One health surveillance strategy for coronaviruses in Italian wildlife.

The recent reinforcement of CoV surveillance in animals fuelled by the COVID-19 pandemic provided increasing evidence that mammals other than bats might hide further diversity and play critical roles in human infectious diseases. This work describes the results of a two-year survey carried out in Italy with the double objective of uncovering CoV diversity associated with wildlife and of excluding the establishment of a reservoir for SARS-CoV-2 in particularly susceptible or exposed species. The survey targeted hosts from five different orders and was harmonised across the country in terms of sample size, target tissues, and molecular test. Results showed the circulation of 8 CoV species in 13 hosts out of the 42 screened. Coronaviruses were either typical of the host species/genus or normally associated with their domestic counterpart. Two novel viruses likely belonging to a novel CoV genus were found in mustelids. All samples were negative for SARS-CoV-2, with minimum detectable prevalence ranging between 0.49% and 4.78% in the 13 species reaching our threshold sample size of 59 individuals. Considering that within-species transmission in white-tailed deer resulted in raising the prevalence from 5% to 81% within a few months, this result would exclude a sustained cycle after spillback in the tested species.

Viral infections at the animal-human interface-Learning lessons from the SARS-CoV-2 pandemic.

This Lilliput explores the current epidemiological and virological arguments for a zoonotic origin of the COVID-19 pandemic. While the role of bats, pangolins and racoon dogs as viral reservoirs has not yet been proven, a spill-over of a coronavirus infection from animals into humans at the Huanan food market in Wuhan has a much greater plausibility than alternative hypotheses such as a laboratory virus escape, deliberate genetic engineering or introduction by cold chain food products. This Lilliput highlights the dynamic nature of the animal-human interface for viral cross-infections from humans into feral white tail deer or farmed minks (reverse zoonosis). Surveillance of viral infections at the animal-human interface is an urgent task since live animal markets are not the only risks for future viral spill-overs. Climate change will induce animal migration which leads to viral exchanges between animal species that have not met in the past. Environmental change and deforestation will also increase contact between animals and humans. Developing an early warning system for emerging viral infections becomes thus a societal necessity not only for human but also for animal and environmental health (One Health concept). Microbiologists have developed tools ranging from virome analysis in key suspects such as viral reservoirs (bats, wild game animals, bushmeat) and in humans exposed to wild animals, to wastewater analysis to detect known and unknown viruses circulating in the human population and sentinel studies in animal-exposed patients with fever. Criteria need to be developed to assess the virulence and transmissibility of zoonotic viruses. An early virus warning system is costly and will need political lobbying. The accelerating number of viral infections with pandemic potential over the last decades should provide the public pressure to extend pandemic preparedness for the inclusion of early viral alert systems.

Impacts of the novel coronavirus SARS-CoV-2 on wildlife behaviour via human activities.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the pandemic of the coronavirus disease 2019 (COVID-19), resulting in a global lockdown in 2020. This stagnation in human activities ('anthropause') has been reported to affect the behaviour of wildlife in various ways. The sika deer Cervus nippon in Nara Park, central Japan, has had a unique relationship with humans, especially tourists, in which the deer bow to receive food and sometimes attack if they do not receive it. We investigated how a decrease and subsequent increase in the number of tourists visiting Nara Park affects the number of deer observed in the park and their behaviour (bows and attacks against humans). Compared with the pre-pandemic years, the number of deer in the study site decreased from an average of 167 deer in 2019 to 65 (39%) in 2020 during the pandemic period. Likewise, the number of deer bows decreased from 10.2 per deer in 2016-2017 to 6.4 (62%) in 2020-2021, whereas the proportion of deer showing aggressive behaviour did not change significantly. Moreover, the monthly numbers of deer and their bows both corresponded with the fluctuation in the number of tourists during the pandemic period of 2020 and 2021, whereas the number of attacks did not. Thus, the anthropause caused by the coronavirus altered the habitat use and behaviour of deer that have continuous interactions with humans.

