Discovery of a Novel Coronavirus in Swedish Bank Voles (Myodes glareolus)

Authors:  Anishia Wasberg 1,Jayna Raghwani 2,Jinlin Li 3,John H.-O. Pettersson 1,4,Johanna F. Lindahl 1,5,6,Åke Lundkvist 1 andJiaxin Ling 1,*

Abstract

The unprecedented pandemic COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with bats as original reservoirs, has once again highlighted the importance of exploring the interface of wildlife diseases and human health. In this study, we identified a novel Betacoronavirus from bank voles (Myodes glareolus) in Grimsö, Sweden, and this virus is designated as Grimso virus. Repeated detection over three years and an overall prevalence of 3.4% suggest that the virus commonly occurs in bank voles. Furthermore, phylogenetic analyses indicate that the Grimso virus belongs to a highly divergent Embecovirus lineage predominantly associated with bank voles. Given that bank voles are one of the most common rodent species in Sweden and Europe, our findings indicate that Grimso virus might be circulating widely in bank voles and further point out the importance of sentinel surveillance of coronaviruses in wild small mammalian animals, especially in wild rodents.

Keywords: coronavirusbank volesRNA-sequencingprevalence

1. Introduction

Coronaviruses (CoVs) have a high plasticity in host infection which produces a great diversity of CoVs and a complexity evolution. Zoonotic resources of human pathogenic CoVs are mostly from bats or rodents. Coronavirus (CoV) belongs to the Coronaviridae family in the Nidovirales order. They are the largest known RNA viruses with a genome size of 26–32 kb. CoVs have the ability of zoonotic transmission to humans or livestock, and they can cause respiratory or enteric diseases. There are four genera within the coronavirus family, the alpha-, beta-, gamma- and delta-coronaviruses. The genera alpha- and beta-coronaviruses are usually associated with human health of importance [1].

Before the emergence of SARS-CoV, MERS-CoV and SARS-CoV2, four human CoVs HCoV-OC43, HCoV-HKU1, HCoV-NL63, and HCoV-229E, have been adapted to the human population, which were responsible for approximately 30% of endemic common colds in human [1]. Two of these human pathogenic CoVs, HCoV-OC43 and HCoV-HKU1, have the most common ancestry with rodent-borne CoVs based on phylogenetic analyses, indicating that rodents are most likely the reservoirs, and more importantly, the plausibility for rodent CoVs spilling over and infecting humans [2].

Bank voles (M. glareolus) is one of the most common rodent species in Europe and a known reservoir for several zoonotic pathogens, such as Puumala orthohantavirus and Francisella tularensis. Previous studies have detected various Alphacoronavirus and Betacoronavirus in bank voles in the UK, Poland, Germany, and France [3,4]. Here, following a virome investigation of Swedish bank voles collected in Grimsö, Sweden, we report two complete genome sequences of the Grimso virus, along with its evolutionary relationship to other rodent CoVs and its prevalence over a three-year study period.

2. Materials and Methods

2.1. Rodent Samples Collection

A total of 450 bank voles were sampled at the same site in Grimsö, Sweden (59°43′ N, 15°28′ E) between 2015 and 2017. All trapping and sampling were approved by the Animal Experiment Ethical Committee, Umeå (Reference: A12–14), and followed the Swedish Board of Agriculture regulations. Animal species were identified in the laboratory, and lung tissues were harvested and subsequently stored at −80 °C until further investigation, including molecular species identification as described in [5].

2.2. RNA Extraction and Reverse Transcription-PCR (RT-PCR)

Total RNA was extracted using Qiagen RNeasy mini kit (Qiagen, Hilden, Germany). Specific primers targeting the spike protein gene (CoVF: 5′-Ggtcaaactactgaatttattg-3′, CoVR: 5′-Aatccatcagaaccaacgac-3′) were designed based on a virome investigation of Swedish bank voles previously. This primer set was used for screening coronaviruses in 266 bank voles captured from Grimsö between 2015 and 2017. In parallel, we also screened the same samples using a published pan-coronavirus RT-PCR [6]. Positive samples were sent for Sanger sequencing at Macrogen Europe (https://dna.macrogen-europe.com/eng/ (accessed on 1 June 2021)).

