Detection of SARS-CoV-2 clade B.1.2 in three snow leopards.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is one of seven coronaviruses known to infect humans. Different from other concerned coronavirus and influenza viruses, SARS-CoV-2 has a higher basic reproduction number and thus transmits more efficiently among hosts. Testing animals for SARS-CoV-2 may help decipher virus reservoirs, transmission and pathogenesis. Here, we report the first detection of SARS-CoV-2 in three snow leopards (Panthera uncia) in a zoo in Kentucky in 2020, the first year of the pandemic. Sequence analysis revealed that snow leopard SARS-CoV-2 strains were non-variant B.1.2 lineage and closely correlated with human strains. One snow leopard shed SARS-CoV-2 in faeces up to 4 weeks. Based on clinical signs and viral shedding periods and levels in the three snow leopards, animal-to-animal transmission events could not be excluded. Further testing of SARS-CoV-2 in animals is needed.

An Evaluation of Avian Influenza Virus Whole-Genome Sequencing Approaches Using Nanopore Technology.

As exemplified by the global response to the SARS-CoV-2 pandemic, whole-genome sequencing played an important role in monitoring the evolution of novel viral variants and provided guidance on potential antiviral treatments. The recent rapid and extensive introduction and spread of highly pathogenic avian influenza virus in Europe, North America, and elsewhere raises the need for similarly rapid sequencing to aid in appropriate response and mitigation activities. To facilitate this objective, we investigate a next-generation sequencing platform that uses a portable nanopore sequencing device to generate and present data in real time. This platform offers the potential to extend in-house sequencing capacities to laboratories that may otherwise lack resources to adopt sequencing technologies requiring large benchtop instruments. We evaluate this platform for routine use in a diagnostic laboratory. In this study, we evaluate different primer sets for the whole genome amplification of influenza A virus and evaluate five different library preparation approaches for sequencing on the nanopore platform using the MinION flow cell. A limited amplification procedure and a rapid procedure are found to be best among the approaches taken.

Multi-species outbreak of SARS-CoV-2 Delta variant in a zoological institution, with the detection in two new families of carnivores.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a worldwide distribution in humans and many other mammalian species. In late September 2021, 12 animals maintained by the Chicago Zoological Society's Brookfield Zoo were observed with variable clinical signs. The Delta variant of SARS-CoV-2 was detected in faeces and nasal swabs by qRT-PCR, including the first detection in animals from the families Procyonidae and Viverridae. Test positivity rate was 12.5% for 35 animals tested. All animals had been vaccinated with at least one dose of a recombinant vaccine designed for animals and all recovered with variable supportive treatment. Sequence analysis showed that six zoo animal strains were closely correlated with 18 human SARS-CoV-2 strains, suggestive of potential human-to-animal transmission events. This report documents the expanding host range of COVID-19 during the ongoing pandemic.

Investigation of SARS-CoV-2 infection and associated lesions in exotic and companion animals.

Documented natural infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in exotic and companion animals following human exposures are uncommon. Those documented in animals are typically mild and self-limiting, and infected animals have only infrequently died or been euthanized. Through a coordinated One Health initiative, necropsies were conducted on 5 animals from different premises that were exposed to humans with laboratory-confirmed SARS-CoV-2 infection. The combination of epidemiologic evidence of exposure and confirmatory real-time reverse transcriptase-polymerase chain reaction testing confirmed infection in 3 cats and a tiger. A dog was a suspect case based on epidemiologic evidence of exposure but tested negative for SARS-CoV-2. Four animals had respiratory clinical signs that developed 2 to 12 days after exposure. The dog had bronchointerstitial pneumonia and the tiger had bronchopneumonia; both had syncytial-like cells with no detection of SARS-CoV-2. Individual findings in the 3 cats included metastatic mammary carcinoma, congenital renal disease, and myocardial disease. Based on the necropsy findings and a standardized algorithm, SARS-CoV-2 infection was not considered the cause of death in any of the cases. Continued surveillance and necropsy examination of animals with fatal outcomes will further our understanding of natural SARS-CoV-2 infection in animals and the potential role of the virus in development of lesions.

SARS-CoV-2 infection in free-ranging white-tailed deer.