Serosurveillance for Severe Acute Respiratory Syndrome Coronavirus 2 Antibody in Feral Swine and White-Tailed Deer in Texas.

Background: Serological evidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been reported in white-tailed deer (WTD) in the United States and Canada. Even though WTD are susceptible to SARS-CoV-2 infection, there is no evidence of infection by this virus in other mammalian species that might interact with WTD in nature. Similar to WTD, feral swine are widely distributed and generally occupy the same range as WTD in Texas. The objective of this study was to determine the prevalence of SARS-CoV-2 neutralizing antibody in WTD during 2020 and 2021 and determine the prevalence of SARS-CoV-2 neutralizing antibody in feral swine during 2018 (prepandemic period) and from March 2020 to February 2021 (pandemic period) in Travis County, Texas. Materials and Methods: Sera samples were collected from hunter-killed WTD and feral swine during the prepandemic and pandemic period and tested for SARS-CoV-2 antibody by a plaque reduction neutralization assay in Vero cells. Results: SARS-CoV-2 antibody was not detected in any of the 166 feral swine sera samples, including 24 samples collected during the prepandemic and 142 samples collected during the pandemic period. Furthermore, SARS-CoV-2 antibody was not detected in the 115 WTD samples collected during late 2020, but antibody was detected in WTD in early 2021. Conclusions: The results indicated that SARS-CoV-2 infection of WTD occurred during early 2021 in Travis County, Texas, but serological evidence of SARS-CoV-2 infection was not detected in the feral swine samples collected from the same locality and during the same time period of the collection of WTD samples.

Cervids ACE2 Residues that Bind the Spike Protein can Provide Susceptibility to SARS-CoV-2.

The susceptibility of the white-tailed deer (WTD; Odocoileus virginianus) to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted cervids as coronavirus reservoirs. This study aimed to evaluate the angiotensin-converting enzyme 2 (ACE2) residues which bind the spike protein of SARS-CoV-2 from 16 cervids to predict their potential susceptibility to SARS-CoV-2 infection. Eleven out of 16 species presented identical ACE2 key residues to WTD ACE2. Four cervids presented K31N, a variant associated with low SARS-CoV-2 susceptibility. Large herding of cervids with ACE2 key residues identical to that of the WTD can result in extensive reservoirs of SARS-CoV-2. Cervids as potential reservoirs could favor SARS-CoV-2 adaptation and the emergence of new coronavirus strains.

Stability of SARS-CoV-2 in Biological Fluids of Animals.

Since its first emergence in 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued to evolve genetically, jump species barriers, and expand its host range. There is growing evidence of interspecies transmission including infection of domestic animals and widespread circulation in wildlife. However, knowledge of SARS-CoV-2 stability in animal biological fluids and their role in transmission is still limited as previous studies focused on human biological fluids. Therefore, this study aimed to determine the SARS-CoV-2 stability in biological fluids from three animal species, cats, sheep and white-tailed deer (WTD). Saliva, feces, 10% fecal suspensions, and urine of cats, sheep, and WTD were mixed with a known concentration of virus and incubated under indoor and three different climatic conditions. Our results show that the virus was stable for up to 1 day in the saliva of cats, sheep, and WTD regardless of the environmental conditions. The virus remained infectious for up to 6 days in feces and 15 days in fecal suspension of WTD, whereas the virus was rather unstable in cat and sheep feces and fecal suspensions. We found the longest survival of SARS-CoV-2 in the urine of cats, sheep, and WTD. Furthermore, side-by-side comparison with different SARS-CoV-2 strains showed that the Alpha, Delta, and Omicron variants of concern were less stable than the ancestral Wuhan-like strain in WTD fecal suspension. The results of our study provide valuable information for assessing the potential role of various animal biological fluids in SARS-CoV-2 transmission.

The emergence of a virus variant: dynamics of a competition model with cross-immunity time-delay validated by wastewater surveillance data for COVID-19.

