Virus gone wild.

An ambitious U.S. project aims to sample more than 50 animal species to clarify how the COVID-19 virus moves between people and wildlife.

Zoonotic and Zooanthroponotic Potential of Monkeypox.

The current multicounty outbreak of monkeypox virus (MPXV) posed an emerging and continued challenge to already strained public healthcare sector, around the globe. Since its first identification, monkeypox disease (mpox) remained enzootic in Central and West African countries where reports of human cases are sporadically described. Recent trends in mpox spread outside the Africa have highlighted increased incidence of spillover of the MPXV from animal to humans. While nature of established animal reservoirs remained undefined, several small mammals including rodents, carnivores, lagomorphs, insectivores, non-human primates, domestic/farm animals, and several species of wildlife are proposed to be carrier of the MPXV infection. There are established records of animal-to-human (zoonotic) spread of MPXV through close interaction of humans with animals by eating bushmeat, contracting bodily fluids or trading possibly infected animals. In contrast, there are reports and increasing possibilities of human-to-animal (zooanthroponotic) spread of the MPXV through petting and close interaction with pet owners and animal care workers. We describe here the rationales and molecular factors which predispose the spread of MPXV not only amongst humans but also from animals to humans. A range of continuing opportunities for the spread and evolution of MPXV are discussed to consider risks beyond the currently identified groups. With the possibility of MPXV establishing itself in animal reservoirs, continued and broad surveillance, investigation into unconventional transmissions, and exploration of spillover events are warranted.

Antibodies to Influenza A(H5N1) Virus in Hunting Dogs Retrieving Wild Fowl, Washington, USA.

We detected antibodies to H5 and N1 subtype influenza A viruses in 4/194 (2%) dogs from Washington, USA, that hunted or engaged in hunt tests and training with wild birds. Historical data provided by dog owners showed seropositive dogs had high levels of exposure to waterfowl.

Nine-year seroepidemiological study of severe fever with thrombocytopenia syndrome virus infection in feral horses in Cape Toi, Japan.

Severe fever with thrombocytopenia syndrome (SFTS) is a fatal zoonosis caused by ticks in East Asia. As SFTS virus (SFTSV) is maintained between wildlife and ticks, seroepidemiological studies in wildlife are important to understand the behavior of SFTSV in the environment. Miyazaki Prefecture, Japan, is an SFTS-endemic area, and approximately 100 feral horses, called Misaki horses (Equus caballus), inhabit Cape Toi in Miyazaki Prefecture. While these animals are managed in a wild-like manner, their ages are ascertainable due to individual identification. In the present study, we conducted a seroepidemiological survey of SFTSV in Misaki horses between 2015 and 2023. This study aimed to understand SFTSV infection in horses and its transmission to wildlife. A total of 707 samples from 180 feral horses were used to determine the seroprevalence of SFTSV using enzyme-linked immunosorbent assay (ELISA). Neutralization testing was performed on 118 samples. In addition, SFTS viral RNA was detected in ticks from Cape Toi and feral horses. The overall seroprevalence between 2015 and 2023 was 78.5% (555/707). The lowest seroprevalence was 55% (44/80) in 2016 and the highest was 92% (76/83) in 2018. Seroprevalence was significantly affected by age, with 11% (8/71) in those less than one year of age and 96.7% (435/450) in those four years of age and older (p < 0.0001). The concordance between ELISA and neutralization test results was 88.9% (105/118). SFTS viral RNA was not detected in ticks (n = 516) or feral horses. This study demonstrated that horses can be infected with SFTSV and that age is a significant factor in seroprevalence in wildlife. This study provides insights into SFTSV infection not only in horses but also in wildlife in SFTS-endemic areas.

Novel Avian Influenza A(H5N6) Virus in Wild Birds, South Korea, 2023.

We isolated novel reassortant avian influenza A(H5N6) viruses containing genes from clade 2.3.4.4b H5N1 virus and low pathogenicity avian influenza viruses in carcasses of whooper swans and bean geese in South Korea during December 2023. Neuraminidase gene was from a clade 2.3.4.4b H5N6 virus infecting poultry and humans in China.

Analysis the molecular similarity of least common amino acid sites in ACE2 receptor to predict the potential susceptible species for SARS-CoV-2.

