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).

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.

Clinical and Molecular Relationships between COVID-19 and Feline Infectious Peritonitis (FIP).

The emergence of severe acute respiratory syndrome 2 (SARS-CoV-2) has led the medical and scientific community to address questions surrounding the pathogenesis and clinical presentation of COVID-19; however, relevant clinical models outside of humans are still lacking. In felines, a ubiquitous coronavirus, described as feline coronavirus (FCoV), can present as feline infectious peritonitis (FIP)-a leading cause of mortality in young cats that is characterized as a severe, systemic inflammation. The diverse extrapulmonary signs of FIP and rapidly progressive disease course, coupled with a closely related etiologic agent, present a degree of overlap with COVID-19. This paper will explore the molecular and clinical relationships between FIP and COVID-19. While key differences between the two syndromes exist, these similarities support further examination of feline coronaviruses as a naturally occurring clinical model for coronavirus disease in humans.

Viral and Host Attributes Underlying the Origins of Zoonotic Coronaviruses in Bats.

With a presumed origin in bats, the COVID-19 pandemic has been a major source of morbidity and mortality in the hu- man population, and the causative agent, SARS-CoV-2, aligns most closely at the genome level with the bat coronaviruses RaBtCoV4991/RaTG13 and RmYN02. The ability of bats to provide reservoirs of numerous viruses in addition to coronaviruses remains an active area of research. Unique aspects of the physiology of the chiropteran immune system may contribute to the ability of bats to serve as viral reservoirs. The coronavirus spike protein plays important roles in viral pathogenesis and the immune response. Although much attention has focused on the spike receptor-binding domain, a unique aspect of SARS-CoV-2 as compared with its closest relatives is the presence of a furin cleavage site in the S1-S2 region of the spike protein. Proteolytic activation is likely an important feature that allows SARS-CoV-2-and other coronaviruses-to overcome the species barriers and thus cause human disease. The diversity of bat species limits the ability to draw broad conclusions about viral pathogenesis, but comparisons across species and with reference to humans and other susceptible mammals may guide future research in this regard.

Furin cleavage sites in the spike proteins of bat and rodent coronaviruses: Implications for virus evolution and zoonotic transfer from rodent species.

Bats and rodents comprise two of the world's largest orders of mammals and the order Chiroptera (bats) has been implicated as a major reservoir of coronaviruses in nature and a source of zoonotic transfer to humans. However, the order Rodentia (rodents) also harbors coronaviruses, with two human coronaviruses (HCoV-OC43 and HCoV-HKU1) considered to have rodent origins. The coronavirus spike protein mediates viral entry and is a major determinant of viral tropism; importantly, the spike protein is activated by host cell proteases at two distinct sites, designated as S1/S2 and S2'. SARS-CoV-2, which is considered to be of bat origin, contains a cleavage site for the protease furin at S1/S2, absent from the rest of the currently known betacoronavirus lineage 2b coronaviruses (Sarbecoviruses). This cleavage site is thought to be critical to its replication and pathogenesis, with a notable link to virus transmission. Here, we examine the spike protein across coronaviruses identified in both bat and rodent species and address the role of furin as an activating protease. Utilizing two publicly available furin prediction algorithms (ProP and PiTou) and based on spike sequences reported in GenBank, we show that the S1/S2 furin cleavage site is typically not present in bat virus spike proteins but is common in rodent-associated sequences, and suggest this may have implications for zoonotic transfer. We provide a phylogenetic history of the Embecoviruses (betacoronavirus lineage 2a), including context for the use of furin as an activating protease for the viral spike protein. From a One Health perspective, continued rodent surveillance should be an important consideration in uncovering novel circulating coronaviruses.

FELINE CORONAVIRUS AND FELINE INFECTIOUS PERITONITIS IN NONDOMESTIC FELID SPECIES.

Feline coronavirus (FCoV) is reported worldwide and known to cause disease in domestic and nondomestic felid species. Although FCoV often results in mild to inapparent disease, a small subset of cats succumb to the fatal, systemic disease feline infectious peritonitis (FIP). An outbreak of FIP in Cheetahs (Acinonyx jubatus) in a zoological collection demonstrated the devastating effect of FCoV introduction into a naïve group of animals. In addition to cheetahs, FIP has been described in European wildcats (Felis silvestris), a tiger (Panthera tigris), a mountain lion (Puma concolor), and lion (Panthera leo). This paper reviews the reported cases of FIP in nondomestic felid species and highlights the surveys of FCoV in populations of nondomestic felids.

