Severe acute respiratory syndrome coronavirus‐2 natural animal reservoirs and experimental models: systematic review

Summary The current severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) outbreak has been rapidly spreading worldwide, causing serious global concern. The role that animal hosts play in disease transmission is still understudied and researchers wish to find suitable animal models for fundamental research and drug discovery. In this systematic review, we aimed to compile and discuss all articles that describe experimental or natural infections with SARS‐CoV‐2, from the initial discovery of the virus in December 2019 through to October 2020. We systematically searched four databases (Scopus, PubMed, Science Direct and Web of Science). The following data were extracted from the included studies: type of infection (natural or experimental), age, sample numbers, dose, route of inoculation, viral replication, detection method, clinical symptoms and transmission. Fifty‐four studies were included, of which 34 were conducted on animal reservoirs (naturally or experimentally infected), and 20 involved models for testing vaccines and therapeutics. Our search revealed that Rousettus aegyptiacus (fruit bats), pangolins, felines, mink, ferrets and rabbits were all susceptible to SARS‐CoV‐2, while dogs were weakly susceptible and pigs, poultry, and tree shrews were not. In addition, virus replication in mice, mink, hamsters and ferrets resembled subclinical human infection, so these animals might serve as useful models for future studies to evaluate vaccines or antiviral agents and to study host‐pathogen interactions. Our review comprehensively summarized current evidence on SARS‐CoV‐2 infection in animals and their usefulness as models for studying vaccines and antiviral drugs. Our findings may direct future studies for vaccine development, antiviral drugs and therapeutic agents to manage SARS‐CoV‐2‐caused diseases.


| INTRODUCTION
The outbreak of coronavirus disease 2019  caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has triggered a serious global public health problem and is spreading in almost all countries around the globe with 45,797,600 confirmed cases and 1,191,670 mortalities, as of 30 October 2020. 1 The current outbreak of SARS-CoV-2 is the third outbreak of animal coronavirus transmission to humans in the past 2 decades. SARS-CoV-2 belongs to the coronaviruses (CoVs), a genus in the Coronaviridae family. Most known CoVs infect animals, are associated with self-limiting diseases and have a narrow host range. 2 However, some zoonotic CoVs have a high capability to mutate, which enables the virus to cross the species barrier to infect humans, such as SARS-CoV, middle east respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2 zoonotic viruses, which have caused severe acute respiratory diseases in humans. [3][4][5] These zoonotic CoVs have claimed thousands of lives in the last 2 decades. 4 Fortunately, compared to SARS-CoV and MERS-CoV, SARS-CoV-2 is less pathogenic; however, the concern lies in the fact that it is highly infectious and transmissible. Moreover, asymptomatic Covid-19 patients have been shown to contribute to the transmission of the infection around the world. 6,7 Recent reports and studies of SARS-CoV-2 in animals have caused unnecessary fear and panic among the population, which have adversely affected the wellbeing and health of animals. Most importantly, it is unethical to cull any animals (wild or domestic) by declaring they are 'potential' reservoir hosts without any conclusive evidence that they can transmit SARS-CoV-2 to humans. Furthermore, finding the best animal models that mimic human Covid-19 infection is vital for scientists to understand the pathogenesis of SARS-CoV-2 infection. In addition, animal models are critical for testing potential vaccines and therapeutic agents. 8 A suitable animal model is one that can mimic human disease in terms of viral load, morbidity, clinical signs, and the immune response. Due to the urgency and demand in the current crisis, there is a pressing need to search for a good animal model to control the SARS-CoV-2 outbreak. 9 In this systematic review, we provide a summary of the current evidence on natural and experimental infections of SARS-CoV-2 in animals. We compare different animals for their susceptibility, clinical signs, transmission dynamics, and immune response. We also discuss the ability of these animals to mimic Covid-19 pathogenesis in humans, as well as their usefulness as ideal models for preclinical testing of vaccine candidates and therapeutic agents.

| METHODOLOGY
This systematic review was performed according to the preferred reporting items for systematic reviews and meta-analyses (PRISMA) statement. 10

| Eligibility criteria
We included published articles that reported infection in domestic or wild animals with serological or molecular confirmation of SARS-CoV-2. Inclusion criteria included: (1) investigation of the management of SARS-CoV-2 and (2) natural or experimentally induced SARS-CoV-2-infected animal species. Exclusion criteria included (1) infection not clearly defined as SARS-CoV-2, (2) non-animal models, (3) reviews, meta-analyses, book reviews, letters, abstracts and cases and (4) non-English reports.

