key: cord-0846604-e93zdnms authors: Logeot, Myriam; Mauroy, Axel; Thiry, Etienne; De Regge, Nick; Vervaeke, Muriel; Beck, Olivier; De Waele, Valérie; Van den Berg, Thierry title: Risk assessment of SARS‐CoV‐2 infection in free‐ranging wild animals in Belgium date: 2021-05-26 journal: Transbound Emerg Dis DOI: 10.1111/tbed.14131 sha: d1a4722c5838c96155c3a902de0fecd87f3eb933 doc_id: 846604 cord_uid: e93zdnms The aim of this review paper is to evaluate the putative susceptibilities of different free‐ranging wild animal species in Belgium to SARS‐CoV‐2 and provide a risk assessment of SARS‐CoV‐2 infection in those animals. Since the onset of the COVID‐19 pandemic, natural SARS‐CoV‐2 infections have mainly been confirmed in domestic and production animals, and in wild animals kept in captivity, although the numbers remain limited when compared to human cases. Recently, the first SARS‐CoV‐2 infections in presumably escaped minks found in the wild have been detected, further addressing the much‐feared scenario of transmission of the virus to animals living in the wild and its consequences. Considering the most likely origin of the virus being a wild animal and the putative susceptibilities of free‐ranging wild animal species to SARS‐CoV‐2, the risk of infection with possible establishment of the virus in these populations has to be investigated closely. The authors conclude that most attention should be given to surveillance and awareness‐raising activities for SARS‐CoV‐2 infection in wild mustelids, bats, wild canids and felids, particularly these collected in wildlife rescue centres. People involved in frequent and close contact with wild animals should take all necessary precautionary measures to protect wild animals against exposure to the virus. More than one year after the first detection of SARS‐CoV‐2 in humans, the time has come to increase investments in research and surveillance activities in animals, including in free‐ranging wild animals, as part of a One Health control of this pandemic. This study focussing on Belgium could be helpful for other countries with similar animal densities and ecosystems. original natural hosts for a precursor of SARS-CoV-2 has been put forward with transmission to humans occurring most probably through an intermediate host (Zhao et al., 2020) . Within a few weeks following the outbreak in China, SARS-CoV-2 human infections were reported all over the world. In response to the rapid spread of the virus and the increasing number of cases and deaths, WHO officially declared COVID-19 (Coronavirus disease 2019) as a pandemic at the beginning of March 2020 (WHO, 2020) . Meanwhile, compared to the human cases, a significantly lower number of infections with SARS-CoV-2 has been diagnosed in pets and production animals, in wild animals kept in captivity and more recently in presumably escaped minks found in the wild (OIE, 2020b) . However, anthropozoonotic events (SARS-CoV-2 transmission from humans to animals) raise new concerns for animal health risk or for the establishment of a new animal reservoir . Furthermore, considering particularly the first cases of SARS-CoV-2 infection reported in presumably escaped minks in Utah (Promed, 2020a) and the possibility of mustelids as intermediate host (Zhao et al., 2020) , avoiding the possible establishment of a wild animal reservoir is of great importance. In order to safeguard the health and wellbeing of affected animal species, and of our own, there is need to get a closer look at the risk of SARS-CoV-2 infection in free-ranging wild animal species. The aim of this review is thus to evaluate the putative susceptibilities of different free-ranging wild animal species in Belgium to SARS-CoV-2 and provide a risk assessment of SARS-CoV-2 infection in those animals. The susceptibility of different animal species to SARS-CoV-2 infection has been studied by means of experimental infections, functional and in silico analysis. Angiotensin-converting enzyme 2 (ACE2) is a functional receptor for SARS-CoV-2 (Wan et al., 2020) . Depending on sequence similarity of their respective ACE2 orthologues, several animal species should be susceptible to SARS-CoV-2 infection. Functional analysis on various animal cell lines also revealed a relatively broad host species for SARS-CoV-2 in vitro (Tang et al., 2020) ; but with several species experimentally proven to be not susceptible in vivo, indicating that the receptor is necessary but not sufficient to mediate infection. Endogenous proteases, such as TMPRSS2, with proteolytic activity on S have indeed been shown to participate in membrane fusion activity . At the day of submission (27 January 2021), a limited number of animal species have been shown to be susceptible at variable degree in experimental infection, including non-human primate, ferret, cat, dog, deer mice, raccoon