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.

Bioinformatic analysis of the spike protein cleavage sites of coronaviruses in the mammalian order Eulipotyphla (preprint)

The mammalian order Eulipotyphla, including hedgehogs and shrews, represent a poorly understood reservoir of coronaviruses with zoonotic potential. Here, we carried out a bioinformatic analyses of these viruses based on the viral spike protein - to illustrate the complexity of coronavirus evolutionary history and the diversity of viruses from these host species, with a focus on the presence of possible furin cleavage sites within the spike protein. We found no evidence for cleavage by furin itself; however, certain strains of Wencheng Sm Shrew coronavirus were shown to have a predicted cleavage site for other member of the proprotein convertases, which are furin family members - suggesting their spillover potential. As the expanding urbanization and the trade of small mammals in the wet markets enhance the wildlife-human interactions, this may increase pathogen spillover risks. Therefore, we should implement broad wild animal surveillance and be vigilant of contact with these small wild mammals in light of one-health perspectives

Intrinsic furin-mediated cleavability of the spike S1/S2 site from SARS-CoV-2 variant B.1.529 (Omicron) (preprint)

The ability of SARS-CoV-2 to be primed for viral entry by the host cell protease furin has become one of the most investigated of the numerous transmission and pathogenicity features of the virus. Here, we analyzed the S1/S2 cleavage site (also called furin cleavage site) of the spike protein of SARS-CoV-2 B.1.529 (Omicron variant) in vitro, to assess the role of two key mutations (spike gene, N679K and P681H) compared to the ancestral B.1 virus. We observed significantly increased intrinsic cleavability with furin compared to an original B lineage virus (Wuhan-Hu-1) and two variants, B.1.1.7 (Alpha) and B.1.617 (Delta), that subsequently had wide circulation. Increased furin-mediated cleavage was attributed to the N679K mutation, which lies outside the conventional furin binding pocket. Our findings suggest that B.1.529 (Omicron variant) has gained genetic features linked to intrinsic furin cleavability, in line with its evolution within the population as the COVID-19 pandemic has proceeded.

Characterization of Hydrophobic Interactions of SARS-CoV-2 and MERS-CoV Spike Protein Fusion Peptides Using Single Molecule Force Measurements (preprint)

We address the challenge of understanding how hydrophobic interactions are encoded by fusion peptide sequences within coronavirus (CoV) spike proteins. Within the fusion peptides of SARS-CoV-2 and MERS-CoV, a largely conserved peptide sequence called FP1 (SFIEDLLFNK and SAIEDLLFDK in SARS-2 and MERS, respectively) has been proposed to play a key role in encoding hydrophobic interactions that drive viral-host cell membrane fusion. While a non-polar triad (LLF) is common to both FP1 sequences, and thought to dominate the encoding of hydrophobic interactions, FP1 from SARS and MERS differ in two residues (Phe 2 versus Ala 2 and Asn 9 versus Asp 9, respectively). Here we explore if single molecule force measurements can quantify hydrophobic interactions encoded by FP1 sequences, and then ask if sequence variations between FP1 from SARS and MERS lead to significant differences in hydrophobic interactions. We find that both SARS-2 and MERS wild-type FP1 generate measurable hydrophobic interactions at the single molecule level, but that SARS-2 FP1 encodes a substantially stronger hydrophobic interaction than its MERS counterpart (1.91 {+/-} 0.03 nN versus 0.68 {+/-} 0.03 nN, respectively). By performing force measurements with FP1 sequences with single amino acid substitutions, we determine that a single residue mutation (Phe 2 versus Ala 2) causes the almost threefold difference in the hydrophobic interaction strength generated by the FP1 of SARS-2 versus MERS, despite the presence of LLF in both sequences. Infrared spectroscopy and circular dichroism measurements support the proposal that the outsized influence of Phe 2 versus Ala 2 on the hydrophobic interaction arises from variation in the secondary structure adopted by FP1. Overall, these insights reveal how single residue diversity in viral fusion peptides, including FP1 of SARS-CoV-2 and MERS-CoV, can lead to substantial changes in intermolecular interactions proposed to play a key role in viral fusion, and hint at strategies for regulating hydrophobic interactions of peptides in a range of contexts.