| Study selection
Four reviewers participated in screening and study selection, of which, three reviewers individually reviewed the identified studies (Salma Younes, Nadin Younes and Farah Shurrab), and a fourth reviewer (Gheyath K. Nasrallah) resolved any discrepancies. All studies retrieved from our initial search strategy were imported to Endnote X9 library, after which, duplicates were removed. Figure 1 illustrates the search strategy. Studies were screened in two stages. The first stage of screening involved screening of titles, abstracts, and keywords. The

| Data collection
Two reviewers (Salma Younes and Nadin Younes) extracted the following data from each included study: animal used, age, number of animals used, type of infection (natural/experimental), inoculation dose, route of challenge, sample, method of detection, immune response, disease and pathology (symptoms), and the possibility of animal-animal transmission (Table S1). For vaccine and therapeutic studies on animal models infected with SARS-CoV-2; the following information were extracted and recorded: animal model, age, number of animals used, inoculation dose, route of challenge, vaccine/antiviral agent, dose, route of inoculation, sample type, method of detection, disease and pathology (symptoms), and the main outcomes of each study (Table S2). The information collected was reviewed independently by all authors to ensure accurate data collection.

| Search findings and study characteristics
We identified 19,012 articles, of which 14,068 remained after the removal of duplicates. Thirteen thousand eight hundred eighty-one irrelevant articles were excluded during the initial stage of screening and 148 articles were excluded during the second stage ( Figure 1).
The majority of the excluded studies were mainly in vivo human studies with no data on experimental or natural animal species. A total of 54 articles were included in the systematic review.

| Animal species and characteristics
The studies collected using the systematic search used various animal species infected with SARS-CoV-2 experimentally or naturally. Most of the studies were conducted on mice (37%) and NHPs (30%), fewer studies were conducted on felines, poultry, dogs, ferrets, fruit bats, minks, hamsters, pigs, rabbits, and tree shrews as shown in Figure 2.
A consolidative summary of the data gathered from all studies F I G U R E 1 Prisma flow diagram of the systematic review study selection process. The search strategy yielded 19,012 studies. A total of 54 studies were included in the systematic review. SARS-CoV-2, severe acute respiratory syndrome coronavirus-2 YOUNES ET AL. included in this systematic review is given in Table 1. The data collected from each study included in this systematic review are presented in Table S1 and Table S2.

| SARS-CoV-2 infection in animals
There is a strong evidence that SARS-CoV-2 virus originated in bats 64 ; however, the intermediate animal host is still unknown ( Figure 3). Several studies showed that pangolins, snakes, turtles, and mink are all possible intermediate hosts, however, further investigations are needed. [66][67][68] It is worth mentioning that recent findings that analyse the probable animal reservoir to Covid-19 suggest the snake as a reservoir, based on relative synonymous codon usage bias. 67 However, the missing link or intermediate link for animal to human transmission of SARS-CoV-2 from a recent study suggests the DNA and protein sequence of Malayan pangolins. 69 Studies included in our systematic search revealed low SARS-CoV-2 replication in pigs, dogs, chickens, ducks, rabbits and treeshrews ( Table 1, Table S1 and Figure 4). However, Rousettus aegyptiacus (fruit bats), pangolins, felines, mink, ferrets and rabbits were all susceptible to SARS-CoV-2 ( Table 1, Table S1 and Figure 4).
Additionally, tigers, ferrets, mink, macaques, lions, hamsters and cats are susceptible to animal-to-animal transmission through the airborne route (Table 1 and Table S1). Moreover, susceptible animals are capable of producing neutralizing antibodies (NAbs) that can offer protection against reinfection (Table S1); however, it remains unclear how long protection will last.