We consider the dynamics of a virus spreading through a population that produces a mutant strain with the ability to infect individuals that were infected with the established strain. Temporary cross-immunity is included using a time delay, but is found to be a harmless delay. We provide some sufficient conditions that guarantee local and global asymptotic stability of the disease-free equilibrium and the two boundary equilibria when the two strains outcompete one another. It is shown that, due to the immune evasion of the emerging strain, the reproduction number of the emerging strain must be significantly lower than that of the established strain for the local stability of the established-strain-only boundary equilibrium. To analyze the unique coexistence equilibrium we apply a quasi steady-state argument to reduce the full model to a two-dimensional one that exhibits a global asymptotically stable established-strain-only equilibrium or global asymptotically stable coexistence equilibrium. Our results indicate that the basic reproduction numbers of both strains govern the overall dynamics, but in nontrivial ways due to the inclusion of cross-immunity. The model is applied to study the emergence of the SARS-CoV-2 Delta variant in the presence of the Alpha variant using wastewater surveillance data from the Deer Island Treatment Plant in Massachusetts, USA.

Cross-species transmission of coronaviruses with a focus on severe acute respiratory syndrome coronavirus 2 infection in animals: a review for the veterinary practitioner.

In 2019 a novel coronavirus termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged from an unidentified source and spread rapidly among humans worldwide. While many human infections are mild, some result in severe clinical disease that in a small proportion of infected people is fatal. The pandemic spread of SARS-CoV-2 has been facilitated by efficient human-to-human transmission of the virus, with no data to indicate that animals contributed to this global health crisis. However, a range of domesticated and wild animals are also susceptible to SARS-CoV-2 infection under both experimental and natural conditions. Humans are presumed to be the source of most animal infections thus far, although natural transmission between mink and between free-ranging deer has occurred, and occasional natural transmission between cats cannot be fully excluded. Considering the ongoing circulation of the virus among people, together with its capacity to evolve through mutation and recombination, the risk of the emergence of animal-adapted variants is not negligible. If such variants remain infectious to humans, this could lead to the establishment of an animal reservoir for the virus, which would complicate control efforts. As such, minimising human-to-animal transmission of SARS-CoV-2 should be considered as part of infection control efforts. The aim of this review is to summarise what is currently known about the species specificity of animal coronaviruses, with an emphasis on SARS-CoV-2, in the broader context of factors that facilitate cross-species transmission of viruses.

Zooanthroponotic transmission of SARS-CoV-2 and host-specific viral mutations revealed by genome-wide phylogenetic analysis.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a generalist virus, infecting and evolving in numerous mammals, including captive and companion animals, free-ranging wildlife, and humans. Transmission among non-human species poses a risk for the establishment of SARS-CoV-2 reservoirs, makes eradication difficult, and provides the virus with opportunities for new evolutionary trajectories, including the selection of adaptive mutations and the emergence of new variant lineages. Here, we use publicly available viral genome sequences and phylogenetic analysis to systematically investigate the transmission of SARS-CoV-2 between human and non-human species and to identify mutations associated with each species. We found the highest frequency of animal-to-human transmission from mink, compared with lower transmission from other sampled species (cat, dog, and deer). Although inferred transmission events could be limited by sampling biases, our results provide a useful baseline for further studies. Using genome-wide association studies, no single nucleotide variants (SNVs) were significantly associated with cats and dogs, potentially due to small sample sizes. However, we identified three SNVs statistically associated with mink and 26 with deer. Of these SNVs, ~⅔ were plausibly introduced into these animal species from local human populations, while the remaining ~⅓ were more likely derived in animal populations and are thus top candidates for experimental studies of species-specific adaptation. Together, our results highlight the importance of studying animal-associated SARS-CoV-2 mutations to assess their potential impact on human and animal health.

Molecular phylogeny of coronaviruses and host receptors among domestic and close-contact animals reveals subgenome-level conservation, crossover, and divergence.