dog, rabbit, hamster, bank voles, white-tailed deer and Egyptian fruit bat (OIE, 2020c) (Fagre et al., 2020; Palmer et al., 2021; Ulrich, Michelitsch, et al., 2020) . Natural infections have been only diagnosed in pet cats and dogs, captive big cats and great apes, in several minks kept in holdings for fur production in different countries and recently in presumably escaped minks (OIE, 2020b). In order to be able to perform a risk assessment of SARS-CoV-2 infection in free-ranging wild animals, we first provide an overview of the several families present in Belgium and their putative susceptibility for infection with SARS-CoV-2. Considering the very limited natural and experimental cases of SARS-CoV-2 infection in wild animals, this information has been deduced from the knowledge from their domestic, production and laboratory counterparts. The wild animal species of the family mustelids (Mustelidae) present in Belgium are the European pine marten (Martes martes), the stone marten (Martes foina), the ermine (Mustela erminea), the badger (Meles meles), the weasel (Mustela nivalis), the polecat (Mustela putorius) and the otter (Lutra lutra) (Libois, 1982; Vercayie et al., 2017; Verkem et al., 2003) . Among these, the stone marten and the badger are the species which are the most brought to rescue centres (source : surveillance network wild fauna University of Liège, Agency Nature and Forest Brussels Region and Brussels Environment). The susceptibility of the ferret (Mustela putorius furo) to SARS-CoV-2 infection has been confirmed by several experimental studies Shi et al., 2020) . Their results showed viral replication at the level of the upper respiratory tract with possibility of isolation of the infectious virus, detection of SARS-CoV-2-genome in the faeces and the lung (without confirmation of infectious virus) and a rise of neutralizing antibodies (titre of 1/128). Beside the transmission of the virus occurring through direct contact between the animals, Kim et al., (2020) suggest the possibility of an airborne transmission. The susceptibility of the farmed mink (Neovison vison) for the virus was confirmed by the first natural infections of minks on a holding in the Netherlands on 23 April 2020 (Promed, 2020b) . Since, SARS-CoV-2 infections in mink holdings have been reported in Denmark, Spain, the United States, Sweden, Italy, Greece, France, Lithuania, Canada and Poland (OIE, 2020b-latest update on 23 February 2021). Independent human-to-mink transmission has been identified as the most probable route of SARS-CoV-2 infection in mink instead of farm to farm transmission. For the infected minks in the Netherlands, besides developing respiratory disease symptoms, a slightly increased mortality was also detected on the farms . At the onset of the outbreak in mink holdings in the Netherlands, the Belgian authorities The wild animal species of the felids family (Felidae) present in Belgium are mostly the stray and feral cat (Felis Catus) with a few European wild cats (Felis sylvestris), and lynxes (Felis lynx) (Libois, 1982; Vercayie et al., 2017; Verkem et al., 2003) . Cats (Felis catus) seem susceptible to SARS-CoV-2 according to studies from Shi et al., (2020) and Halfmann et al., (2020) . A natural infection of a domestic cat has been confirmed in Belgium (Garigliany et al., 2020) and worldwide (OIE, 2020b) . Transmission from infected humans to domestic cats and wild big cats kept in captivity is the most probable route of SARS-CoV-2 infection. The wild animal species of the canids family (Canidae) present in Belgium are mostly the red fox (Vulpes Vulpes), but several raccoon dogs (Nyctereutes procyonoides), and a few grey wolves (Canis lupus) and stray dogs (Canis lupus familiaris) have also been documented (Libois, 1982; Vercayie et al., 2017; Verkem et al., 2003) . Domestic dogs (Canis lupus familiaris) have a very low susceptibility for the virus . In Hong Kong, two dogs (from a cohort of 17) which had been in contact with SARS-CoV-2 positive patients were confirmed positive but did not show any signs of the disease nor did they transmit the virus (Promed, 2020c, d) . However, detection of SARS-CoV-2 infection has also been de- The wild animal species of the bats family (Chiroptera) present in Belgium are listed in Table 1. There is currently no evidence of a natural infection with SARS-CoV-2 in bats. An experimental study using Egyptian fruit bats (Rousettus aegyptiacus), belonging to a family (Pteropodidae) of chiroptera not present in Europe, showed that the animals became infected but did not show any clinical sign of disease. A transient infection of the respiratory tract has been detected, and infectious virus was confirmed in one of the 9 inoculated bats. Intraspecies transmission of the virus to in-contact animals also occurred. The authors therefore stress the risk of SARS-CoV-2 