Potential zoonotic sources of SARS‐CoV‐2 infections

Abstract The severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) causing coronavirus disease‐2019 (COVID‐19) likely has evolutionary origins in other animals than humans based on genetically related viruses existing in rhinolophid bats and pangolins. Similar to other animal coronaviruses, SARS‐CoV‐2 contains a functional furin cleavage site in its spike protein, which may broaden the SARS‐CoV‐2 host range and affect pathogenesis. Whether ongoing zoonotic infections are possible in addition to efficient human‐to‐human transmission remains unclear. In contrast, human‐to‐animal transmission can occur based on evidence provided from natural and experimental settings. Carnivores, including domestic cats, ferrets and minks, appear to be particularly susceptible to SARS‐CoV‐2 in contrast to poultry and other animals reared as livestock such as cattle and swine. Epidemiologic evidence supported by genomic sequencing corroborated mink‐to‐human transmission events in farm settings. Airborne transmission of SARS‐CoV‐2 between experimentally infected cats additionally substantiates the possibility of cat‐to‐human transmission. To evaluate the COVID‐19 risk represented by domestic and farmed carnivores, experimental assessments should include surveillance and health assessment of domestic and farmed carnivores, characterization of the immune interplay between SARS‐CoV‐2 and carnivore coronaviruses, determination of the SARS‐CoV‐2 host range beyond carnivores and identification of human risk groups such as veterinarians and farm workers. Strategies to mitigate the risk of zoonotic SARS‐CoV‐2 infections may have to be developed in a One Health framework and non‐pharmaceutical interventions may have to consider free‐roaming animals and the animal farming industry.

emerged coronavirus termed severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) . A conspecific coronavirus termed SARS-CoV caused an outbreak in 2003-2004, infecting about 8,000 people mainly in China, of which about 10% died (Cheng et al., 2013). Control of SARS in 2003-2004 by strict non-pharmaceutical interventions was facilitated by predominant SARS-CoV replication in the lower respiratory tract (Gu & Korteweg, 2007), as opposed to efficient replication of SARS-CoV-2 in the upper respiratory tract (Wolfel et al., 2020) despite usage of the same receptor molecule angiotensin-converting enzyme 2 (ACE2) Li et al., 2003). Here, we discuss the evidence for the zoonotic origins of human coronaviruses, identify epidemiologically relevant animal hosts and discuss their impact on containment strategies of COVID-19 under a One Health approach, a concept that is gaining recognition which addresses health issues, such as zoonotic diseases, at the human-animal-environment interface in a collaborative effort among multiple relevant disciplines and sectors (FAO-OIE-WHO, 2019).