2.3. Next-Generation Sequencing and Sequence Assembly

Two positive RNA samples extracted from lung tissues (Grimso215 and Grimso2306) were sent for RNA sequencing, at the Illumina NovaSeq 6000 sequencing platform, from Novogene Hong Kong (https://en.novogene.com/ (accessed on 1 May 2021)). The number of raw reads reached around 50 million pair-end reads of 150 base-pairs (bp). A data analysis pipeline was used to trim and assemble the reads from the sequencing results as described in [7]. We obtained 86,322,748 (96.89% of raw reads) and 105,068,356 (98.81% of raw reads) clean pair-end reads of 150 base-pairs (bp) after filtering from Grimso215 and Grimso2306 samples, respectively. Full-genome and subgenomic sequences of the strain Grimso215 were obtained through de novo assembly using Trinity v2.13.2 with default settings [8]. Using the de novo assembled full genome sequence as a reference, we mapped the reads from the two samples separately (strain Grimso215: 104,891 reads; strain Grimso2306: 2700 reads). As a result, we obtained complete and near-complete coronavirus genome sequences from the strain Grimso215 (100%; 31,317 nt, with a mean coverage value of 502) and strain Grimso2306 (98.2%; 30,767 nt, with a mean coverage of 12.9), respectively. The genome sequences are available via NCBI GenBank (accession number: OM373090 and OM373091).

2.4. Phylogenetic Analysis

For the recombination and phylogenetic analyses, the reference rodent CoVs were downloaded from the NCBI RefSeq database, and multiple sequence alignment was obtained using MAFFT v7.490, which was refined using trimAl v.1.4.1 [9,10]. Pairwise genetic distances were obtained using Geneious Prime v.2019.2.1. Potential recombination events were detected by using Simplot v. 3.5.1 and RDP3 [11]. We assembled six multiple nucleotide sequences alignments for the tree inference, including (A) thirty-one partial spike protein gene sequences from NCBI RefSeq viral database and seven sequences from this study (Figure 1), (B) ORF1b gene, (C) spike protein gene, and (D) nucleocapsid protein gene from forty-two coronavirus nucleotide and amino acid sequences from the database and two genome sequences from this study (Figure 2B,C); (E) thirty partial RdRp gene sequences from alphacoronavirus genome sequences retrieved from the database (Figure 3A); (F) sixty-nine partial RdRp gene sequences from betacoronavirus genome sequences retrieved from the database and two sequences from this study (Figure 3B). The substitution model was determined by using jModelTest2 [12]. Phylogenetic trees were built using MrBayes rooted on the midpoint [13]. All computational calculations from this study were performed using the UPPMAX service from Uppsala University (https://www.uppmax.uu.se/ (accessed on 20 April 2022) (Project ID: SNIC 2019/8-68)).

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Figure 1. (A) Geographic map showing the province Örebro, Sweden and the site of sampling (Grimsö) where bank voles were captured. Table demonstrating the prevalence of Grimso virus from 2015 to 2017. (B) MrBayes midpoint root tree based on the 252 nt of the spike gene. The scale bar indicates the nt substitution per site. The numbers above the branches indicate the posterior probability. Grimso virus samples are highlighted in blue.

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Figure 2. (A) Genomic RNA and subgenomic mRNAs organizations of Grimso virus, Grimso215 strain. MrBayes trees based on the complete nucleotide sequences (B) and amino acid sequences (C) of ORF1b, S, and N genes of CoVs. The red color shows Grimso virus. The scale bar indicates the nt and aa substitution per site, respectively. The numbers above the branches indicate the posterior probability.

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Figure 3. MrBayes tree based on 441nt of partial RdRp gene of CoVs. (A) Phylogeny of alphacoronaviruses and (B) betacoronaviruses. The red color shows CoVs carried by bank voles across Europe. The scale bar indicates the nt substitution per site. The numbers above the branches indicate the posterior probability.

2.5. Protein Domain Analysis

To analyze possible receptor usages, the sequences of spike protein and hemagglutinin esterase genes were examined by searching in the blastx with default settings.

3. Results

3.1. Prevalence of Grimso Virus

Initially, we screened 266 bank voles collected during 2015–2017 at the same site in Grimsö, Sweden (59°43′ N, 15°28′ E) for coronaviruses using an in-house PCR method based on custom primers targeting the spike gene (Figure 1). We detected nine positive samples, which we further characterized using Sanger sequencing. We also screened the same samples using a published pan-coronavirus RT-PCR [6], although this failed to detect any coronaviruses.