Coronaviruses Associated with the Superfamily Musteloidea.

Among the animal superfamily Musteloidea, which includes those commonly known as mustelids, naturally occurring and species-specific alphacoronavirus infections have been observed in both mink (Mustela vison/Neovison vison) and domestic ferrets (Mustela putorius furo). Ferret systemic coronavirus (FRSCV), in particular, has been associated with a rare but fatal systemic disease. In recent months, it has become apparent that both minks and ferrets are susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a betacoronavirus and the cause of the coronavirus disease 2019 (COVID-19) pandemic. Several mink farms have experienced SARS-CoV-2 outbreaks, and experimental models have demonstrated susceptibility of ferrets to SARS-CoV-2. The potential for pet ferrets to become infected with SARS-CoV-2, however, remains elusive. During the 2002-2003 SARS epidemic, it was also apparent that ferrets were susceptible to SARS-CoV and could be utilized in vaccine development. From a comparative standpoint, understanding the relationships between different infections and disease pathogenesis in the animal superfamily Musteloidea may help elucidate viral infection and transmission mechanisms, as well as treatment and prevention strategies for coronaviruses.

Infectious disease surveillance of apparently healthy horses at a multi-day show using a novel nanoscale real-time PCR panel.

In the United States, horses are used for a variety of purposes including recreation, exhibition, and racing. As farm, performance, and companion animals, horses are a unique species from a zoonotic disease risk perspective, and the risks of subclinical infections spreading among horses can pose challenges. Using a nanoscale real-time PCR platform, we investigated the prevalence of 14 enteric pathogens, 11 Escherichia coli genes, and 9 respiratory pathogens in fecal samples from 97 apparently healthy horses at a multi-day horse event. In addition, sugar flotation test was performed for fecal parasites. E. coli f17 was commonly detected, prevalent in 59% of horses, followed closely by Streptococcus equi subsp. zooepidemicus (55%). Additional pathogens recognized included betacoronavirus, Campylobacter jejuni, Cryptosporidium sp., E. coli O157, equine adenovirus 1, equine rhinitis B virus, and others. The use of PCR data may overestimate the true prevalence of these pathogens but provides a sensitive overview of common pathogens present in healthy horses. Our results prompt the continued need for practical biosecurity measures at horse shows, both to protect individuals interacting with these horses and to minimize transmission among horses.

Coronaviruses in cats and other companion animals: Where does SARS-CoV-2/COVID-19 fit?

Coronaviruses (CoVs) cause disease in a range of agricultural and companion animal species, and can be important causes of zoonotic infections. In humans, several coronaviruses circulate seasonally. Recently, a novel zoonotic CoV named SARS-CoV-2 emerged from a bat reservoir, resulting in the COVID-19 pandemic. With a focus on felines, we review here the evidence for SARS-CoV-2 infection in cats, ferrets and dogs, describe the relationship between SARS-CoV-2 and the natural coronaviruses known to infect these species, and provide a rationale for the relative susceptibility of these species to SARS-CoV-2 through comparative analysis of the ACE-2 receptor.

Persistent infection and pancytopenia associated with ferret systemic coronaviral disease in a domestic ferret.

Ferret systemic coronaviral disease (FSCD) is a well-established cause of mortality in domestic ferrets. We describe herein novel findings in a case of FSCD that was diagnosed and medically managed following virus detection by immunohistochemical (IHC) staining of surgical biopsy samples. Hematologic changes in this ferret suggested spread of the virus to the bone marrow, which was confirmed by IHC staining of a postmortem sample. Genotyping of the virus indicated that the virus grouped with alphacoronaviruses and was most closely related to ferret enteric coronavirus (FRECV) MSU-2. Our clinical case demonstrates that a FRECV MSU-2-like ferret coronavirus associated previously with the enteric pathotype may cause systemic disease, including bone marrow involvement causing persistent pancytopenia.

A Tale of Two Viruses: The Distinct Spike Glycoproteins of Feline Coronaviruses.