SARS-CoV-2 infections in animals have been reported globally. However, the understanding of the complete spectrum of animals susceptible to SARS-CoV-2 remains limited. The virus's dynamic nature and its potential to infect a wide range of animals are crucial considerations for a One Health approach that integrates both human and animal health. This study introduces a bioinformatic approach to predict potential susceptibility to SARS-CoV-2 in both domestic and wild animals. By examining genomic sequencing, we establish phylogenetic relationships between the virus and its potential hosts. We focus on the interaction between the SARS-CoV-2 genome sequence and specific regions of the host species' ACE2 receptor. We analyzed and compared ACE2 receptor sequences from 29 species known to be infected, selecting 10 least common amino acid sites (LCAS) from key binding domains based on similarity patterns. Our analysis included 49 species across primates, carnivores, rodents, and artiodactyls, revealing complete consistency in the LCAS and identifying them as potentially susceptible. We employed the LCAS similarity pattern to predict the likelihood of SARS-CoV-2 infection in unexamined species. This method serves as a valuable screening tool for assessing infection risks in domestic and wild animals, aiding in the prevention of disease outbreaks.

SARS-CoV-2 Surveillance of Wild Mice and Rats in North American Cities.

From July 2020 to June 2021, 248 wild house mice (Mus musculus), deer mice (Peromyscus maniculatus), brown rats (Rattus norvegicus), and black rats (Rattus rattus) from Texas and Washington, USA, and British Columbia, Canada, were tested for SARS-CoV-2 exposure and infection. Two brown rats and 11 house mice were positive for neutralizing antibodies using a surrogate virus neutralization test, but negative or indeterminate with the Multiplexed Fluorometric ImmunoAssay COVID-Plex, which targets full-length spike and nuclear proteins. Oro-nasopharyngeal swabs and fecal samples tested negative by RT-qPCR, with an indeterminate fecal sample in one house mouse. Continued surveillance of SARS-CoV-2 in wild rodents is warranted.

Two Lineages of Papillomaviruses Identified from Caracals (Caracal caracal) in South Africa.

Papillomaviruses (PV) infect epithelial cells and can cause hyperplastic or neoplastic lesions. In felids, most described PVs are from domestic cats (Felis catus; n = 7 types), with one type identified in each of the five wild felid species studied to date (Panthera uncia, Puma concolor, Leopardus wiedii, Panthera leo persica and Lynx rufus). PVs from domestic cats are highly diverse and are currently classified into three genera (Lambdapapillomavirus, Dyothetapapillomavirus, and Taupapillomavirus), whereas those from wild felids, although diverse, are all classified into the Lambdapapillomavirus genus. In this study, we used a metagenomic approach to identify ten novel PV genomes from rectal swabs of five deceased caracals (Caracal caracal) living in the greater Cape Town area, South Africa. These are the first PVs to be described from caracals, and represent six new PV types, i.e., Caracal caracal papillomavirus (CcarPV) 1-6. These CcarPV fall into two phylogenetically distinct genera: Lambdapapillomavirus, and Treisetapapillomavirus. Two or more PV types were identified in a single individual for three of the five caracals, and four caracals shared at least one of the same PV types with another caracal. This study broadens our understanding of wild felid PVs and provides evidence that there may be several wild felid PV lineages.

Culicoides Midge Abundance across Years: Modeling Inter-Annual Variation for an Avian Feeder and a Candidate Vector of Hemorrhagic Diseases in Farmed Wildlife.

(1) Background: Epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV) are orbiviruses that cause hemorrhagic disease (HD) with significant economic and population health impacts on domestic livestock and wildlife. In the United States, white-tailed deer (Odocoileus virginianus) are particularly susceptible to these viruses and are a frequent blood meal host for various species of Culicoides biting midges (Diptera: Ceratopogonidae) that transmit orbiviruses. The species of Culicoides that transmit EHDV and BTV vary between regions, and larval habitats can differ widely between vector species. Understanding how midges are distributed across landscapes can inform HD virus transmission risk on a local scale, allowing for improved animal management plans to avoid suspected high-risk areas or target these areas for insecticide control. (2) Methods: We used occupancy modeling to estimate the abundance of gravid (egg-laden) and parous (most likely to transmit the virus) females of two putative vector species, C. stellifer and C. venustus, and one species, C. haematopotus, that was not considered a putative vector. We developed a universal model to determine habitat preferences, then mapped a predicted weekly midge abundance during the HD transmission seasons in 2015 (July-October) and 2016 (May-October) in Florida. (3) Results: We found differences in habitat preferences and spatial distribution between the parous and gravid states for C. haematopotus and C. stellifer. Gravid midges preferred areas close to water on the border of well and poorly drained soil. They also preferred mixed bottomland hardwood habitats, whereas parous midges appeared less selective of habitat. (4) Conclusions: If C. stellifer is confirmed as an EHDV vector in this region, the distinct spatial and abundance patterns between species and physiological states suggest that the HD risk is non-random across the study area.