| Zoonotic links of SARS-CoV-2 outbreak
SARS-CoV-2 was first identified in Wuhan city in China in hospitalized patients who previously visited the Huanan wet seafood market where various animals including chickens, pigs, pangolins, bats, snakes, frogs, rabbits, and marmots are sold for human consumption, proposing a suitable environment for zoonotic infection spill-over to humans. 67,[70][71][72][73] Scientists believe that these traditional Chinese practices might be responsible for the SARS-CoV-2 pandemic in humans and that the recurrent interactions between humans and animals without proper biosafety measures present a substantial risk for the occurrence of zoonotic diseases. 74 Zoonotic CoVs have crossed the species barrier twice in the past 2 decades (SARS-CoV; 2002 and MERS-CoV; 2012). Thus, scientists F I G U R E 2 Characteristics of the included studies. Articles obtained from the systematic search were categorized based on the animal used in each study. Most of the studies were conducted on mice and NHPs. Fewer studies were conducted on felines, poultry, dogs, ferrets, fruit bats, minks, hamsters, pigs, rabbits, and tree shrews. Some studies included more than one animal; NHPs, non-human primates have speculated that SARS-CoV-2 resulted from a zoonotic spillover event as well. Zoonotic CoVs need to propagate in their zoonotic reservoirs, and then, seek the chances to spillover via intermediate hosts into susceptible human targets, where they can maintain humanto-human transmission. Bats have been revealed as the natural hosts for several human CoVs, including HCoV-NL63, HCoV-229E, SARS-CoV, and MERS-CoV. 75,76 Genome sequence analysis confirmed that SARS-CoV-2 is 96% identical to the bat CoV RaTG13 at the wholegenomic level, 77 and hence bats are believed to be the primary source of origin for the novel SARS-CoV-2. However, the intermediate host that is yet to be elucidated. 11,67,70,72,78 Researchers proposed two hypotheses for the emergence of SARS-CoV-2: (1) Natural selection may have occurred in an animal host before transmission to mankind; and (2) natural selection of viruses may have occurred in humans after zoonotic transmission. 79 In this regard, studies involving the use of animal models or cell culture are in need to help clarify these two scenarios. 79

| SARS-CoV-2 infection in animals
Infections with SARS-CoV-2 have been documented in various animal species naturally or by experimental infection (Table 1 and   Table S1). Our systematic search revealed that R. aegyptiacus fruit bats, pangolins, felines, minks, NHPs, hamsters, ferrets and rabbits are all susceptible to SARS-CoV-2, dogs are weakly susceptible, whereas pigs, poultry, and tree shrews are not susceptible to SARS-CoV-2 infection (Table 1 and Table S1).

| Animals that are susceptible to SARS-CoV-2
Bats SARS-CoV and MERS-CoV originated in bats and were capable of infecting humans. 80,81 Bats are known to be the ideal hosts for pathogenic CoVs, as viruses remain persistent in asymptomatic T A B L E 1 Summary of findings of SARS-CoV-2 infection in animals bats. 82 Accordingly, researchers predicted the role of bats as the origin of the current SARS-CoV-2 outbreak. 68,[82][83][84] Several studies showed that SARS-CoV-2 is virtually identical to the bat CoV on a genomic level, hence it is assumed that bats are the primary source of the SARS-CoV-2 spillover. 83,85 Experimental studies revealed that R. aegyptiacus fruit bats are susceptible to SARS-CoV-2, as detected by RT-qPCR, immunohistochemistry, and in situ hybridisation in the nasal cavity of intranasally SARS-CoV-2-inoculated fruit bats 86 (Table 1 and Table S1).

Pangolins
Several studies showed evidence based on metagenomic sequencing analysis that pangolins (Manis javanica), a group of endangered mammals, may harbour the ancestral beta-CoVs, which is the second closest relative SARS-CoV-2 virus. 29

Felines
There have been several reports of human-to-feline transmission of SARS-CoV-2 (Table 1 and Table S1). Cats from Belgium and Hong Kong tested SARS-CoV-2-positive. 87 Cats were shown to be susceptible to SARS-CoV-2 and that they may transmit the virus to other cats in close-contact. 88 (Table 1 and Table S1).

Mink
To date, there has been one report of mink infection with SARS-CoV-2. 13  including interstitial pneumonia. 13 Investigations later revealed that workers at the farm had previously tested positive for Covid-19, suggestive of human-to-animal transmission ( Table 1 and Table S1).