BACKGROUND: Coronaviruses have the potential to cross species barriers. To learn the molecular intersections among the most common coronaviruses of domestic and close-contact animals, we analyzed representative coronavirus genera infecting mouse, rat, rabbit, dog, cat, cattle, white-tailed deer, swine, ferret, mink, alpaca, Rhinolophus bat, dolphin, whale, chicken, duck and turkey hosts; reference or complete genome sequences were available for most of these coronavirus genera. Protein sequence alignments and phylogenetic trees were built for the spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins. The host receptors and enzymes aminopeptidase N (APN), angiotensin converting enzyme 2 (ACE2), sialic acid synthase (SAS), transmembrane serine protease 2 (TMPRSS2), dipeptidyl peptidase 4 (DPP4), cathepsin L (and its analogs) and furin were also compared. RESULTS: Overall, the S, E, M, and N proteins segregated according to their viral genera (α, ß, or γ), but the S proteins of alphacoronaviruses lacked conservation of phylogeny. Interestingly, the unique polybasic furin cleavage motif found in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) but not in severe acute respiratory syndrome coronavirus (SARS-CoV) or Middle East respiratory syndrome coronavirus (MERS-CoV) exists in several ß-coronaviruses and a few α- or γ-coronaviruses. Receptors and enzymes retained host species-dependent relationships with one another. Among the hosts, critical ACE2 residues essential for SARS-CoV-2 spike protein binding were most conserved in white-tailed deer and cattle. CONCLUSION: The polybasic furin cleavage motif found in several ß- and other coronaviruses of animals points to the existence of an intermediate host for SARS-CoV-2, and it also offers a counternarrative to the theory of a laboratory-engineered virus. Generally, the S proteins of coronaviruses show crossovers of phylogenies indicative of recombination events. Additionally, the consistency in the segregation of viral proteins of the MERS-like coronavirus (NC_034440.1) from pipistrelle bat supports its classification as a ß-coronavirus. Finally, similarities in host enzymes and receptors did not always explain natural cross-infections. More studies are therefore needed to identify factors that determine the cross-species infectivity of coronaviruses.

White-tailed deer (Odocoileus virginianus) may serve as a wildlife reservoir for nearly extinct SARS-CoV-2 variants of concern.

The spillover of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from humans to white-tailed deer (WTD) and its ability to transmit from deer to deer raised concerns about the role of WTD in the epidemiology and ecology of the virus. Here, we present a comprehensive cross-sectional study assessing the prevalence, genetic diversity, and evolution of SARS-CoV-2 in WTD in the State of New York (NY). A total of 5,462 retropharyngeal lymph node samples collected from free-ranging hunter-harvested WTD during the hunting seasons of 2020 (Season 1, September to December 2020, n = 2,700) and 2021 (Season 2, September to December 2021, n = 2,762) were tested by SARS-CoV-2 real-time RT-PCR (rRT-PCR). SARS-CoV-2 RNA was detected in 17 samples (0.6%) from Season 1 and in 583 samples (21.1%) from Season 2. Hotspots of infection were identified in multiple confined geographic areas of NY. Sequence analysis of SARS-CoV-2 genomes from 164 samples demonstrated the presence of multiple SARS-CoV-2 lineages and the cocirculation of three major variants of concern (VOCs) (Alpha, Gamma, and Delta) in WTD. Our analysis suggests the occurrence of multiple spillover events (human to deer) of the Alpha and Delta lineages with subsequent deer-to-deer transmission and adaptation of the viruses. Detection of Alpha and Gamma variants in WTD long after their broad circulation in humans in NY suggests that WTD may serve as a wildlife reservoir for VOCs no longer circulating in humans. Thus, implementation of continuous surveillance programs to monitor SARS-CoV-2 dynamics in WTD is warranted, and measures to minimize virus transmission between humans and animals are urgently needed.

SARS-CoV-2 Omicron (B.1.1.529) Infection of Wild White-Tailed Deer in New York City.

