Adaptive Mutation in the Main Protease Cleavage Site of Feline Coronavirus Renders the Virus More Resistant to Main Protease Inhibitors.

The rapid global emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused serious health problems, highlighting the urgent need for antiviral drugs. The viral main protease (Mpro) plays an important role in viral replication and thus remains the target of choice for the prevention or treatment of several viral diseases due to high sequence and structural conservation. Prolonged use of viral protease inhibitors can lead to the development of mutants resistant to those inhibitors and to many of the available antiviral drugs. Here, we used feline infectious peritonitis virus (FIPV) as a model to investigate its development of resistance under pressure from the Mpro inhibitor GC376. Passage of wild-type (WT) FIPV in the presence of GC376 selected for a mutation in the nsp12 region where Mpro cleaves the substrate between nsp12 and nsp13. This mutation confers up to 3-fold resistance to GC376 and nirmatrelvir, as determined by EC50 assay. In vitro biochemical and cellular experiments confirmed that FIPV adapts to the stress of GC376 by mutating the nsp12 and nsp13 hydrolysis site to facilitate cleavage by Mpro and release to mediate replication and transcription. Finally, we demonstrate that GC376 cannot treat FIP-resistant mutants that cause FIP in animals. Taken together, these results suggest that Mpro affects the replication of coronaviruses (CoVs) and the drug resistance to GC376 by regulating the amount of RdRp from a distant site. These findings provide further support for the use of an antiviral drug combination as a broad-spectrum therapy to protect against contemporary and emerging CoVs. IMPORTANCE CoVs cause serious human infections, and antiviral drugs are currently approved to treat these infections. The development of protease-targeting therapeutics for CoV infection is hindered by resistance mutations. Therefore, we should pay attention to its resistance to antiviral drugs. Here, we identified possible mutations that lead to relapse after clinical treatment of FIP. One amino acid substitution in the nsp12 polymerase at the Mpro cleavage site provided low-level resistance to GC376 after selection exposure to the GC376 parental nucleoside. Resistance mutations enhanced FIPV viral fitness in vitro and attenuated the therapeutic effect of GC376 in an animal model of FIPV infection. Our research explains the evolutionary characteristics of coronaviruses under antiviral drugs, which is helpful for a more comprehensive understanding of the molecular basis of virus resistance and provides important basic data for the effective prevention and control of CoVs.

Possible Cross-Reactivity of Feline and White-Tailed Deer Antibodies against the SARS-CoV-2 Receptor Binding Domain.

In late 2019, a novel coronavirus began circulating within humans in central China. It was designated SARS-CoV-2 because of its genetic similarities to the 2003 SARS coronavirus (SARS-CoV). Now that SARS-CoV-2 has spread worldwide, there is a risk of it establishing new animal reservoirs and recombination with native circulating coronaviruses. To screen local animal populations in the United States for exposure to SARS-like coronaviruses, we developed a serological assay using the receptor binding domain (RBD) from SARS-CoV-2. SARS-CoV-2's RBD is antigenically distinct from common human and animal coronaviruses, allowing us to identify animals previously infected with SARS-CoV or SARS-CoV-2. Using an indirect enzyme-linked immunosorbent assay (ELISA) for SARS-CoV-2's RBD, we screened serum from wild and domestic animals for the presence of antibodies against SARS-CoV-2's RBD. Surprisingly prepandemic feline serum samples submitted to the University of Tennessee Veterinary Hospital were ∼50% positive for anti-SARS RBD antibodies. Some of these samples were serologically negative for feline coronavirus (FCoV), raising the question of the etiological agent generating anti-SARS-CoV-2 RBD cross-reactivity. We also identified several white-tailed deer from South Carolina with anti-SARS-CoV-2 antibodies. These results are intriguing, as cross-reactive antibodies toward SARS-CoV-2 RBD have not been reported to date. The etiological agent responsible for seropositivity was not readily apparent, but finding seropositive cats prior to the current SARS-CoV-2 pandemic highlights our lack of information about circulating coronaviruses in other species. IMPORTANCE We report cross-reactive antibodies from prepandemic cats and postpandemic South Carolina white-tailed deer that are specific for that SARS-CoV RBD. There are several potential explanations for this cross-reactivity, each with important implications to coronavirus disease surveillance. Perhaps the most intriguing possibility is the existence and transmission of an etiological agent (such as another coronavirus) with similarity to SARS-CoV-2's RBD region. However, we lack conclusive evidence of prepandemic transmission of a SARS-like virus. Our findings provide impetus for the adoption of a One Health Initiative focusing on infectious disease surveillance of multiple animal species to predict the next zoonotic transmission to humans and future pandemics.