Humans have infected a wide range of animals with SARS-CoV-21-5, but the establishment of a new natural animal reservoir has not been observed. Here we document that free-ranging white-tailed deer (Odocoileus virginianus) are highly susceptible to infection with SARS-CoV-2, are exposed to multiple SARS-CoV-2 variants from humans and are capable of sustaining transmission in nature. Using real-time PCR with reverse transcription, we detected SARS-CoV-2 in more than one-third (129 out of 360, 35.8%) of nasal swabs obtained from O. virginianus in northeast Ohio in the USA during January to March 2021. Deer in six locations were infected with three SARS-CoV-2 lineages (B.1.2, B.1.582 and B.1.596). The B.1.2 viruses, dominant in humans in Ohio at the time, infected deer in four locations. We detected probable deer-to-deer transmission of B.1.2, B.1.582 and B.1.596 viruses, enabling the virus to acquire amino acid substitutions in the spike protein (including the receptor-binding domain) and ORF1 that are observed infrequently in humans. No spillback to humans was observed, but these findings demonstrate that SARS-CoV-2 viruses have been transmitted in wildlife in the USA, potentially opening new pathways for evolution. There is an urgent need to establish comprehensive 'One Health' programmes to monitor the environment, deer and other wildlife hosts globally.

Transmission of SARS-CoV-2 from humans to a 16-year-old domestic cat with comorbidities in Pennsylvania, USA.

BACKGROUND AND OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), besides causing human infection, has been shown to naturally infect several susceptible animal species including large cats (tigers, lions, pumas, spotted leopards), dogs, cats, ferrets, gorillas and minks. Cats and minks are continuing to be the most reported species with SARS-CoV-2 infections among animals but it needs to be investigated further. METHODS AND RESULTS: We report the detection of SARS-CoV-2 from a domestic cat that exhibited respiratory disease after being exposed to SARS-CoV-2 virus from humans in the same household. SARS-CoV-2 RNA was detected in two oropharyngeal swabs collected at two time points, 11 days apart; the first, when the cat was reported to be sick and the second, before euthanasia due to poor prognosis. The viral nucleic acid detected at two time points showed no genomic variation and resembled the clade GH circulating in humans in the United States. Clinical and pathological findings noted in this 16-year-old cat were consistent with respiratory and cardiac insufficiency. CONCLUSIONS: SARS-CoV-2 viral infection was likely an incidental clinical finding, as the virus was not detected in fixed lungs, heart, or kidney tissues. Only fresh lung tissue collected at necropsy showed the presence of viral nucleic acid, albeit at a very low level. Further research is needed to clarify the clinical course of SARS-CoV-2 in companion animals of advanced age and underlying cardiac disease.

SARS-CoV-2 exposure in wild white-tailed deer (Odocoileus virginianus).

Widespread human SARS-CoV-2 infections combined with human-wildlife interactions create the potential for reverse zoonosis from humans to wildlife. We targeted white-tailed deer (Odocoileus virginianus) for serosurveillance based on evidence these deer have angiotensin-converting enzyme 2 receptors with high affinity for SARS-CoV-2, are permissive to infection, exhibit sustained viral shedding, can transmit to conspecifics, exhibit social behavior, and can be abundant near urban centers. We evaluated 624 prepandemic and postpandemic serum samples from wild deer from four US states for SARS-CoV-2 exposure. Antibodies were detected in 152 samples (40%) from 2021 using a surrogate virus neutralization test. A subset of samples tested with a SARS-CoV-2 virus neutralization test showed high concordance between tests. These data suggest white-tailed deer in the populations assessed have been exposed to SARS-CoV-2.

Determining the role of natural SARS-CoV-2 infection in the death of domestic pets: 10 cases (2020-2021).

OBJECTIVE: To establish a pathoepidemiological model to evaluate the role of SARS-CoV-2 infection in the first 10 companion animals that died while infected with SARS-CoV-2 in the US. ANIMALS: 10 cats and dogs that tested positive for SARS-CoV-2 and died or were euthanized in the US between March 2020 and January 2021. PROCEDURES: A standardized algorithm was developed to direct case investigations, determine the necessity of certain diagnostic procedures, and evaluate the role, if any, that SARS-CoV-2 infection played in the animals' course of disease and death. Using clinical and diagnostic information collected by state animal health officials, state public health veterinarians, and other state and local partners, this algorithm was applied to each animal case. RESULTS: SARS-CoV-2 was an incidental finding in 8 animals, was suspected to have contributed to the severity of clinical signs leading to euthanasia in 1 dog, and was the primary reason for death for 1 cat. CONCLUSIONS AND CLINICAL RELEVANCE: This report provides the global community with a standardized process for directing case investigations, determining the necessity of certain diagnostic procedures, and determining the clinical significance of SARS-CoV-2 infections in animals with fatal outcomes and provides evidence that SARS-CoV-2 can, in rare circumstances, cause or contribute to death in pets.