Figure 1 shows the location of the study site and demonstrates the prevalence of the Grimso virus between 2015 and 2017. We obtained partial spike gene sequences (252 nt) from seven out of nine positive samples using Sanger sequencing. Pairwise genetic analyses indicated that the partial Grimso virus sequences shared 98.0–100% and 94.0–100% sequence identity at the nucleotide and amino acid levels, respectively. Strikingly, Grimso virus sequences showed less than 60% and less than 50% sequence identity at both the nucleotide and amino acid levels, respectively, with other rodent Betacoronaviruses. Bayesian phylogenetic analysis of partial spike gene sequences from thirty-one reference CoV genome sequences indicates that the Grimso virus sequences form a distinct monophyletic group that cluster with other rodent-borne Embecoviruses with strong statistical support (posterior probability = 1.0).

3.2. Genome Organization of Grimso Virus

Thereafter, we selected two samples collected in 2015 (Grimso215) and 2017 (Grimso2306) for RNA-sequencing to characterize the complete genome. We obtained a full-length coronavirus sequence from the bank vole sample Grimso215 (31,317 nt) and a near-complete coronavirus sequence from the bank vole sample Grimso2306 (98%; 30,767 nt). The in-depth phylogeny of Grimso virus is demonstrated in Figure 2 where Figure 2A outlines the whole genome, including seven subgenomic regions, of the Grimso virus, strain Grimso215. The Grimso virus genome encodes for hemagglutinin esterase (HE), spike protein (S), envelop protein (E), membrane protein (M), and nucleocapsid protein (N). We found no significant evidence for recombination in the genome of strain Grimso215. Phylogenetic analyses based on the ORF1b, S and N gene sequences consistently showed that the strains Grimso215 and Grimso2306 fell within known rodent Betacoronavirus diversity and formed a distinct lineage within the subgenus Embecovirus (Figure 2B). Additionally, we analyzed the amino acid sequences of ORF1b, S and N genes, which confirmed the phylogenetic relationship (Figure 2C).

Pairwise sequence identity of Grimso virus (strain Grimso215) with other rodent CoVs ranged from 55 to 79% at the nucleotide level and 44 to 67% at the amino acid level, confirming that the Grimso virus is divergent and genetically distinct from earlier described rodent CoVs (Table 1).

Table 1. Identity (%) of Grimso215 strain on the genes across the genome (at nucleotide/amino acid levels) as compared to the representatives of known rodent CoVs.

Table

3.3. Phylogenetic Analysis

To better understand the evolutionary history of rodent CoVs and the phylogenetic positioning of the divergent Grimso strains, we undertook an additional phylogenetic analysis based on 441 nt of the partial RNA-dependent RNA polymerase (RdRp) gene from 101 CoV genome sequences. The results indicated that bank voles harbor at least three virus species across the Alphacoronavirus and Betacoronavirus genera (Figure 3A,B). Specifically, four viruses isolated from bank voles in Germany, Poland, and the UK [14] were closely related to the Lucheng Rn rat coronavirus (genus: Alphacoronavirus) (Figure 3A). In addition, we also found two distinct Embecoviruses associated with bank voles (Figure 3B): (1) a bank vole coronavirus from Germany closely related to Myodes coronavirus 2JL14 (subgenus: Embecovirus) and (2) a cluster of CoVs isolated from bank voles in Germany and France, along with strains Grimso215 and Grimso2306 from this study, which formed separate divergent lineage within the subgenus Embecovirus (Figure 3B).

3.4. Protein Domain Analysis

We examined the spike protein, hemagglutinin esterase and possible receptor usages by searching in the blastx. It had only specific hits in the S2 region but not in the S1 or receptor-binding regions (Figure S1).

4. Discussion

Rodents are the primordial hosts of CoVs, and rodent CoVs constitute at least two subgenera, Luchacovirus and Embecovirus from genera Alphacoronavirus and Betacoronavirus, respectively [14,15]. In this study, we have discovered a highly divergent betacoronavirus (Grimso virus) in the Swedish bank voles. Furthermore, based on our knowledge, this study is the first time to identify full-genome features of Grimso virus, together with the prevalence and diversity of this rodent CoV in Sweden.