SARS-CoV-2 in domestic cats (Felis catus) in the northwest of Iran: Evidence for SARS-CoV-2 circulating between human and cats.

This study aimed to investigate the prevalence of COVID-19 in domestic cats, focusing on the disease in the northwest of Iran and then showing the natural transmission of SARS-COV-2 circulating between domestic cats and humans. After receiving ethic codes from Tehran University of Medical Sciences (IR.TUMS.VCR.REC.1399.303) and confirmed by the Center of Communicable Diseases Control (CDC) of Iran, 124 domestic cats were collected from the homes and only one hospital of Meshkin -Shahr district from northwestern Iran where SARS-CoV-2 patients were hospitalized and quarantined during 2020. Samples were prepared from fluid materials of oropharynx and nasopharynx. All samples were tested by real-time PCR (RT-PCR) using specific genes N and ORF1ab in Pasteur Institute of Iran, and then partial sequence analyses of S gene were performed. All collected cats were kept in separated cages until SARS-COV-2 infection was confirmed with the RT-PCR. RT- PCR Ct values of 123 collected cats were ≥40; thus, all of them showed negative results, but one of the collected cats with close contact with its owner, whom confirmed SARS-CoV-2 showed positive results with gene N(Ct=30) and gene ORF1ab (Ct=32). Furthermore, the positive pet cat showed respiratory and gastro-intestinal clinical manifestations, and its owner was infected with SARS-CoV-2 two weeks ago. Cats are susceptible animals to SARS-CoV-2 infection. Epidemiological evidence showed that SARS-COV-2 is able to transmit to healthy cats due to having close contact with its owner as a reverse zoonosis.

First genome sequencing of SARS-CoV-2 recovered from an infected cat and its owner in Latin America.

An 11-year-old male mixed-breed cat, with exclusively indoor life, presented 3 cough episodes after the owners tested positive by RT-PCR for SARS-CoV-2. The house is inhabited by 5 people (3 adults and 2 children), and 2 of the adults have shown mild symptoms associated with throat discomfort. The cat was vaccinated, had no history of any previous disease, and tested negative for feline coronavirus (FCoV), feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV). Rectal sample collected from the cat was positive for SARS-CoV-2 by RT-PCR. Viral genome sequences recovered from human and cat samples showed an average 99.4% sequence identity. This is the first report of genome sequences of SARS-CoV-2 recovered from a cat and its owner in Latin America.

The Upper Respiratory Tract of Felids Is Highly Susceptible to SARS-CoV-2 Infection.

Natural or experimental infection of domestic cats and virus transmission from humans to captive predatory cats suggest that felids are highly susceptible to SARS-CoV-2 infection. However, it is unclear which cells and compartments of the respiratory tract are infected. To address this question, primary cell cultures derived from the nose, trachea, and lungs of cat and lion were inoculated with SARS-CoV-2. Strong viral replication was observed for nasal mucosa explants and tracheal air-liquid interface cultures, whereas replication in lung slices was less efficient. Infection was mainly restricted to epithelial cells and did not cause major pathological changes. Detection of high ACE2 levels in the nose and trachea but not lung further suggests that susceptibility of feline tissues to SARS-CoV-2 correlates with ACE2 expression. Collectively, this study demonstrates that SARS-CoV-2 can efficiently replicate in the feline upper respiratory tract ex vivo and thus highlights the risk of SARS-CoV-2 spillover from humans to felids.

Cell Entry of Animal Coronaviruses.