Prevalence and molecular characterization of hepatitis E virus (HEV) from wild rodents in Hubei Province, China.

Hepatitis E, caused by the hepatitis E virus (HEV), is a global public health issue. Low similarity between the gene sequences of mouse and human HEV led to the belief that the risk of human infection was low. Recent reports of chronic and acute hepatitis E caused by murine HEV infection in humans in Hong Kong have raised global concerns. Therefore, it is crucial to investigate the epidemiology and prevalence of HEV in China. We comprehensively analyzed different rodent HEV strains to understand rocahepevirus occurrence in Hubei Province, China. The HEV positivity rate for was 6.43% (73/1136). We identified seven near-full-length rocahepevirus strains and detected rat HEV antigens in tissues from different mouse species. HEV has extensive tissue tropism and a high viral load in the liver. We highlight the genetic diversity of HEVs in rodents and underscore the importance of paying attention to their variation and evolution.

Iceland: an underestimated hub for the spread of high-pathogenicity avian influenza viruses in the North Atlantic.

High-pathogenicity avian influenza viruses (HPAIVs) of the goose/Guangdong lineage are enzootically circulating in wild bird populations worldwide. This increases the risk of entry into poultry production and spill-over to mammalian species, including humans. Better understanding of the ecological and epizootiological networks of these viruses is essential to optimize mitigation measures. Based on full genome sequences of 26 HPAIV samples from Iceland, which were collected between spring and autumn 2022, as well as 1 sample from the 2023 summer period, we show that 3 different genotypes of HPAIV H5N1 clade 2.3.4.4b were circulating within the wild bird population in Iceland in 2022. Furthermore, in 2023 we observed a novel introduction of HPAIV H5N5 of the same clade to Iceland. The data support the role of Iceland as an utmost northwestern distribution area in Europe that might act also as a potential bridging point for intercontinental spread of HPAIV across the North Atlantic.

Surveillance for highly pathogenic avian influenza A (H5N1) in a raptor rehabilitation center-2022.

An ongoing, severe outbreak of highly pathogenic avian influenza virus (HPAI) A H5N1 clade 2.3.4.4b has been circulating in wild and domestic bird populations throughout the world, reaching North America in 2021. This HPAI outbreak has exhibited unique characteristics when compared to previous outbreaks. The global distribution of disease, prolonged duration, extensive number of species and individual wild birds affected, and the large impact on the global poultry industry have all exceeded historical impacts of previous outbreaks in North America. In this study, we describe the results of HPAI surveillance conducted at The Raptor Center, a wildlife rehabilitation hospital at University of Minnesota (Saint Paul, MN, U.S.A.), from March 28th-December 31, 2022. All wild raptors admitted to the facility were tested for avian influenza viruses using polymerase chain reaction (PCR) testing. All non-negative samples were submitted to the United States Department of Agriculture (USDA) Animal and Plant Health Inspection Service (APHIS) National Veterinary Services Laboratories for confirmatory HPAI testing and genetic sequencing. During the study period, 996 individual birds representing 20 different species were tested for avian influenza, and 213 birds were confirmed HPAI positive. Highly pathogenic avian influenza surveillance conducted at The Raptor Center contributed 75% of the HPAI positive raptor detections within the state of Minnesota, located within the Mississippi flyway, significantly augmenting state wildlife surveillance efforts. The viral genotypes observed in birds sampled at The Raptor Center were representative of what was seen in wild bird surveillance within the Mississippi flyway during the same time frame. Wildlife rehabilitation centers provide an opportune situation to augment disease surveillance at the human, wildlife and domestic animal interface during ongoing infectious disease outbreaks.

The virus is out of the barn: the emergence of HPAI as a pathogen of avian and mammalian wildlife around the globe.