The Grimso virus is highly divergent and genetically distinct from earlier described rodent CoVs based on the RNA-sequencing results, which also explains that we failed to detect any CoVs by using a published pan-coronavirus RT-PCR. By using the specific primers targeted to the spike gene, we have discovered nine samples which were positive in the PCR screening, with a continuous positivity for three years, which are 2/48 in 2015, 1/61 in 2016, and 6/157 in 2017.

We recovered two genome sequences from two strains of Grismo virus. The genomes of strains Grimso215 and Grimso2306 shared 95.6% sequence identity at the nucleotide level, with 1338 site differences. This divergence is notably higher than the expected differences based on a typical substitution rate for coronaviruses of 0.001 substitutions per site per year [16,17], which under Poisson distribution predicts an accumulation of 61–121 substitutions over three years. This observation suggests that either multiple strains of Grimso-like viruses are co-circulating in bank voles in Grimsö or that these viruses are transmitted regularly to bank voles from other species. One could also contemplate that the observed divergence could be associated with the temporal fluctuations in bank vole population density, leaving room for an increased viral transmission in a cyclic peak preceded by population turnover, similarly as previously described for Puumla Hantavirus dynamics in bank voles [18]. Nevertheless, with a prevalence of around 3.4% (9/266), we hypothesize that Swedish bank voles are competent hosts for the Grimso virus.

The wild animals provide pools of divergent virus species for interspecies transmission. The studies on the discovery of animal CoVs have been especially important since the emergence of human coronaviruses. The hidden diversity of CoVs has been explored in at least 37 rodent species distributed in Asia and Europe [3,4,15,19,20,21]. These studies point out that rodent CoVs have undergone frequent recombination and cross-species transmission events [15,20]. Previous studies found distinct CoVs in bank voles based on the partial sequences of the RdRp gene in the UK, Poland, Germany, and France [3,4]. Our phylogenetic analyses based on complete sequences of ORF1b, S, and N genes, as well as a partial RdRp gene, all suggested that bank voles carry one more divergent CoV, Grimso virus. Together, these observations suggest a relatively broad geographic distribution of CoVs in bank voles in Europe, which is indicative of possible long-term host–virus association. Furthermore, as coronaviruses closely related to the Grimso virus have been detected in bank voles elsewhere in Europe, it further supports that this divergent coronavirus infects and circulates in Swedish bank voles.

To understand the zoonotic risks of Grismo virus, we analyzed the protein domain of the spike protein and hemagglutinin esterase. Due to the high divergence and the lack of isolated live virus, we could not yet identify the host receptor usage for this novel rodent CoV. However, we cannot neglect the zoonotic potential of Grimso virus to livestock or humans. Together with mapping and continuous monitoring of the Grimso virus, further studies will aim to isolate the virus and assess the pathogenic profile.

5. Conclusions

We identified a highly divergent Embecovirus, named the Grimso virus, in Swedish bank voles. Our analyses suggest that multiple distinct viral strains co-circulate in this population, although further investigation will be necessary to fully understand the transmission ecology. Furthermore, we found that closely related coronaviruses are broadly distributed across Europe and exclusively associated with bank voles and other vole species, indicating that bank voles are likely natural reservoirs of the Grimso virus. While the potential threat posed by the virus to human and animal health is unknown, our findings underscore the importance of longitudinal surveillance of CoVs in wild rodents in advancing current knowledge on the ecology of CoVs in reservoir populations.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/v14061205/s1, Figure S1: Conserved domains on the hemagglutinin esterase (A) and spike (B) of Grimso virus.

Author Contributions

Conceptualization: Å.L., J.L. (Jiaxin Ling) and J.L. (Jinlin Li). Formal analysis: J.L. (Jiaxin Ling), J.R. and A.W. Funding acquisition: J.F.L. and Å.L. Investigation: J.L. (Jiaxin Ling), A.W., J.R., J.L. (Jinlin Li) and J.H.-O.P. Writing—original draft: J.L. (Jiaxin Ling), J.R. and A.W. Writing—review & editing: J.L. (Jiaxin Ling), A.W., J.R., J.L. (Jinlin Li), J.H.-O.P., J.F.L. and Å.L. All authors have read and agreed to the published version of the manuscript.