The interaction of calcium ions with specific residues in the SARS-CoV fusion peptide and the regulation of viral infectivity (preprint)

Viral envelope fusion with the host cell membrane is dependent on a specific viral fusion peptide (FP) or loop, which becomes exposed during virus entry to drive the process of membrane fusion. In coronaviruses, the FP is a highly conserved domain that sits in the center of spike protein and in SARS-CoV, is adjacent to the S2' proteolytic cleavage site. This peptide contains a hydrophobic LLF motif, as well as several conserved negatively charged amino acids that interact with Ca2+ ions to promote membrane fusion. In this work we perform a systematic mutagenesis study of the negatively charged amino acids within the SARS-CoV fusion peptide (FP1/FP2) and combine this with molecular dynamics simulations to define the membrane interactions that regulate virus infectivity. We show that the E801/D802 amino acid pair in the SARS-CoV FP likely binds to one Ca2+ ion to promote FP-membrane interaction, with a second Ca2+ ion likely pairing residue D812 with either E821 or D825. The D812/D821 residue pair promotes membrane interaction, whereas the D821/D825 is inhibitory to membrane insertion. Taken together, our results demonstrate the dynamic nature of the coronavirus FP region that likely facilitates its interactions with host cell membranes.

In vitro and computational analysis of the putative furin cleavage site (RRARS) in the divergent spike protein of the rodent coronavirus AcCoV-JC34 (sub-genus luchacovirus) (preprint)

The Coronaviridae is a highly diverse virus family, with reservoir hosts in a variety of wildlife species that encompass bats, birds and small mammals, including rodents. Within the taxonomic group alphacoronavirus, certain sub-genera (including the luchacoviruses) have phylogenetically distinct spike proteins, which remain essentially uncharacterized. Using in vitro and computational techniques, we analyzed the spike protein of the rodent coronavirus AcCoV-JC34 from the sub-genus luchacovirus, previously identified in Apodemus chevrieri (Chevriers field mouse). We show that AcCoV-JC34, unlike the other luchacoviruses, has a putative furin cleavage site (FCS) within its spike S1 domain, close to the S1/S2 interface. The pattern of basic amino acids within the AcCoV-JC34 FCS (-RR-R-) is identical to that found in pre-variant SARS-CoV-2, which is in itself atypical for an FCS, and suboptimal for furin cleavage. Our analysis shows that, while containing an -RR-R- motif, the AcCoVJC34 spike FCS is not cleaved by furin (unlike for SARS-CoV-2), suggesting the possible presence of a progenitor sequence for viral emergence from a distinct wildlife host.

Identification of pathogens in respiratory samples of shelter cats in New York State (preprint)

Objectives: Upper respiratory tract disease (URTD) is common across feline populations. Feline coronavirus (FCoV) frequently circulates widely and has occasionally been noted to have a potential role in respiratory disease. The aim of this cross-sectional pilot study was to investigate common respiratory pathogens and FCoV in shelter cats. Methods: Cats were enrolled at two animal shelters in New York state between November 2018 and March 2020 and considered either clinical for URTD or apparently healthy. Respiratory samples were submitted to the Cornell Animal Health Diagnostic Center for routine upper respiratory diagnostic testing. Additional qRT-PCR was performed on respiratory and fecal samples to investigate the presence of FCoV. Results: Five pathogens were identified in this population: Bordetella, feline calicivirus (FCV), M. felis, panleukopenia, and pneumovirus. FCV was the only pathogen associated with URTD signs. Pneumovirus was identified in two cats. FCoV was present in respiratory samples from one cat, who later developed gastrointestinal disease. Fecal shedding of FCoV was observed in 30% of URTD cases, compared to 11% of cats without URTD, but was not statistically significant. Conclusions: and relevance Common respiratory pathogens were identified in cats with and without URTD. One cat tested positive for FCoV in a respiratory sample; FCoV shedding the feces was common, but not statistically significant, in cats with respiratory disease. Two cats were identified as shedding pneumovirus. The significance of pneumovirus to feline respiratory disease remains unknown and in further need of study.