| ZOONOTI C ORI G IN OF H UMAN CORONAVIRUS E S
Endemic human coronaviruses (HCoVs) include the alphacoronavi-  (Drexler et al., 2014). In the case of SARS-related CoVs, rhinolophid bats (Rhinolophus spp., also termed horseshoe bats) are particularly relevant natural hosts, as exemplified by the extent of genetically divergent SARS-related CoVs found in those bats across Asia, Africa and Europe (Drexler et al., 2010;Hu et al., 2017;Li et al., 2005). In the tropics, direct contact between humans and bats potentially facilitating human infection is more frequent than in temperate climates, including consumption of bats as bushmeat and their use for traditional medicine (Anti et al., 2015;Mildenstein et al., 2016). However, other animals than bats have been implicated as intermediate hosts of and source of infection with HCoVs ( Figure 1a) . For instance, alphacoronaviruses found in camels (Camelus dromedarius) and alpacas (Vicugna pacos) share a common ancestor with HCoV-229E , whereas HCoV-OC43 shares a common ancestor with coronaviruses infecting cattle (Bos taurus) (Vijgen et al., 2006). The emerging MERS-CoV is enzootic in camels (mainly C. dromedarius) and causes regular spill-over infections into humans (WHO, 2020). In the case of SARS-CoV, genetically closely related viruses were found in masked palm civets (Paguma larvata) and raccoon dogs (Nyctereutes procyonoides) sold in a live-animal market in the Guangdong province (Guan et al., 2003;Kan et al., 2005). Genomic comparisons of full-length SARS-CoV sequences from human and civets showed high nucleotide identity of approximately 99.6%. In 2005, genetically highly diverse SARS-related CoVs were found in horseshoe bats from China (Li et al., 2005). Masked palm civets were thus likely intermediate hosts, while horseshoe bats were the likely evolutionary origin of SARS-CoV. It has been speculated that mutations in the SARS-CoV receptor-binding domain may have arisen in palm civets that facilitated infection efficiency by increasing the affinity to the human ACE2 receptor (Graham & Baric, 2010;Song et al., 2005).
Efforts to identify potential SARS-CoV-2 intermediate hosts have not been successful yet. The origin of the outbreak was associated with the Huanan seafood wholesale market in Wuhan during epidemiologic investigations of the first cases , which was supported by SARS-CoV-2-positive environmental samples from that market (Zhang & Holmes, 2020). However, retrospective F I G U R E 1 Mammals as reservoirs and intermediary hosts of endemic and emerging human coronaviruses. (a) Animal reservoirs and intermediary hosts of human coronaviruses. (b) Cladogram of mammalian orders adapted from (Foley et al., 2016). Hosts of coronaviruses are depicted by pictograms (teal). Squares depict families susceptible to SARS-CoV (orange), SARS-CoV-2 (purple) and other SARS-related CoVs (grey) [Colour figure can be viewed at wileyonlinelibrary.com] epidemiologic analyses indicated that an earlier COVID-19 case had no exposure to that seafood market . The origins of the pandemic thus remain unclear. The closest relatives to human SARS-CoV-2 known so far are a coronavirus coming from the intermediate horseshoe bat (R. affinis) from China with approximately 96% nucleotide identity, and two sub-lineages of SARS-related CoVs found in Malayan pangolins (Manis javanica) with 85.5%-92.4% nucleotide identity averaged over the whole viral genome (Figure 2a) (Lam et al., 2020;Xiao et al., 2020). Moreover, phylogenetic analyses using a large subgenomic data set of bat coronaviruses from China indicate that alike SARS-CoV, SARS-CoV-2 likely originated in horseshoe bats (Latinne et al., 2020). However, whereas zoonotic infection of humans with civet-associated SARS-CoV strains was likely according to the genetic relatedness of those CoVs (Figure 2a
Other reports on SARS-CoV-2-positive dogs in the Netherlands and  No symptoms, effective virus production and seroconversion in 6/9 animals, effective transmission to contact animals, higher viral shedding in nose and throat than rectum, mild rhinitis (Freuling et al., 2020) Chiroptera Egyptian fruit bat Experimental High 10 5 TCID 50 , intranasal No symptoms, viral shedding in 9/9 inoculated animals and 2/3 contact animals, low titres of neutralizing antibodies, mild rhinitis and infiltrating lymphocytes and neutrophils (Schlottau et al., 2020) (Continues) the USA indicated that different symptoms can occur in infected dogs, ranging from mild to severe respiratory distress symptoms (Daly, 2020;Sterling, 2020). In sum, the impact of SARS-CoV-2 infections on domestic animal health is unclear, as both dogs and cats were reported with and without clinical signs.

TA B L E 1 (Continued)
hamsters has also been reported (Halfmann et al., 2020;Shi et al., 2020;Sia et al., 2020). In the case of non-human primates, given their genetic relatedness to humans and occurrences of transmission of other human respiratory viruses such as HCoV-OC43 (Patrono et al., 2018) and respiratory syncytial virus (Grutzmacher et al., 2016), susceptibility to SARS-CoV-2 was not unexpected (Munster et al., 2020;Rockx et al., 2020). CoV-2 infection, 7 of which were confirmed by a highly specific microneutralization assay and one by PCR (Oreshkova et al., 2020). Virus transmission from infected minks back to humans was corroborated by phylogenetic comparison between human-and mink-derived SARS-CoV-2 sequences, which grouped together . Zoonotic infections are common occupational hazards in individuals who are frequently exposed to animals or animal products such as veterinarians, zoo and abattoir workers, breeders and farmers (Epp & Waldner, 2012). Risk assessments should be carried out to identify occupational groups that are disproportionately exposed to potentially SARS-CoV-2 infected animals, as was exemplified by a novel squirrel bornavirus, which caused fatal encephalitis among squirrel breeders and a zoo worker (Tappe et al., 2018).
The combined data from natural and experimental infections together with preliminary epidemiologic investigations highlight the potential transmission of SARS-CoV-2 from humans to different mammalian orders, particularly members of the order Carnivora ( Figure 3a, Table 1). Comparing the ACE2 residues involved in SARS-CoV-2 entry in humans (Shang et al., 2020) with the sequences from different carnivore species revealed only few amino acid changes, which is consistent with SARS-CoV-2 infection in those mammals (Table 2). It is unclear whether mutations in the SARS-CoV-2 spike receptor-binding domain are needed to increase infection efficiency in different carnivore hosts or if an ACE2-independent pathway, potentially mediated by the SARS-CoV-2 furin cleavage site, is F I G U R E 3 Susceptibility of carnivore hosts to coronaviruses (a) Carnivores susceptible to SARS-related CoVs are dispersed across the family tree. Cladogram of carnivore families adapted from (Foley et al., 2016). (b) Furin cleavage site between the spike subunits S1 and S2 is predominantly present in betacoronaviruses. Scheme of SARS-CoV-2 genome organization with a magnified S1/ S2 furin cleavage site within carnivore and human coronaviruses. employed in parallel to receptor-mediated entry .