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Tracking coronavirus in animals takes on new urgency

Authors: Ariana Eunjung Cha  May 20, 2022 The Washington Post

Researchers Sarah Hamer and Lisa Auckland donned their masks and gowns as they pulled up to the suburban home in College Station, Tex. The family of three inside had had covid a few weeks earlier, and now it was time to check on the pets.

Oreo the rabbit was his usual chill self, and Duke the golden retriever was a model patient, lying on his back as Hamer and Auckland swabbed their throats and took blood samples. But Ellie, a Jack Russell terrier, wiggled and barked in protest. “She was not exactly happy with us,” Auckland recalled. “But we’re trying to understand how transmission works within a household, so we needed samples from everyone.”

The questions driving the researchers goes far beyond pet welfare. They’re investigating whether animals infected with the coronavirus might become reservoirs for the evolution of new variants that might jump back into humans — an issue with huge implications for both human and animal health.

In year three of the pandemic, scientists have confirmed that the virus believed to have first spilled over to humans from bats or possibly pangolins has already spread to at least 20 other animal species, including big cats, ferrets, North American white-tailed deer and great apes. To date, incidents of animals infecting humans are rare. Only three species — hamsters in Hong Kong, mink in the Netherlands and, possibly, also white-tailed deer in the United States and Canada — have transmitted a mutated, albeit mostly benign, version of the virus back to humans. But those cases are spurring concern.

The search for infected animals in Texas — led by Texas A&M University in conjunction with the U.S. Centers for Disease Control and Prevention — is part of a scattered but growing global effort to monitor pets, livestock and wildlife for new, potentially more dangerous coronavirus variants and stop them from wreaking havoc on humans.

Tracking coronavirus in animals takes on new urgency

The World Health Organization warned in March that animal reservoirs could lead to “potential acceleration of virus evolution” and new variants. The agency noted the large numbers of infected animals, and it urged countries to increase their monitoring of mammalian species for SARS-CoV-2 and suspend the sale of live, wild mammals in food markets as an emergency measure. The CDC this year also endorsed efforts to track the virus in animals, even as it described the risk of transmission to humans as “low.”

In March, a city in China ordered the killing of pets whose owners had tested positive, but the action was put on hold after a public outcry. (There was no evidence these pets were spreading the virus.) Earlier this year, Hong Kong’s first outbreak in months is believed to have been caused by hamsters imported from Europe.

Meanwhile, U.S. scientists estimated late last year that a third of white-tailed deer in several states appear to carry antibodies to the coronavirus, suggesting recent infection, and three snow leopards at the Lincoln Children’s Zoo in Nebraska died of covid-19. In 2020, Denmark had culled millions of mink after they were infected and the virus spilled back into humans.

Most new variants are simply scientific curiosities, and they die out. The challenge for scientists is to create a system to identify the dangerous ones — the ones that are more transmissible, more deadly or more likely to break through vaccines — and attempt to halt their transmission.

“We need to be very aware there are going to be more epidemics and pandemics and plan ahead,” said Pamela Bjorkman, a professor at the California Institute of Technology.

In the span of more than two years, the coronavirus itself has been evolving faster than most anyone expected — creating an evolutionary “super-tree” with major new branches seeming to sprout every few months. The environment in which these variants are forming, researchers surmise, is likely one that allows the virus to live longer and thereby make more copies of itself, increasing the prospect of new mutations.

One leading theory is immunocompromised patients, such as those with cancer or HIV, who can harbor the infection for many weeks or months, as compared with mere days for most people. But another more daunting possibility is that the virus is finding hosts among the more than 1 million animal species, many still not catalogued, that inhabit Earth.

“It’s a scary thing.” Bjorkman, who has been working on a universal coronavirus vaccine, said there has long been viral transmission between humans and animals that nobody pays attention to.

The problem is that “every once in a while, there is transmission that catches on” and will explode if it spills into the human population, she said.

Animal reservoirs

Scientists believe most major outbreaks of disease serious enough to be deemed epidemics or pandemics have begun with animals.

H5N1, a highly pathogenic flu that occurs in wild birds, sent fear through the medical community after a young boy died of it in Hong Kong in May 1997. (The first U.S. case was reported in Colorado in April.) SARS1, which caused an outbreak in Asia from 2002 to 2004, infecting more than 8,000 people, is believed to have jumped from civets, a catlike mammal, to humans.