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.

A high throughput screening assay for inhibitors of SARS-CoV-2 pseudotyped particle entry (preprint)

Effective small molecule therapies to combat the SARS-CoV-2 infection are still lacking as the COVID-19 pandemic continues globally. High throughput screening assays are needed for lead discovery and optimization of small molecule SARS-CoV-2 inhibitors. In this work, we have applied viral pseudotyping to establish a cell-based SARS-CoV-2 entry assay. Here, the pseudotyped particles (PP) contain SARS-CoV-2 spike in a membrane enveloping both the murine leukemia virus (MLV) gag-pol polyprotein and luciferase reporter RNA. Upon addition of PP to HEK293-ACE2 cells, the SARS-CoV-2 spike protein binds to the ACE2 receptor on the cell surface, resulting in priming by host proteases to trigger endocytosis of these particles, and membrane fusion between the particle envelope and the cell membrane. The internalized luciferase reporter gene is then expressed in cells, resulting in a luminescent readout as a surrogate for spike-mediated entry into cells. This SARS-CoV-2 PP entry assay can be executed in a biosafety level 2 containment lab for high throughput screening. From a collection of 5,158 approved drugs and drug candidates, our screening efforts identified 7 active compounds that inhibited the SARS-CoV-2-S PP entry. Of these seven, six compounds were active against live replicating SARS-CoV-2 virus in a cytopathic effect assay. Our results demonstrated the utility of this assay in the discovery and development of SARS-CoV-2 entry inhibitors as well as the mechanistic study of anti-SARS-CoV-2 compounds. Additionally, particles pseudotyped with spike proteins from SARS-CoV-2 B.1.1.7 and B.1.351 variants were prepared and used to evaluate the therapeutic effects of viral entry inhibitors.

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 is currently notable as 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 a Ugandan prison in July 2020, and then spilled into the general population adding additional spike mutations (R102I, L141F, E484K and P681R) to comprise lineage A.23.1 by September 2020, with this virus being designated a variant of interest (VOI) in Africa and with subsequent spread to 26 other countries. The P681R spike mutation of the A.23.1 VOI 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 mutation may affect viral replication, transmissibility or pathogenic properties. Here, we performed assays using fluorogenic peptides mimicking the S1/S2 sequence from A.23.1 and observed significantly increased cleavability with furin, compared to sequences matching Wuhan-Hu1 S1/S2. We performed functional infectivity assays using pseudotyped MLV particles harboring SARS-CoV-2 spike proteins and observed an increase in transduction for A.23.1-pseudotyped particles in Vero-TMPRSS2 and Calu-3 cells, compared to Wuhan-Hu1, and a lowered infection in Vero E6 cells. However, these changes in infectivity were not reproduced in a P681R point mutant of Wuhan-Hu1 spike. Our findings suggest that while A.23.1 has increased furin-mediated cleavage linked to the P681R mutation, which may affect viral infection and transmissibility, this mutation needs to occur on the background of other spike protein changes to enable its functional consequences.

Functional evaluation of proteolytic activation for the SARS-CoV-2 variant B.1.1.7: role of the P681H mutation (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent behind the current COVID-19 pandemic having emerged in Wuhan China in late 2019 from a yet to be determined animal reservoir. SARS-CoV-2 B.1.1.7, a variant identified in the UK in late 2020, contains a higher than typical level of point mutants across its genome, including P681H in the spike S1/S2 cleavage site. Here, we performed assays using fluorogenic peptides mimicking the S1/S2 sequence from Wuhan-Hu1 and B.1.1.7 and observed no definitive difference in furin cleavage between Wuhan-Hu1 and B.1.1.7 in vitro. We performed functional assays using pseudo-typed particles harboring SARS-CoV-2 spike proteins and observed no significant differences between Wuhan-Hu1, Wuhan-Hu1 P681H or B.1.1.7 spike-carrying pseudo-typed particles in VeroE6 or Vero-TMPRSS2 cells, despite the spikes containing P681H being more efficiently cleaved. Likewise, we or show no differences in cell-cell fusion assays using the spike P681H-expressing cells. Our findings suggest that while the introduction of P681H in the SARS-CoV-2 B.1.1.7 variant may increase spike cleavage by furin-like proteases, this does not significantly impact viral entry or cell-cell spread. We consider that other factors are at play to account for the increased in transmission and disease severity attributed to this variant of concern (VOC).