An Opportunistic Survey Reveals an Unexpected Coronavirus Diversity Hotspot in North America.

In summer 2020, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was detected on mink farms in Utah. An interagency One Health response was initiated to assess the extent of the outbreak and included sampling animals from on or near affected mink farms and testing them for SARS-CoV-2 and non-SARS coronaviruses. Among the 365 animals sampled, including domestic cats, mink, rodents, raccoons, and skunks, 261 (72%) of the animals harbored at least one coronavirus. Among the samples that could be further characterized, 127 alphacoronaviruses and 88 betacoronaviruses (including 74 detections of SARS-CoV-2 in mink) were identified. Moreover, at least 10% (n = 27) of the coronavirus-positive animals were found to be co-infected with more than one coronavirus. Our findings indicate an unexpectedly high prevalence of coronavirus among the domestic and wild free-roaming animals tested on mink farms. These results raise the possibility that mink farms could be potential hot spots for future trans-species viral spillover and the emergence of new pandemic coronaviruses.

One Health Investigation of SARS-CoV-2 Infection and Seropositivity among Pets in Households with Confirmed Human COVID-19 Cases-Utah and Wisconsin, 2020.

Approximately 67% of U.S. households have pets. Limited data are available on SARS-CoV-2 in pets. We assessed SARS-CoV-2 infection in pets during a COVID-19 household transmission investigation. Pets from households with ≥1 person with laboratory-confirmed COVID-19 were eligible for inclusion from April-May 2020. We enrolled 37 dogs and 19 cats from 34 households. All oropharyngeal, nasal, and rectal swabs tested negative by rRT-PCR; one dog's fur swabs (2%) tested positive by rRT-PCR at the first sampling. Among 47 pets with serological results, eight (17%) pets (four dogs, four cats) from 6/30 (20%) households had detectable SARS-CoV-2 neutralizing antibodies. In households with a seropositive pet, the proportion of people with laboratory-confirmed COVID-19 was greater (median 79%; range: 40-100%) compared to households with no seropositive pet (median 37%; range: 13-100%) (p = 0.01). Thirty-three pets with serologic results had frequent daily contact (≥1 h) with the index patient before the person's COVID-19 diagnosis. Of these 33 pets, 14 (42%) had decreased contact with the index patient after diagnosis and none were seropositive; of the 19 (58%) pets with continued contact, four (21%) were seropositive. Seropositive pets likely acquired infection after contact with people with COVID-19. People with COVID-19 should restrict contact with pets and other animals.

First Reported Cases of SARS-CoV-2 Infection in Companion Animals - New York, March-April 2020.

On April 22, CDC and the U.S. Department of Agriculture (USDA) reported cases of two domestic cats with confirmed infection with SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19). These are the first reported companion animals (including pets and service animals) with SARS-CoV-2 infection in the United States, and among the first findings of SARS-CoV-2 symptomatic companion animals reported worldwide. These feline cases originated from separate households and were epidemiologically linked to suspected or confirmed human COVID-19 cases in their respective households. Notification of presumptive positive animal test results triggered a One Health* investigation by state and federal partners, who determined that no further transmission events to other animals or persons had occurred. Both cats fully recovered. Although there is currently no evidence that animals play a substantial role in spreading COVID-19, CDC advises persons with suspected or confirmed COVID-19 to restrict contact with animals during their illness and to monitor any animals with confirmed SARS-CoV-2 infection and separate them from other persons and animals at home (1).

Antibodies to SARS-CoV-2 in dogs and cats, USA.