There is mounting evidence of SARS-CoV-2 spillover from humans into many domestic, companion, and wild animal species. Research indicates that humans have infected white-tailed deer, and that deer-to-deer transmission has occurred, indicating that deer could be a wildlife reservoir and a source of novel SARS-CoV-2 variants. We examined the hypothesis that the Omicron variant is actively and asymptomatically infecting the free-ranging deer of New York City. Between December 2021 and February 2022, 155 deer on Staten Island, New York, were anesthetized and examined for gross abnormalities and illnesses. Paired nasopharyngeal swabs and blood samples were collected and analyzed for the presence of SARS-CoV-2 RNA and antibodies. Of 135 serum samples, 19 (14.1%) indicated SARS-CoV-2 exposure, and 11 reacted most strongly to the wild-type B.1 lineage. Of the 71 swabs, 8 were positive for SARS-CoV-2 RNA (4 Omicron and 4 Delta). Two of the animals had active infections and robust neutralizing antibodies, revealing evidence of reinfection or early seroconversion in deer. Variants of concern continue to circulate among and may reinfect US deer populations, and establish enzootic transmission cycles in the wild: this warrants a coordinated One Health response, to proactively surveil, identify, and curtail variants of concern before they can spill back into humans.

Divergent SARS-CoV-2 variant emerges in white-tailed deer with deer-to-human transmission.

Wildlife reservoirs of broad-host-range viruses have the potential to enable evolution of viral variants that can emerge to infect humans. In North America, there is phylogenomic evidence of continual transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from humans to white-tailed deer (Odocoileus virginianus) through unknown means, but no evidence of transmission from deer to humans. We carried out an observational surveillance study in Ontario, Canada during November and December 2021 (n = 300 deer) and identified a highly divergent lineage of SARS-CoV-2 in white-tailed deer (B.1.641). This lineage is one of the most divergent SARS-CoV-2 lineages identified so far, with 76 mutations (including 37 previously associated with non-human mammalian hosts). From a set of five complete and two partial deer-derived viral genomes we applied phylogenomic, recombination, selection and mutation spectrum analyses, which provided evidence for evolution and transmission in deer and a shared ancestry with mink-derived virus. Our analysis also revealed an epidemiologically linked human infection. Taken together, our findings provide evidence for sustained evolution of SARS-CoV-2 in white-tailed deer and of deer-to-human transmission.

Wild Red Deer (Cervus elaphus) Do Not Play a Role as Vectors or Reservoirs of SARS-CoV-2 in North-Eastern Poland.

Several studies reported a high prevalence of SARS-CoV-2 among white-tailed deer in North America. Monitoring cervids in all regions to better understand SARS-CoV-2 infection and circulation in other deer populations has been urged. To evaluate deer exposure and/or infection to/by SARS-CoV-2 in Poland, we sampled 90 red deer shot by hunters in five hunting districts in north-eastern Poland. Serum and nasopharyngeal swabs were collected, and then an immunofluorescent assay (IFA) to detect anti-SARS-CoV-2 antibodies was performed as well as real-time PCR with reverse transcription for direct virus detection. No positive samples were detected. There is no evidence of spillover of SARS-CoV-2 from the human to deer population in Poland.

Multiple Introductions of SARS-CoV-2 Alpha and Delta Variants into White-Tailed Deer in Pennsylvania.