β-Coronaviruses use lysosomal organelles for cellular egress. (preprint)

{beta}-Coronaviruses are a family of positive-strand enveloped RNA viruses that include the severe acute respiratory syndrome-CoV2 (SARS-CoV2). While much is known regarding their cellular entry and replication pathways, their mode of egress remains uncertain; however, this is assumed to be via the biosynthetic secretory pathway by analogy to other enveloped viruses. Using imaging methodologies in combination with virus-specific reporters, we demonstrate that {beta}-Coronaviruses utilize lysosomal trafficking for egress from cells. This pathway is regulated by the Arf-like small GTPase Arl8b; thus, virus egress is insensitive to inhibitors of the biosynthetic secretory pathway. Coronavirus infection results in lysosome deacidification, inactivation of lysosomal degradation and disruption of antigen presentation pathways. This coronavirus-induced exploitation of lysosomes provides insights into the cellular and immunological abnormalities observed in patients and suggests new therapeutic modalities.

FDA approved calcium channel blockers inhibit SARS CoV 2 infectivity in epithelial lung cells (preprint)

Covid-19 has infected more than 14 million people worldwide causing over 600.000 deaths1. The disease is caused by the severe acute respiratory syndrome coronavirus (CoV) 2 (SARS-CoV-2), which shares a high sequence similarity to SARS CoV2. Currently there are no vaccinations available to provide protection and the only antiviral therapy in active use in patients is remdesivir, which currently provides only limited benefit3. Hence, there is an urgent need for antiviral therapies against SARS CoV 2. SARS CoV requires Ca2+ ions for host cell entry4 and based on the similarity between SARS CoV and SARS CoV 25 it is highly likely that the same requirements exist for the two viruses. Here, we tested whether FDA-approved calcium channel blocker (CCB) drugs are efficacious to inhibit the spread of SARS CoV 2 in cell culture. Our data shows that amlodipine, felodipine and nifedipine limit the growth of SARS CoV 2 in epithelial kidney (Vero E6) and epithelial lung (Calu-3) cells. We observed some differences in the inhibition efficacy of the drugs in the two different cell lines, but with felodopine and nifedipine having the greatest effect. Overall, our data suggest that CCBs have a high potential to treat SARS CoV 2 infections and their current FDA approval would allow for a fast repurposing of these drugs.

β-Coronaviruses Use Lysosomal Organelles for Cellular Egress (preprint)

β-Coronaviruses are a family of positive-strand enveloped RNA viruses that include the severe acute respiratory syndrome-CoV2 (SARS-CoV2). While much is known regarding their cellular entry and replication pathways, their mode of egress remains uncertain; however, this is assumed to be via the biosynthetic secretory pathway by analogy to other enveloped viruses. Using imaging methodologies in combination with virus-specific reporters, we demonstrate that β-Coronaviruses utilize lysosomal trafficking for egress from cells. This pathway is regulated by the Arf-like small GTPase Arl8b; thus, virus egress is insensitive to inhibitors of the biosynthetic secretory pathway. Coronavirus infection results in lysosome deacidification, inactivation of lysosomal degradation and disruption of antigen presentation pathways. This coronavirus-induced exploitation of lysosomes provides insights into the cellular and immunological abnormalities observed in patients and suggests new therapeutic modalities.Funding: NAB, SG, TDR, EP, QQ, MF and CB were supported with NHLBI/NIH; GAB and SRA were supported with NCI/NIH intramural funds. PMT was supported by NIH R01 A1091985-05; SP by NIH R01 NS36592 and AF by F32-AI113973; VH by NIH R37GM058615; GW by NIH R01AI35270.Conflict of Interest: None.

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).