To provide more complete data on SARS-CoV-2 infections in dogs and cats in the U.S., we conducted a serosurvey on convenience serum samples from dogs (n=1336) and cats (n=956) collected from 48 states of the USA in 2020. An ELISA targeting the antibody against nucleocapsid identified eleven positive and two doubtful samples in cats, and five positive and five doubtful samples in dogs. A surrogate neutralization assay detecting antibodies blocking the attachment of the spike protein to ACE2 was positive with three of the ELISA positive and doubtful samples, and one of 463 randomly selected ELISA negative samples. These four positive samples were confirmed by SARS-CoV-2 virus neutralization testing. All were from cats, in New York, Florida, and New Jersey (n=2). The serosurvey results, one of the largest yet completed on dogs and cats globally, support the OIE and CDC positions that currently there is no evidence that pets play a role in the spread of SARS CoV-2 in humans.

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.

SARS-CoV-2 B.1.1.7 variant of concern detected in a pet dog and cat after exposure to a person with COVID-19, USA.

As part of a longitudinal household transmission study of pets living with persons with COVID-19 in Texas, two pets were confirmed to be infected with the SARS-CoV-2 B.1.1.7 variant of concern (VOC). The pets were a dog and a cat from the same household, sampled two days after their owner tested positive for COVID-19. The oral, nasal and fur swabs for both pets tested positive for SARS-CoV-2 by qRT-PCR and consensus whole-genome sequences from the dog and cat were 100% identical and matched the B.1.1.7 VOC. Virus was isolated from the cat's nasal swab. One month after initial detection of infection, the pets were re-tested twice at which time only the fur swabs (both pets) and oral swab (dog only) remained positive, and neutralizing antibodies for SARS-CoV-2 were present in both animals. Sneezing by both pets was noted by the owner in the weeks between initial and follow-up testing. This study documents the first detection of B.1.1.7. in companion animals in the United States, and the first genome recovery and isolation of B.1.1.7 variant of concern globally in any animal.

SARS-CoV-2 Exposure in Escaped Mink, Utah, USA.

In August 2020, outbreaks of coronavirus disease were confirmed on mink farms in Utah, USA. We surveyed mammals captured on and around farms for evidence of infection or exposure. Free-ranging mink, presumed domestic escapees, exhibited high antibody titers, suggesting a potential severe acute respiratory syndrome coronavirus 2 transmission pathway to native wildlife.

From People to Panthera: Natural SARS-CoV-2 Infection in Tigers and Lions at the Bronx Zoo.

Despite numerous barriers to transmission, zoonoses are the major cause of emerging infectious diseases in humans. Among these, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and ebolaviruses have killed thousands; the human immunodeficiency virus (HIV) has killed millions. Zoonoses and human-to-animal cross-species transmission are driven by human actions and have important management, conservation, and public health implications. The current SARS-CoV-2 pandemic, which presumably originated from an animal reservoir, has killed more than half a million people around the world and cases continue to rise. In March 2020, New York City was a global epicenter for SARS-CoV-2 infections. During this time, four tigers and three lions at the Bronx Zoo, NY, developed mild, abnormal respiratory signs. We detected SARS-CoV-2 RNA in respiratory secretions and/or feces from all seven animals, live virus in three, and colocalized viral RNA with cellular damage in one. We produced nine whole SARS-CoV-2 genomes from the animals and keepers and identified different SARS-CoV-2 genotypes in the tigers and lions. Epidemiologic and genomic data indicated human-to-tiger transmission. These were the first confirmed cases of natural SARS-CoV-2 animal infections in the United States and the first in nondomestic species in the world. We highlight disease transmission at a nontraditional interface and provide information that contributes to understanding SARS-CoV-2 transmission across species.IMPORTANCE The human-animal-environment interface of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important aspect of the coronavirus disease 2019 (COVID-19) pandemic that requires robust One Health-based investigations. Despite this, few reports describe natural infections in animals or directly link them to human infections using genomic data. In the present study, we describe the first cases of natural SARS-CoV-2 infection in tigers and lions in the United States and provide epidemiological and genetic evidence for human-to-animal transmission of the virus. Our data show that tigers and lions were infected with different genotypes of SARS-CoV-2, indicating two independent transmission events to the animals. Importantly, infected animals shed infectious virus in respiratory secretions and feces. A better understanding of the susceptibility of animal species to SARS-CoV-2 may help to elucidate transmission mechanisms and identify potential reservoirs and sources of infection that are important in both animal and human health.

Complete Genome Sequence of SARS-CoV-2 in a Tiger from a U.S. Zoological Collection.

This report describes the identification and characterization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a Malayan tiger in a U.S. zoo.