Coronaviruses (CoVs) are a group of enveloped positive-sense RNA viruses and can cause deadly diseases in animals and humans. Cell entry is the first and essential step of successful virus infection and can be divided into two ongoing steps: cell binding and membrane fusion. Over the past two decades, stimulated by the global outbreak of SARS-CoV and pandemic of SARS-CoV-2, numerous efforts have been made in the CoV research. As a result, significant progress has been achieved in our understanding of the cell entry process. Here, we review the current knowledge of this essential process, including the viral and host components involved in cell binding and membrane fusion, molecular mechanisms of their interactions, and the sites of virus entry. We highlight the recent findings of host restriction factors that inhibit CoVs entry. This knowledge not only enhances our understanding of the cell entry process, pathogenesis, tissue tropism, host range, and interspecies-transmission of CoVs but also provides a theoretical basis to design effective preventive and therapeutic strategies to control CoVs infection.

Predicting hosts based on early SARS-CoV-2 samples and analyzing the 2020 pandemic.

The SARS-CoV-2 pandemic has raised concerns in the identification of the hosts of the virus since the early stages of the outbreak. To address this problem, we proposed a deep learning method, DeepHoF, based on extracting viral genomic features automatically, to predict the host likelihood scores on five host types, including plant, germ, invertebrate, non-human vertebrate and human, for novel viruses. DeepHoF made up for the lack of an accurate tool, reaching a satisfactory AUC of 0.975 in the five-classification, and could make a reliable prediction for the novel viruses without close neighbors in phylogeny. Additionally, to fill the gap in the efficient inference of host species for SARS-CoV-2 using existing tools, we conducted a deep analysis on the host likelihood profile calculated by DeepHoF. Using the isolates sequenced in the earliest stage of the COVID-19 pandemic, we inferred that minks, bats, dogs and cats were potential hosts of SARS-CoV-2, while minks might be one of the most noteworthy hosts. Several genes of SARS-CoV-2 demonstrated their significance in determining the host range. Furthermore, a large-scale genome analysis, based on DeepHoF's computation for the later pandemic in 2020, disclosed the uniformity of host range among SARS-CoV-2 samples and the strong association of SARS-CoV-2 between humans and minks.

First direct human-to-cat transmission of the SARS-CoV-2 B.1.1.7 variant.

A highly transmissible severe acute respiratory coronavirus 2 (SARS-CoV-2) caused the coronavirus diseases 2019 (COVID-19) pandemic, which resulted the highest morbidity and mortality rates among SARS-CoV and MERS-CoV. SARS-CoV-2 B.1.1.7 variant indicated the higher transmission among human-to-human and increasing hospitalisation. SARS-CoV-2 infection was observed in domestic animals showing human-to-pet transmission. In the current study, we report the first direct known human-to-cat transmission of the SARS-CoV-2 B.1.1.7 variant within the same family. Previous findings showed that companion animals can get infected by COVID-19 patients after 3-6 weeks; however, according to our molecular findings, the cat was infected by the viral variant at the same period. Moreover, B.1.1.7 infection caused and developed several clinical symptoms including cardiac and ocular abnormalities. Overall, our findings determined the first direct and high transmission ability of the B.1.1.7 variant from COVID-19 affected family members to cat. This result showed that the SARS-CoV-2 B.1.1.7 variant could have the highest transition capacity from human to domestic cat as shown for human-to-human. The governmental or worldwide policies should consider more detailed against the war with COVID-19 pandemic.

Current State of Knowledge about Role of Pets in Zoonotic Transmission of SARS-CoV-2.

Pets play a crucial role in the development of human feelings, social life, and care. However, in the era of the prevailing global pandemic of COVID-19 disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), many questions addressing the routes of the virus spread and transmission to humans are dramatically emerging. Although cases of SARS-CoV-2 infection have been found in pets including dogs, cats, and ferrets, to date there is no strong evidence for pet-to-human transmission or sustained pet-to-pet transmission of SARS-CoV-2. However, an increasing number of studies reporting detection of SARS-CoV-2 in farmed minks raises suspicion of potential viral transmission from these animals to humans. Furthermore, due to the high susceptibility of cats, ferrets, minks and hamsters to COVID-19 infection under natural and/or experimental conditions, these animals have been extensively explored as animal models to study the SARS-CoV-2 pathogenesis and transmission. In this review, we present the latest reports focusing on SARS-CoV-2 detection, isolation, and characterization in pets. Moreover, based on the current literature, we document studies aiming to broaden the knowledge about pathogenicity and transmissibility of SARS-CoV-2, and the development of viral therapeutics, drugs and vaccines. Lastly, considering the high rate of SARS-CoV-2 evolution and replication, we also suggest routes of protection against the virus.