| FURIN CLE AVAG E S ITE IN H UMAN AND C ARNIVORE CORONAVIRUS E S
Different from SARS-CoV, SARS-CoV-2 contains a furin protease cleavage site in the viral spike protein, which is responsible for attachment and entry into cells (Coutard et al., 2020). In MERS-CoV and influenza viruses, presence of a furin cleavage site has been associated with enhanced transmissibility, increased virulence and broader host range (Andersen et al., 2020). It has been speculated that furin-mediated spike cleavage may also affect SARS-CoV-2 pathogenesis (Andersen et al., 2020). Several feline coronavirus

| CORONAVIRUS E S NATUR ALLY INFEC TING C ARNIVORE S
Besides from FCoV, which was already introduced in the section above, enzootic carnivore coronaviruses include canine coronavirus Modified live attenuated FIPV and inactivated CCoV vaccines are commercially available. However, the use of FIPV vaccine has been controversial, as antibody-dependent enhancement of infection has been reported in experimental settings, albeit not in the field (Takano et al., 2008). It is unclear what immunity to enzootic cat and dog CoVs elicited either by vaccination or natural infection will play in SARS-CoV-2 transmission between carnivores.
Currently, it is not known whether pre-existing antibodies against human-endemic CoVs afford cross-protection against or immune enhancement of SARS-CoV-2 infection. Despite decades of investigation into endemic HCoVs, no vaccine has been developed to prevent infections with HCoV-229E, HCoV-OC43, HCoV-NL63 and HKU1, as these viruses mostly generate mild disease . For SARS-CoV, translational research has been limited as the importance of the virus decreased due to the successful containment of the virus by timely public health interventions (Cheng et al., 2013). In contrast, several vaccine candidates have been tested against the recently emerged MERS-CoV, including vectored vaccines based on adenovirus and poxvirus backbones (Alharbi, 2017). Because MERS-CoV naturally infects camels, and human infections are almost exclusively zoonotic outside of outbreaks in hospital settings , MERS-CoV vaccines were developed following a One Health approach considering applicability to both humans and camels (Alharbi, 2017). A vaccine candidate using a poxvirus vector is now in phase I trial as of December 2019 after tests in dromedary camels were successful (Alharbi et al., 2019;NLM, 2019).

Further investigation into animal coronavirus immune responses
will be required, particularly into the implications of pre-existing carnivore coronavirus immunity on SARS-CoV-2 infections including both cross-protection and potential immune enhancement, which can shed light on the immune interplay in HCoV infections and potentially inform SARS-CoV-2 vaccine development.

| CON CLUS ION
Pandemic SARS-CoV-2 spread combined with over 800 million cats and dogs kept as pets worldwide (Bedford, 2020), and the large extent of the animal farming industry (Ritchie, 2017)

ACK N OWLED G EM ENT
Open access funding enabled and organized by Projekt DEAL.

CO N FLI C T O F I NTE R E S T
Authors declare no competing interests.

E TH I C A L A PPROVA L
No ethical approval was required as this is a review article with no original research data.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable as no new data was created.