Highly pathogenic avian influenza (HPAI) has persisted as a One Health threat whose current circulation and impact are addressed in the companion Currents in One Health by Puryear and Runstadler, JAVMA, May 2024. Highly pathogenic avian influenza emerged as a by-product of agricultural practices and adapted to endemic circulation in wild bird species. Over more than 20 years, continued evolution in a complex ecology involving multiple hosts has produced a lineage that expanded globally over the last 2 years. Understanding the continued evolution and movement of HPAI relies on understanding how the virus is infecting different hosts in different contexts. This includes understanding the environmental factors and the natural ecology of viral transmission that impact host exposure and ultimately evolutionary trajectories. Particularly with the rapid host expansion, increased spillover to mammalian hosts, and novel clinical phenotypes in infected hosts, despite progress in understanding the impact of specific mutations to HPAI viruses that are associated with spillover potential, the threat to public health is poorly understood. Active research is focusing on new approaches to understanding the relationship of viral genotype to phenotype and the implementation of research and surveillance pipelines to make sense of the enormous potential for diverse HPAI viruses to emerge from wild reservoirs amid global circulation.

Abundant Intra-Subtype Reassortment Revealed in H13N8 Influenza Viruses.

Influenza A viruses (IAVs) pose a serious threat to global health. On the one hand, these viruses cause seasonal flu outbreaks in humans. On the other hand, they are a zoonotic infection that has the potential to cause a pandemic. The most important natural reservoir of IAVs are waterfowl. In this study, we investigated the occurrence of IAV in birds in the Republic of Buryatia (region in Russia). In 2020, a total of 3018 fecal samples were collected from wild migratory birds near Lake Baikal. Of these samples, 11 were found to be positive for the H13N8 subtype and whole-genome sequencing was performed on them. All samples contained the same virus with the designation A/Unknown/Buryatia/Arangatui-1/2020. To our knowledge, virus A/Unknown/Buryatia/Arangatui-1/2020 is the first representative of the H13N8 subtype collected on the territory of Russia, the sequence of which is available in the GenBank database. An analysis of reassortments based on the genome sequences of other known viruses has shown that A/Unknown/Buryatia/Arangatui-1/2020 arose as a result of reassortment. In addition, a reassortment most likely occurred several decades ago between the ancestors of the viruses recently collected in China, the Netherlands, the United States and Chile. The presence of such reassortment emphasizes the ongoing evolution of the H13N8 viruses distributed in Europe, North and East Asia, North and South America and Australia. This study underscores the importance of the continued surveillance and research of less-studied influenza subtypes.

Avian Influenza Virus and Avian Paramyxoviruses in Wild Waterfowl of the Western Coast of the Caspian Sea (2017-2020).

The flyways of many different wild waterfowl pass through the Caspian Sea region. The western coast of the middle Caspian Sea is an area with many wetlands, where wintering grounds with large concentrations of birds are located. It is known that wild waterfowl are a natural reservoir of the influenza A virus. In the mid-2000s, in the north of this region, the mass deaths of swans, gulls, and pelicans from high pathogenicity avian influenza virus (HPAIV) were noted. At present, there is still little known about the presence of avian influenza virus (AIVs) and different avian paramyxoviruses (APMVs) in the region's waterfowl bird populations. Here, we report the results of monitoring these viruses in the wild waterfowl of the western coast of the middle Caspian Sea from 2017 to 2020. Samples from 1438 individuals of 26 bird species of 7 orders were collected, from which 21 strains of AIV were isolated, amounting to a 1.46% isolation rate of the total number of samples analyzed (none of these birds exhibited external signs of disease). The following subtypes were determined and whole-genome nucleotide sequences of the isolated strains were obtained: H1N1 (n = 2), H3N8 (n = 8), H4N6 (n = 2), H7N3 (n = 2), H8N4 (n = 1), H10N5 (n = 1), and H12N5 (n = 1). No high pathogenicity influenza virus H5 subtype was detected. Phylogenetic analysis of AIV genomes did not reveal any specific pattern for viruses in the Caspian Sea region, showing that all segments belong to the Eurasian clades of classic avian-like influenza viruses. We also did not find the amino acid substitutions in the polymerase complex (PA, PB1, and PB2) that are critical for the increase in virulence or adaptation to mammals. In total, 23 hemagglutinating viruses not related to influenza A virus were also isolated, of which 15 belonged to avian paramyxoviruses. We were able to sequence 12 avian paramyxoviruses of three species, as follows: Newcastle disease virus (n = 4); Avian paramyxovirus 4 (n = 5); and Avian paramyxovirus 6 (n = 3). In the Russian Federation, the Newcastle disease virus of the VII.1.1 sub-genotype was first isolated from a wild bird (common pheasant) in the Caspian Sea region. The five avian paramyxovirus 4 isolates obtained belonged to the common clade in Genotype I, whereas phylogenetic analysis of three isolates of Avian paramyxovirus 6 showed that two isolates, isolated in 2017, belonged to Genotype I and that an isolate identified in 2020 belonged to Genotype II. The continued regular monitoring of AIVs and APMVs, the obtaining of data on the biological properties of isolated strains, and the accumulation of information on virus host species will allow for the adequate planning of epidemiological measures, suggest the most likely routes of spread of the virus, and assist in the prediction of the introduction of the viruses in the western coastal region of the middle Caspian Sea.