transmission to some free-living bat populations (Schlottau et al., 2020) . As the Egyptian fruit bats are not present in Europe, indigenous bat species in Europe should first be tested for SARS-CoV-2 susceptibility. Furthermore, the Big Brown Bat (Eptesicus fuscus), a North American bat species, was experimentally challenged with SARS-CoV-2 but showed no susceptibility for infection (Hall et al., 2020) . The wild animal species of the lagomorphs family (Lagomorpha) present in Belgium are the Brown Hare (Lepus europaeus) and the Wild Rabbit (Oryctolagus cuniculus) (Libois, 1982; Vercayie et al., 2017; Verkem et al., 2003) . The New-Zealand White rabbit has a low susceptibility for infection with SARS-CoV-2 under experimental conditions (Mykytyn et al., 2020) . The rabbits showed limited clinical signs and the virus was detected with PCR at the level of the nose, throat and rectum while the longest infectious virus shedding was taking place in the nose. Neither virus replication nor transmission between animals was evaluated. The wild animal species of the rodents family (Rodentia) present in Belgium are listed in Table 2 . Syrian or golden hamsters (Mesocricetus auratus) Sia et al., 2020) , deer mice (Peromyscus maniculatus), a New World species not present in Europe (Fagre et al., 2020) and transgenic mice for the receptor ACE2 of human origin (Gu et al., 2020) are very susceptible to the virus. The Syrian or golden hamster is, together with the non-human primates and the ferret, an animal which shows clinical signs very similar to those observed in humans. After experimental infection (through the nose with a viral inoculum of 10 5 plaque-forming units), shallow breathing and weight loss appear as clinical signs. The experiments showed transmission of the virus between hamsters, but this was not investigated in mice. Furthermore, experimental infection of the bank vole showed a very low susceptibility for SARS-CoV-2 infection and no transmission to contact animals (Ulrich, Michelitsch, et al., 2020) . The wild animal species of the Erinaceidae family present in Belgium is the hedgehog (Erinaceus europaeus) (Libois, 1982; Vercayie et al., 2017; Verkem et al., 2003) . for SARS-CoV-2 has not yet been studied. This would be useful since this species is highly present in the rescue centres in Belgium, and it is known that they are susceptible to another Betacoronavirus, being the Erinaceus coronavirus (EriCov). The latter belongs to the same genus Betacoronavirus as SARS-CoV-2 but to a different subgenus, A very low susceptibility for cattle for SARS-CoV-2 infection was confirmed based on the information from the experimental study done by Ulrich et al. (2020) . Also, a recent preprint publication has shown that the white-tailed deer (Odocoileus virginianus) is susceptible to SARS-CoV-2 via intranasal inoculation and can efficiently transmit the virus to other white-tailed deer through indirect contact. However, infected animals do not present clinical disease signs. Their potential status as a reservoir species is unknown and should therefore be investigated (Palmer et al., 2021) . The deer species most related to white-tailed deer in Belgium is the roe deer (Capreolus capreolus). Risk assessments for animal health usually consider three main steps (Dufour et al., 2011) , namely assessment of the release of the hazard from its source, of exposure to the hazard (while release assessment and exposure assessment are further combined to provide with occurrence probability) and the consequence of the occurrence of the hazard. Considering SARS-CoV-2 as the hazard, occurrence of the hazard should be considered as SARS-CoV-2 infection in freeranging wild animals. As such, the risk is mainly based on literature survey and experts advice and can be scaled in a qualitative way from 'very low' to 'high', based on a combination of a 'likelihood assessment scale' and a 'consequence assessment scale'. The infectious virus is mainly present in respiratory secretions of SARS-CoV-2-infected humans, but also from some pets and some production animal species (minks). Isolation of the virus has also been achieved from urine and stool samples from SARS-CoV-2-infected humans (Cuicchi et al., 2020; Jeong et al., 2020) and from stool from experimentally infected ferrets . Therefore, the possibility of presence of SARS-CoV-2 in the natural aquatic environment through waste water treatment plants has also been con- Free-ranging wild animal species are likely to be most exposed to SARS-CoV-2 when in direct or indirect contact with infected humans. Transmission of this respiratory virus mainly occurs through respiratory droplets or airborne aerosols (R. . In comparison with pet and production animals, direct contact between humans (including those infected with SARS-CoV-2) and free-ranging wild animals is very limited. Rodents