The SARS-CoV-2 pandemic began by viral spillover from animals to humans; today multiple animal species are known to be susceptible to infection. White-tailed deer, Odocoileus virginianus, are infected in North America at substantial levels, and genomic data suggests that a variant in deer may have spilled back to humans. Here, we characterize SARS-CoV-2 in deer from Pennsylvania (PA) sampled during fall and winter 2021. Of 123 nasal swab samples analyzed by RT-qPCR, 20 (16.3%) were positive for SARS-CoV-2. Seven whole genome sequences were obtained, together with six more partial spike gene sequences. These annotated as alpha and delta variants, the first reported observations of these lineages in deer, documenting multiple new jumps from humans to deer. The alpha lineage persisted in deer after its displacement by delta in humans, and deer-derived alpha variants diverged significantly from those in humans, consistent with a distinctive evolutionary trajectory in deer. IMPORTANCE Coronaviruses have been documented to replicate in numerous species of vertebrates, and multiple spillovers of coronaviruses from animals into humans have founded human epidemics. The COVID-19 epidemic likely derived from a spillover of SARS-CoV-2 from bats into humans, possibly via an intermediate host. There are now several examples of SARS-CoV-2 jumping from humans into other mammals, including mink and deer, creating the potential for new animal reservoirs from which spillback into humans could occur. For this reason, data on formation of new animal reservoirs is of great importance for understanding possible sources of future infection. Here, we identify extensive infection in white-tailed deer in Pennsylvania, including what appear to be multiple independent transmissions. Data further suggests possible transmission among deer. These data thus help identify a potential new animal reservoir and provide background information relevant to its management.

Persistence of SARS-CoV-2 neutralizing antibodies longer than 13 months in naturally infected, captive white-tailed deer (Odocoileus virginianus), Texas.

After identifying a captive herd of white-tailed deer in central Texas with >94% seroprevalence with SARS-CoV-2 neutralizing antibodies in September 2021, we worked retrospectively through archived serum samples of 21 deer and detected seroconversion of all animals between December 2020 and January 2021. We then collected prospective samples to conclude that the duration of persistence of neutralizing antibodies is at least 13 months for 19 (90.5%) of the animals, with two animals converting to seronegative after six and eight months. Antibody titres generally waned over this time frame, but three deer had a temporary 4- to 8-fold increases in plaque reduction neutralization test titres over a month after seroconversion; anamnestic response cannot be ruled out.

Transmission history of SARS-CoV-2 in humans and white-tailed deer.

The emergence of a novel pathogen in a susceptible population can cause rapid spread of infection. High prevalence of SARS-CoV-2 infection in white-tailed deer (Odocoileus virginianus) has been reported in multiple locations, likely resulting from several human-to-deer spillover events followed by deer-to-deer transmission. Knowledge of the risk and direction of SARS-CoV-2 transmission between humans and potential reservoir hosts is essential for effective disease control and prioritisation of interventions. Using genomic data, we reconstruct the transmission history of SARS-CoV-2 in humans and deer, estimate the case finding rate and attempt to infer relative rates of transmission between species. We found no evidence of direct or indirect transmission from deer to human. However, with an estimated case finding rate of only 4.2%, spillback to humans cannot be ruled out. The extensive transmission of SARS-CoV-2 within deer populations and the large number of unsampled cases highlights the need for active surveillance at the human-animal interface.

Transmission of SARS-CoV-2 from humans to animals and potential host adaptation.

SARS-CoV-2, the causative agent of the COVID-19 pandemic, can infect a wide range of mammals. Since its spread in humans, secondary host jumps of SARS-CoV-2 from humans to multiple domestic and wild populations of mammals have been documented. Understanding the extent of adaptation to these animal hosts is critical for assessing the threat that the spillback of animal-adapted SARS-CoV-2 into humans poses. We compare the genomic landscapes of SARS-CoV-2 isolated from animal species to that in humans, profiling the mutational biases indicative of potentially different selective pressures in animals. We focus on viral genomes isolated from mink (Neovison vison) and white-tailed deer (Odocoileus virginianus) for which multiple independent outbreaks driven by onward animal-to-animal transmission have been reported. We identify five candidate mutations for animal-specific adaptation in mink (NSP9_G37E, Spike_F486L, Spike_N501T, Spike_Y453F, ORF3a_L219V), and one in deer (NSP3a_L1035F), though they appear to confer a minimal advantage for human-to-human transmission. No considerable changes to the mutation rate or evolutionary trajectory of SARS-CoV-2 has resulted from circulation in mink and deer thus far. Our findings suggest that minimal adaptation was required for onward transmission in mink and deer following human-to-animal spillover, highlighting the 'generalist' nature of SARS-CoV-2 as a mammalian pathogen.