The H1N1 influenza virus that hit the world in 2009 and is estimated to have infected as many as 1.4 billion people is believed to be what scientists call a “reassortment” of flu that has been found in birds, pigs and humans. The MERS virus, first reported in Saudi Arabia in 2012 with a fatality rate as high as 35 percent, is believed to have emerged from camels.

SARS-CoV-2, the pathogen terrorizing the world since early 2020, has higher potential for transmission to animals than many other known viruses because it invades the body by latching onto a receptor known as ACE2, which is found in a number of species. In recent weeks, researchers reported evidence to support early suspicions that the original coronavirus that jumped into humans sometime before January 2020 may have come from wet market animals, perhaps bats, perhaps raccoon dogs, or another animal used for food or fur in Wuhan, China.

As of April, scientists had logged 675 coronavirus outbreaks in animals, affecting 23 species in 36 countries, and other species have been shown to be vulnerable in lab experiments. But there are likely many thousands more that are susceptible. A University of California at Davis study of the potential vulnerability of different species to coronavirus infection — based on modeling of which ones had ACE2 cellular receptors similar to those in humans, because that’s how the virus enters the body — ranked animals as diverse as giant anteaters and bottlenose dolphins as high risk, and Siberian tigers, sheep and cattle as medium risk.

So far, most of the coronavirus transmission appears to have jumped from animals to humans. Scientists have documented infection going in the other direction only three times: from mink to humans, hamsters to humans, and one likely case of deer to humans. None of those three events is believed to have introduced dangerous variants.

In its monthly situation report in late April, the World Organization for Animal Health said that although the main driver of international viral spread is still human-to-human transmission, animal cases “continue to rise.” The big question is not whether certain animals can be infected, researchers say. It is which might act as so-called reservoirs that can serve as sources of new variants that could pose greater threats to humans.

Hong Kong

Leo Poon recalled feeling immediately uneasy in January when he got word of a new coronavirus infection in the northern part of the island.

Hong Kong had been quiet for months, and the delta wave that had devastated much of the world seemed to wash over the city with few cases. The city had implemented a strict — some it called draconian — “zero covid” policy that had kept the islands infection-free for long periods. Poon, head of the division of public health laboratory science at the University of Hong Kong, had been helping the government sequence SARS-CoV-2 samples from patients to find out where infections originated and which close contacts were at risk.

This new patient was a 23-year-old saleswoman with mild symptoms of headache and fever who had not had contact with anyone with an infection. Nor had she traveled or been in contact with anyone who had. When Poon checked the global databank that scientists are using to track the evolution of the virus, he was surprised to find that some of the genetic mutations in the young woman’s sample appeared to be novel and had never been documented in any other human sample.

As he delved into the case report, Poon noticed the woman worked at a pet shop, and that’s when it hit him: Could she have gotten covid from an animal?

A flurry of hurried phone calls and emails followed, and Hong Kong authorities locked down the store, Little Boss in the shopping district of Causeway Bay, and a related warehouse, swabbing the nearly 200 animals they found.“We really have to highlight the concept of one health. It’s not only about human health. We have to consider health in animals and the environment. And if you don’t look after these areas, we are the one that suffer at the end.” Leo Poon, head of the division of public health laboratory science at the University of Hong Kong

The rabbits, chinchillas and guinea pigs were cleared. But 11 of the hamsters, specifically the golden Syrian hamsters, tested positive for coronavirus, with a variant similar to the one that had infected the woman. The timelines matched: The hamsters had been flown in from the Netherlands on Jan. 7. The woman became ill on Jan. 11.

The young woman, and a second patient believed to have gotten sick directly from a hamster whose case was described in a preprint paper in the Lancet, did not get very ill. But Poon worried that one of the major changes to the virus related to how it attaches to receptors. He feared that if it was allowed to hop back and forth between humans and animals, it might ultimately change into something less benign.

Investigators identified 150 people, mostly customers who had visited the store, who were at risk of being infected and ordered them into quarantine, banned the importation of small mammals, and put to sleep the remaining 2,000 hamsters in city pet stores within days of the discovery of the link. Public health officials “strongly advised” pet owners to turn over any additional hamsters to be euthanized.

