.617 and observed significantly increased cleavability with furin, compared to sequences derived from the original Wuhan-Hu1 S1/S2. We performed cell-cell fusion and functional infectivity assays using pseudotyped particles harboring SARS-CoV-2 spike proteins and observed an increase in transduction for A.23.1-pseudotyped particles compared to Wuhan-Hu-1. However, these changes in activity were not reproduced in the original Wuhan-Hu-1 spike bearing only the P681R substitution. Our findings suggest that while A.23.1 has increased furin-mediated cleavage linked to the P681R substitution—which may affect viral infection and transmissibility—this substitution alone needs to occur on the background of other spike protein changes to enable its full functional consequences.Funding: This work was funded in part by the National Institute of Health research grant R01AI35270 (to GW and SD). We thank the global SARS-CoV-2 sequencing groups for their open and rapid sharing of sequence data and GISAID for providing an effective platform to make these data available. DLB, MVTP and MC were funded by the UK Medical Research Council (MRC/UK Research and Innovation) and the UK Department for International Development (DFID) under the MRC/DFID Concordat agreement (grant agreement no. NC_PC_19060) and Wellcome Trust, UK FCDO—Wellcome Epidemic Preparedness—Coronavirus (grant agreement no. 220977/Z/20/Z). TT was supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. DGE-1650441 and the Samuel C. Fleming Family Graduate Fellowship. Declaration of Interests: The authors manifest no conflict of interest.

Proteolytic Cleavage of the SARS-CoV-2 Spike Protein and the Role of the Novel S1/S2 Site (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 19 (COVID-19) has rapidly spread from an initial outbreak in Wuhan, China in December 2019 to the rest of the world within a few months. On March 11th 2020, the rapidly evolving COVID-19 situation was characterized as a pandemic by the WHO. Much attention has been drawn to the origin of SARS-CoV-2, a virus which is related to the lineage B betacoronavirus SARS-CoV and SARS-related coronaviruses found in bat species. The closest known relative to SARS-CoV-2 is a bat coronavirus named RaTG13 (BatCoV-RaTG13). Early characterizations of the SARS-CoV-2 genome revealed the existence of a distinct 4 amino acid insert (underlined, SPRRAR↓S), found within the spike (S) protein, at a position termed the S1/S2 site located at the interface between the S1 receptor binding subunit and the S2 fusion subunit. Notably, this S1/S2 insert appears to be distinguishing feature among SARS-related sequences and introduces a potential cleavage site for the protease furin. Here, we investigate the potential role of this novel S1/S2 cleavage site and present direct biochemical evidence for proteolytic processing by a variety of proteases, including furin, trypsin-like proteases and cathepsins. We discuss these findings in the broader context of the origin of SARS-CoV-2, viral stability and transmission.Funding: Work in the author’s laboratory is supported by the National Institutes of Health (research grant R01AI35270).