SARS-CoV-2 Infections and Viral Isolations among Serially Tested Cats and Dogs in Households with Infected Owners in Texas, USA.

Understanding the ecological and epidemiological roles of pets in the transmission of SARS-CoV-2 is critical for animal and human health, identifying household reservoirs, and predicting the potential enzootic maintenance of the virus. We conducted a longitudinal household transmission study of 76 dogs and cats living with at least one SARS-CoV-2-infected human in Texas and found that 17 pets from 25.6% of 39 households met the national case definition for SARS-CoV-2 infections in animals. This includes three out of seventeen (17.6%) cats and one out of fifty-nine (1.7%) dogs that were positive by RT-PCR and sequencing, with the virus successfully isolated from the respiratory swabs of one cat and one dog. Whole-genome sequences of SARS-CoV-2 obtained from all four PCR-positive animals were unique variants grouping with genomes circulating among people with COVID-19 in Texas. Re-sampling showed persistence of viral RNA for at least 25 d-post initial test. Additionally, seven out of sixteen (43.8%) cats and seven out of fifty-nine (11.9%) dogs harbored SARS-CoV-2 neutralizing antibodies upon initial sampling, with relatively stable or increasing titers over the 2-3 months of follow-up and no evidence of seroreversion. The majority (82.4%) of infected pets were asymptomatic. 'Reverse zoonotic' transmission of SARS-CoV-2 from infected people to animals may occur more frequently than recognized.

Anthropogenic Infection of Cats during the 2020 COVID-19 Pandemic.

COVID-19 is a severe acute respiratory syndrome (SARS) caused by a new coronavirus (CoV), SARS-CoV-2, which is closely related to SARS-CoV that jumped the animal-human species barrier and caused a disease outbreak in 2003. SARS-CoV-2 is a betacoronavirus that was first described in 2019, unrelated to the commonly occurring feline coronavirus (FCoV) that is an alphacoronavirus associated with feline infectious peritonitis (FIP). SARS-CoV-2 is highly contagious and has spread globally within a few months, resulting in the current pandemic. Felids have been shown to be susceptible to SARS-CoV-2 infection. Particularly in the Western world, many people live in very close contact with their pet cats, and natural infections of cats in COVID-19-positive households have been described in several countries. In this review, the European Advisory Board on Cat Diseases (ABCD), a scientifically independent board of experts in feline medicine from 11 European Countries, discusses the current status of SARS-CoV infections in cats. The review examines the host range of SARS-CoV-2 and human-to-animal transmissions, including infections in domestic and non-domestic felids, as well as mink-to-human/-cat transmission. It summarises current data on SARS-CoV-2 prevalence in domestic cats and the results of experimental infections of cats and provides expert opinions on the clinical relevance and prevention of SARS-CoV-2 infection in cats.

COVID-19, Your Pet and Other Animals: Are You at Risk?

Despite fast-tracked research, the precise origin, transmission and evolution of COVID-19 are still unknown. While the bat genus Rhinolophus is likely the primary source of the zoonotic-origin pathogen SARS-CoV-2 that causes COVID-19, its transmission route into the human population is still being studied.[1,2] Coronaviruses (CoV) affect humans and various animal species. Bats were the original hosts of the CoV that causes Severe Acute Respiratory Syndrome (SARS-CoV) and Middle East Respiratory Syndrome coronavirus (MERS-CoV), for example, with masked palm civet cats and dromedaries, respectively, the intermediate hosts of those two viruses. Research is ongoing regarding intermediate species for SARS-CoV-2, but one possibility is the large stray cat and dog population around the live animal market in Wuhan, China, where the pandemic is thought to have started.