Selective deforestation and exposure of African wildlife to bat-borne viruses.

Proposed mechanisms of zoonotic virus spillover often posit that wildlife transmission and amplification precede human outbreaks. Between 2006 and 2012, the palm Raphia farinifera, a rich source of dietary minerals for wildlife, was nearly extirpated from Budongo Forest, Uganda. Since then, chimpanzees, black-and-white colobus, and red duiker were observed feeding on bat guano, a behavior not previously observed. Here we show that guano consumption may be a response to dietary mineral scarcity and may expose wildlife to bat-borne viruses. Videos from 2017-2019 recorded 839 instances of guano consumption by the aforementioned species. Nutritional analysis of the guano revealed high concentrations of sodium, potassium, magnesium and phosphorus. Metagenomic analyses of the guano identified 27 eukaryotic viruses, including a novel betacoronavirus. Our findings illustrate how "upstream" drivers such as socioeconomics and resource extraction can initiate elaborate chains of causation, ultimately increasing virus spillover risk.

Phylogeographic Dynamics of H9N2 Avian Influenza Viruses in Tunisia.

Avian influenza virus subtype H9N2 is endemic in commercial poultry in Tunisia. This subtype affects poultry and wild birds in Tunisia and poses a potential zoonotic risk. Tunisian H9N2 strains carry, in their hemagglutinins, the human-like marker 226 L that is most influential in avian-to-human viral transmission. For a better understanding of how ecological aspects of the H9N2 virus and its circulation in poultry, migratory birds and environment shapes the spread of the dissemination of H9N2 in Tunisia, herein, we investigate the epidemiological, evolutionary and zoonotic potential of seven H9N2 poultry isolates and sequence their whole genome. Phylogeographic and phylodymanic analysis were used to examine viral spread within and among wild birds, poultry and environment at geographical scales. Genetic evolution results showed that the eight gene sequences of Tunisian H9N2 AIV were characterized by molecular markers involved with virulence and mammalian infections. The geographical distribution of avian influenza virus appears as a network interconnecting countries in Europe, Asia, North Africa and West Africa. The spatiotemporal dynamics analysis showed that the H9N2 virus was transmitted from Tunisia to neighboring countries notably Libya and Algeria. Interestingly, this study also revealed, for the first time, that there was a virus transmission between Tunisia and Morocco. Bayesian analysis showed exchanges between H9N2 strains of Tunisia and those of the Middle Eastern countries, analysis of host traits showed that duck, wild birds and environment were ancestry related to chicken. The subtypes phylodynamic showed that PB1 segment was under multiple inter-subtype reassortment events with H10N7, H12N5, H5N2 and H6N1 and that PB2 was also a subject of inter-subtype reassortment with H10N4.

Multiplex PCR method for MinION sequencing of Bagaza virus isolated from wild caught mosquitoes in South Africa.