families and species present in Belgium (Libois, 1982; Vercayie et al., 2017; Verkem et al., 2003) that certain species such as bats, stone martens and hedgehogs have been observed to make their nesting or resting sites in buildings relatively close to humans. Uncertainties exist for the frequency and closeness of contact moments between free-ranging wild animals and pets and production animals. Particular attention should be given to transmission that could occur through predator-prey interactions and when using ferrets at hunting activities for rabbits for example. Uncertainties The occurrence of SARS-CoV-2 infection in free-ranging wild animals may have an impact on the health of both animals and humans. Then, if a reservoir of SARS-CoV-2 would persistently establish in free-ranging wild animal populations, recurring epidemics in the human population could be even more challenging to control. In the context of the outbreaks in mink farms in the Netherlands, a Dutch research team considered it likely that minks and other mustelids could be a true reservoir of SARS-CoV-2, although further research is needed . Given the high susceptibility of the raccoon dog, this animal species could also become a reservoir. The risk of a SARS-CoV-2 infection is the combined result of the assessment of the likelihood that an infection would occur (4 levels : very low, low, high and very high) and the consequence. Two different consequences were considered, either the severity of clinical consequences for individual human or animal health (4 levels : marginal, minor, medium and major), or the potential that the infection would lead to the installation of a reservoir. The combination of likelihood and consequence led to a final appreciation of the risk for infection going from very low, low and moderate to high (see Table 3 below). The risk assessment was performed for the following five possible transmission routes : a) Risk of transmission of SARS-CoV-2 from an infected person to a free-ranging wild animal (see Figure 1- Note: These results and data allow the qualitative assessment of the 'likelihood of occurrence' of the SARS-CoV-2 infection (infectivity for the species as well as transmission potential to congeners) and the 'consequences' of this infection (sensitivity to the infection at the clinical level as well as putative reservoir establishment). The method is described in the Guidelines for the opinions of the Scientific Committee established by the FASFC (http://www.favv-afsca.fgov.be/ scien tific commi ttee/publi catio ns/broch ures/guide lines opini ons/_docum ents/2017-04-19_Guide lines forth eopin ionso ftheS cient ificC ommit tee_en.pdf, adapted) which is based on classical Zepeda matrix (Zepeda & and, 1998 (Zepeda & and, , 2002 and OIE's method for risk at import in animal Health (OIE, 2016) . Currently, the risk linked to the infection of a free-ranging wild animal through transmission from a domestic animal infected with SARS-CoV-2 is qualified as 'very low' to 'low' (for feral cats and wild mustelids through domestic animals other than production minks) and 'moderate' (for wild mustelids through transmission from escaped production minks). Mink industry usually relies on closed buildings. However, infected mink escaping from a contaminated holding is a continuing risk for susceptible animals in the wild. This risk should be taken into account in every country where minks are used for the fur industry. Attention should also be paid to domestic cats infected with SARS-CoV-2 by their owner as they could possibly transmit the virus to stray or feral cats, which in turn could infect surviving prey animals like rodents or bats if susceptible for the virus F I G U R E 1 Risk assessment of SARS-CoV-2 infection in free-ranging wild animals (Ancillotto et al., 2013 ) (De Bruyn et al., 2020 , or production animals such as minks . Anecdotic infection of stray cats has already been reported during the outbreaks in the mink farms in the Netherlands even if the infection route for these infections has not been clarified (final report Dutch government, 2020). c) Risk of SARS-CoV-2 Transmission from free-ranging wild animals to humans (see Figure 1 -transmission route C): In the context of the current human pandemic, the risk for humans getting infected with SARS-CoV-2 via wild animals is Based on the current state of knowledge, most attention should be paid to wild mustelids, bats, wild felids and canids. Following the reported risk of SARS-CoV-2 infection in free-ranging wild animals, the following recommendations can be made concerning surveillance and awareness raising : • For all activities at risk involving human-to-animal contacts, The authors declare no conflicts of interest. The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. 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