“We really have to highlight the concept of one health,” Poon reflected. “It’s not only about human health. We have to consider health in animals and the environment. And if you don’t look after these areas, we are the one that suffer at the end.”

Ontario, Canada

The first white-tailed deer were tested on a whim.

It was early 2020, and Andrew Bowman, an associate professor of veterinary medicine at Ohio State University, was tracking the animals near Columbus for other purposes and thought he might as well add in one more test. When the results came back positive, he was so taken aback that he rechecked and then triple-checked, and then called in the U.S. Department of Agriculture to verify before announcing the finding to the world.

“At that point, it was a surprise,” he recalled. “But when we step back, we really shouldn’t have been that surprised.”

As suburban communities expand into what was once forest, the population of white-tailed deer living in proximity to humans is increasing. While few people interact directly with deer, scientists are investigating whether the animals might be exposed to the coronavirus through discarded face masks and other trash, contaminated water or, perhaps, some intermediary species. Several scientific teams confirmed the breadth of the cases in deer and found that most of the animals appeared to be asymptomatic.

In August, the USDA announced that its own analysis found antibodies in about one-third of deer in Illinois, Michigan, New York and Pennsylvania. It issued a warning to the public to be cautious in their interactions, and to limit contact between wildlife and domestic animals.

In Canada, Brad Pickering, an animal pathogens expert with the country’s Food Inspection Agency, went further to learn more about the evolution of the virus in deer. Examining samples of 300 animals hunted in Ontario from Nov. 1 to Dec. 31, he was shocked to find 76 mutations in some deer strains.

“That’s a lot, more than omicron,” he said of the number of mutations. “It is showing there seems to be some adaptation to deer or wildlife, in general.”

According to a preprint paper he and a group of more than 30 scientists posted in February, the closest known branches to the deer strain were found in humans in Michigan a year earlier. Those, in turn, were related to mink samples from Michigan previously identified in September/October 2020. Given that the area where the deer samples were taken in southwestern Ontario is adjacent to Michigan, the researchers wondered whether the variant had jumped from humans to mink and then to deer.“Even if it’s left the human population. It doesn’t mean we’re done with it.” Brad Pickering, animal pathogens expert with the country’s Food Inspection Agency

Even more odd, the researchers identified a human sample from Michigan that was very similar to the highly mutated deer sample. It turned out that person had had close contact with deer, and while the information could not conclusively point to deer-to-human transmission, the evidence was strong.

Neither Bowman nor Pickering worries that the new deer strains of coronavirus pose any immediate danger to humans. Bowman said that the threat is “more of a long-game question.” If “it evolves in a different trajectory than humans, how long do we have before it’s diverse enough that the next pandemic strain spills back into humans?”

“Even if it’s left the human population,” Pickering said, “it doesn’t mean we’re done with it.”

Texas

Sarah Hamer’s work at Texas A&M University involves the broad ecology of animals and humans. The future of coronavirus variants, she explained, depends not just on how we interact with a single animal species but also on the web of associations among humans, domestic animals and wildlife.

For cats and dogs, scientists are somewhat confident the transmission has been mostly one-way, from humans to the animals spurred by people snuggling and playing with their pets. Hamer’s research aims to clarify details about the chain of transmission — who brought the infection into the household, who got it next and so forth.

Figuring out what is going on with deer has been much more challenging.

“We cannot explain it as spillover from humans, because not all of them have had a close contact,” Hamer explained. “That’s where it gets pretty interesting to think about.”

On the same weekend that Hamer and Auckland were taking samples from Oreo, Duke, Ellie and their human owners, another team from the same lab was in a nearby forest in eastern Texas, studying small and medium mammals.

After setting a couple of hundred traps that evening, they returned the next morning to find wild mice, wild rats and other mammals. While the primary purpose of the study was to look at vector-borne pathogens such as tick diseases, all the animals were also swabbed for coronavirus before being released back into the woods. The wildlife samples are being stored in a freezer, as Hamer awaits funding to test them.

Right now, such efforts are being conducted mostly piecemeal, often added to non-covid work that is already funded. She and other scientists say a more coordinated global surveillance approach that will target a range of species during different seasons and in different geographic areas is needed to stop a potential new generation of variants.

“There is so much more that should be done,” she said.