Bagaza virus (BAGV) is a mosquito-borne orthoflavivirus known to occur in regions of southern Europe, Africa, India and the Middle East. The virus has been associated with neurological disease and fatalities in various wild bird species. Association with human disease is not confirmed although limited serological evidence has suggested human infection. Surveillance programs for screening mosquitoes for evidence of arbovirus infection play an important role in providing information regarding the circulation and spread of viruses in specific regions. BAGV was detected in a mosquito pool during surveillance of mosquitoes collected in central South Africa between November 2019 and March 2023. Homogenized mosquito pools were screened for flaviviral RNA using conventional RT-PCR and virus isolation was attempted on positive samples. BAGV was detected and subsequently isolated using cell culture. A multiplex tiling PCR method for targeted enrichment using a PCR based or amplicon sequencing approach of the complete genome of BAGV was developed and optimized. Primers were designed using alignment of complete genome sequence data retrieved from GenBank to identify suitable primer sites that would generate overlapping fragments spanning the complete genome. Six forward primers and eight reverse primers were identified that target the complete genome and amplified nine overlapping fragments, that ranged in length from 1954 to 2039 with an overlap ranging from 71 to 711 base pairs. The design strategy included multiple forward and reverse primer pairs for the 5' and 3' ends. Phylogenetic analysis with other isolates was performed and BAGV isolate VBD 74/23/3 was shown to share high similarity with previous BAGV isolates from all regions, with genetic distance ranging from 0.026 to 0.083. VBD 74/23/3 was most closely related to previous isolates from southern Africa, ZRU96/16/2 isolated from a post-mortem sample from a pheasant in 2016 and MP-314-NA-2018 isolated from mosquitoes in northwestern Namibia with genetic distance 0.0085 and 0.016 respectively. Currently there is limited complete genome sequence data available for many of the arboviruses circulating in Africa. The multiplex tiling method provided a simple and cost-effective method for obtaining complete genome sequence. This method can be readily applied to other viruses using sequence data from publicly available databases and would have important application facilitating genomic surveillance of arboviruses in low resource countries.

A Scoping Review on the Epidemiology of Orthobunyaviruses in Canada, in the Context of Human, Wildlife, and Domestic Animal Host Species.

Background: Mosquito-borne orthobunyaviruses in Canada are a growing public health concern. Orthobunyaviral diseases are commonly underdiagnosed and in Canada, likely underreported as surveillance is passive. No vaccines or specific treatments exist for these disease agents. Further, climate change is facilitating habitat expansion for relevant reservoirs and vectors, and it is likely that the majority of the Canadian population is susceptible to these viruses. Methods: A scoping review was conducted to describe the current state of knowledge on orthobunyavirus epidemiology in Canada. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews guideline was used. Literature searches were conducted in six databases and in gray literature. The epidemiology of orthobunyaviruses was characterized for studies focusing on host species, including spatiotemporal patterns, risk factors, and climate change impact. Results: A total of 172 relevant studies were identified from 1734 citations from which 95 addressed host species, including humans, wildlife, and domestic animals including livestock. The orthobunyaviruses-Cache Valley virus (CVV), Jamestown Canyon virus (JCV), Snowshoe Hare virus (SHV), and La Crosse virus (LACV)-were identified, and prevalence was widespread across vertebrate species. CVV, JCV, and SHV were detected across Canada and the United States. LACV was reported only in the United States, predominantly the Mid-Atlantic and Appalachian regions. Disease varied by orthobunyavirus and was associated with age, environment, preexisting compromised immune systems, or livestock breeding schedule. Conclusion: Knowledge gaps included seroprevalence data in Canada, risk factor analyses, particularly for livestock, and disease projections in the context of climate change. Additional surveillance and mitigation strategies, especially accounting for climate change, are needed to guide future public health efforts to prevent orthobunyavirus exposure and disease.

Evolution of SARS-CoV-2 Spikes shapes their binding affinities to animal ACE2 orthologs.

IMPORTANCE: Spike-receptor interaction is a critical determinant for the host range of coronaviruses. In this study, we investigated the SARS-CoV-2 WHU01 strain and five WHO-designated SARS-CoV-2 variants of concern (VOCs), including Alpha, Beta, Gamma, Delta, and the early Omicron variant, for their Spike interactions with ACE2 proteins of 18 animal species. First, the receptor-binding domains (RBDs) of Alpha, Beta, Gamma, and Omicron were found to display progressive gain of affinity to mouse ACE2. More interestingly, these RBDs were also found with progressive loss of affinities to multiple ACE2 orthologs. The Omicron RBD showed decreased or complete loss of affinity to eight tested animal ACE2 orthologs, including that of some livestock animals (horse, donkey, and pig), pet animals (dog and cat), and wild animals (pangolin, American pika, and Rhinolophus sinicus bat). These findings shed light on potential host range shift of SARS-CoV-2 VOCs, especially that of the Omicron variant.