Feline coronavirus (FCoV) is a complex viral agent that causes a variety of clinical manifestations in cats, commonly known as feline infectious peritonitis (FIP). It is recognized that FCoV can occur in two different serotypes. However, differences in the S protein are much more than serological or antigenic variants, resulting in the effective presence of two distinct viruses. Here, we review the distinct differences in the S proteins of these viruses, which are likely to translate into distinct biological outcomes. We introduce a new concept related to the non-taxonomical classification and differentiation among FCoVs by analyzing and comparing the genetic, structural, and functional characteristics of FCoV and the FCoV S protein among the two serotypes and FCoV biotypes. Based on our analysis, we suggest that our understanding of FIP needs to consider whether the presence of these two distinct viruses has implications in clinical settings.

Intranasal IgG4/7 antibody responses protect horses against equid herpesvirus-1 (EHV-1) infection including nasal virus shedding and cell-associated viremia.

Equid herpesvirus-1 (EHV-1) outbreaks continue despite widely used vaccination. We demonstrated previously that an ORF1/ORF71 gene deletion mutant of the EHV-1 strain Ab4 (Ab4ΔORF1/71) is less virulent than its parent Ab4 virus. Here, we describe the Ab4 challenge infection evaluating protection induced by the Ab4ΔORF1/71 vaccine candidate. Susceptible control horses developed respiratory disease, fever, nasal shedding, and viremia. Full protection after challenge infection was observed in 5/5 previously Ab4 infected horses and 3/5 Ab4ΔORF1/71 horses. Two Ab4ΔORF1/71 horses developed short-lasting viremia and/or virus shedding. Protective immunity in the respiratory tract was characterized by pre-existing EHV-1-specific IgG4/7 antibodies, the absence of IFN-α secretion and rapidly increasing IgG4/7 upon challenge infection. Pre-existing systemic EHV-1-specific IgG4/7 highly correlated with protection. T-cell immunity was overall low. In conclusion, protective immunity against EHV-1 infection including prevention of viremia was associated with robust systemic and intranasal IgG4/7 antibodies suggesting immediate virus neutralization at the local site.

The deletion of the ORF1 and ORF71 genes reduces virulence of the neuropathogenic EHV-1 strain Ab4 without compromising host immunity in horses.

The equine herpesvirus type 1 (EHV-1) ORF1 and ORF71 genes have immune modulatory effects in vitro. Experimental infection of horses using virus mutants with multiple deletions including ORF1 and ORF71 showed promise as vaccine candidates against EHV-1. Here, the combined effects of ORF1 and ORF71 deletions from the neuropathogenic EHV-1 strain Ab4 on clinical disease and host immune response were further explored. Three groups of EHV-1 naïve horses were experimentally infected with the ORF1/71 gene deletion mutant (Ab4ΔORF1/71), the parent Ab4 strain, or remained uninfected. In comparison to Ab4, horses infected with Ab4ΔORF1/71 did not show the initial high fever peak characteristic of EHV-1 infection. Ab4ΔORF1/71 infection had reduced nasal shedding (1/5 vs. 5/5) and, simultaneously, decreased intranasal interferon (IFN)-α, interleukin (IL)-10 and soluble CD14 secretion. However, Ab4 and Ab4ΔORF1/71 infection resulted in comparable viremia, suggesting these genes do not regulate the infection of the mononuclear cells and subsequent viremia. Intranasal and serum anti-EHV-1 antibodies to Ab4ΔORF1/71 developed slightly slower than those to Ab4. However, beyond day 12 post infection (d12pi) serum antibodies in both virus-infected groups were similar and remained increased until the end of the study (d114pi). EHV-1 immunoglobulin (Ig) G isotype responses were dominated by short-lasting IgG1 and long-lasting IgG4/7 antibodies. The IgG4/7 response closely resembled the total EHV-1 specific antibody response. Ex vivo re-stimulation of PBMC with Ab4 resulted in IFN-γ and IL-10 secretion by cells from both infected groups within two weeks pi. Flow cytometric analysis showed that IFN-γ producing EHV-1-specific T-cells were mainly CD8+/IFN-γ+ and detectable from d32pi on. Peripheral blood IFN-γ+ T-cell percentages were similar in both infected groups, albeit at low frequency (~0.1%). In summary, the Ab4ΔORF1/71 gene deletion mutant is less virulent but induced antibody responses and cellular immunity similar to the parent Ab4 strain.