A household case evidences shorter shedding of SARS-CoV-2 in naturally infected cats compared to their human owners.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been detected in domestic and wild cats. However, little is known about natural viral infections of domestic cats, although their importance for modelling disease spread, informing strategies for managing positive human-animal relationships and disease prevention. Here, we describe the SARS-CoV-2 infection in a household of two human adults and sibling cats (one male and two females) using real-time RT-PCR, an ELISA test, viral sequencing, and virus isolation. On May 5th, 2020, the cat-owners tested positive for SARS-CoV-2. Two days later, the male cat showed mild respiratory symptoms and tested positive. Four days after the male cat, the two female cats became positive, asymptomatically. Also, one human and one cat showed antibodies against SARS-CoV-2. All cats excreted detectable SARS-CoV-2 RNA for a shorter duration than humans and viral sequences analysis confirmed human-to-cat transmission. We could not determine if cat-to-cat transmission also occurred.

COVID-19, Your Pet and Other Animals: Are You at Risk?

Despite fast-tracked research, the precise origin, transmission and evolution of COVID-19 are still unknown. While the bat genus Rhinolophus is likely the primary source of the zoonotic-origin pathogen SARS-CoV-2 that causes COVID-19, its transmission route into the human population is still being studied.[1,2] Coronaviruses (CoV) affect humans and various animal species. Bats were the original hosts of the CoV that causes Severe Acute Respiratory Syndrome (SARS-CoV) and Middle East Respiratory Syndrome coronavirus (MERS-CoV), for example, with masked palm civet cats and dromedaries, respectively, the intermediate hosts of those two viruses. Research is ongoing regarding intermediate species for SARS-CoV-2, but one possibility is the large stray cat and dog population around the live animal market in Wuhan, China, where the pandemic is thought to have started.

Human animal interface of SARS-CoV-2 (COVID-19) transmission: a critical appraisal of scientific evidence.

Coronaviruses are a large family of viruses that are known to infect both humans and animals. However, the evidence of inter-transmission of coronavirus between humans and companion animals is still a debatable issue. There is substantial evidence that the virus outbreak is fueled by zoonotic transmission because this new virus belongs to the same family of viruses as SARS-CoV associated with civet cats, and MERS-CoV associated with dromedary camels. While the whole world is investigating the possibility about the transmission of this virus, the transmission among humans is established, but the interface between humans and animals is not much evident. Not only are the lives of human beings at risk, but there is an equal potential threat to the animal world. With multiple reports claiming about much possibility of transmission of COVID-19 from humans to animals, there has been a significant increase in the number of pets being abandoned by their owners. Additionally, the risk of reverse transmission of COVID-19 virus from companion pets like cats and dogs at home is yet another area of concern. The present article highlights different evidence of human-animal interface and necessitates the precautionary measures required to combat with the consequences of this interface. The Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) have suggested various ways to promote awareness and corroborate practices for helping people as well as animals to stay secure and healthy.

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.

Infectivity, virulence, pathogenicity, host-pathogen interactions of SARS and SARS-CoV-2 in experimental animals: a systematic review.

The outbreak of the SARS-CoV-2 in mainland China with subsequent human to human transmission worldwide had taken up the shape of a devastating pandemic. The ability of the virus to infect multiple species other than humans has currently been reported in experimental conditions. Non-human primates, felines, ferrets, rodents and host of other animals could previously be infected in experimental conditions with SARS-CoV and recently with SARS-CoV-2, both virus using Angiotensin-converting-enzyme 2 receptor for cellular entry. The variations in sequence homology of ACE2 receptor across species is identified as one of the factors determining virulence and pathogenicity in animals. The infection in experimental animals with SARS-CoV or SARS-CoV-2 on most occasions are asymptomatic, however, the virus could multiply within the respiratory tract and extra-pulmonary organs in most of the species. Here, we discuss about the pathogenicity, transmission, variations in angiotensin-converting-enzyme 2 receptor-binding across species and host pathogen interactions of SARS and SARS-CoV-2 in laboratory animals used in research.