Non-human primates
It has been shown that all NHPs are susceptible to previous SARS-CoV infection where symptoms of fever, diarrhoea and pneumonitis were reported. 91,92 Recent studies have shown that rhesus macaques are susceptible to SARS-CoV-2 infection. The virus was found to cause mild pneumonia similar to that in humans. 30 Additionally, infected rhesus macaques developed NAbs that may protect them from subsequent infections. 30 Other NHPs, such as cynomolgus macaques and common marmosets were shown to be susceptible to SARS-CoV-2 infection 29 (Table 1 and   Table S1). However, comparing the susceptibility of rhesus macaques to cynomolgus macaques and common marmosets, the former showed higher susceptibility to SARS-CoV-2 infection. It is worth mentioning that viral replication of nasopharyngeal swabs, anal swabs and lung in old monkeys was more active than that in young monkeys for 14 days after SARS-CoV-2 challenge. 31 In addition, old monkeys exhibited diffuse severe interstitial pneumonia compared to young monkeys. Interestingly, a notably higher percentage of lymphocytes was observed in younger monkeys F I G U R E 4 Animal host classification according to susceptibility. NHPs, hamsters, transgenic mice, felines, ferrets, minks, pangolins, and rabbits have high susceptibility, dogs have low susceptibility, while pigs, poultry, and tree shrews have no susceptibility to SARS-CoV-2 infection; SARS-CoV-2, severe acute respiratory syndrome coronavirus-2 YOUNES ET AL.
-7 of 13 compared to older monkeys, but the decreased counts of lymphocytes were found in both age groups. 31

Hamsters
Several studies have shown that hamsters are susceptible to SARS-CoV-2 infection (Table 1 and Table S1). Golden Syrian hamsters infected with SARS-CoV-2 developed clinical signs, such as lethargy, ruffled fur, hunched back, abnormal breathing and weight loss. [21][22][23] In addition, viral transmission to naive hamsters through direct contact was described. 22 Of particular interest is the fact that infection with SARS-CoV-2 in hamsters reflects some of the

Rabbits
A recent study showed that SARS-CoV-2 can infect rabbits, and thus they may serve as potential sources of animal to human transmission (Table 1 and Table S1). 45 Nevertheless, whether or not rabbits are naturally susceptible to the virus remains under investigation.

| Animals that are weakly/not susceptible to SARS-CoV-2
Dogs Under natural conditions, there have been few reports of dogs that have tested SARS-CoV-2-positive, of which none reported that dogs could pose as a source of infection for humans (Table 1 and  11 and thus, failed to show potential for serving as animal models of SARS-CoV-2 infection (Table 1 and Table S1).

Pigs
A recent study was conducted to investigate whether pigs are susceptible to SARS-CoV-2 or not (Table 1 and Table S1). Swabs were found to be free from viral RNA, in addition, no viral RNA was detected in any of the naïve animals in contact, indicating that pigs  (Table 1 and Table S1). Viral RNA was not detected in swabs taken from SARS-CoV-2-inoculated chickens and ducks. In addition, viral RNA was not detected in any of the naïve animals that were in close contact, indicating no animal-to-animal transmission.
Furthermore, the inoculated chickens and ducks were found to be seronegative when tested by ELISA. 11

Tree shrews
Experimental infection of tree shrews with SARS-CoV-2 has been described 63 (Table 1 and Table S1). No clinical signs were observed in SARS-CoV-2-inoculated tree shrews except high body temperature, 63 and thus, may not be a suitable animal for Covid-19 research.

| Potential animal models that are still under research
Other animals are still under investigation whether or not they are susceptible to SARS-CoV-2 infection and if they can be useful as models. In Silico Analysis revealed that wild felidae, goats, spotted hyenas and civets may be SARS-CoV-2-susceptible and could potentially act as intermediate hosts to transmit the virus from bats to humans. 95 These findings highlight the usefulness of these animals to be used as experimental animal models for studying SARS-CoV-2 infection directly without the need for transgenic operation. 95 It is noteworthy to mention that rodents, birds, and reptiles are unlikely to be susceptible to SARS-CoV-2, 95 however, further investigation is needed.

| Potential animal models to study vaccine and therapeutic candidates for SARS-CoV-2 infection
Not every susceptible animal can serve as a good model for testing vaccine and therapeutic candidates. A good animal model is defined by its ability to reproduce relevant human physiology to be used for investigating the effectiveness of potential therapeutic agents and pharmacological drugs. Thus, the identification of reliable and

| Transgenic mice
Mouse models are known to be convenient models to study a disease because they are cheap, available and easy to manipulate. However, these models are challenging to study SARS-CoV-2 due to differences in the usage pattern of angiotensin-converting enzyme 2 (ACE2) receptor compared to human, 96

| Ferrets
Ferrets have been widely used to study viral respiratory diseases in humans and remain one of the best candidates as an animal model for respiratory tract infections as they display most of the human-like symptoms. 71 Unlike mice, ferrets can be infected with the virus without prior transgenic adaptation. Ferrets were used as animal models for evaluating the effectiveness of antiviral drugs against influenza, 100 SARS-CoV 101 and Ebola. 102 Thus, researchers believe that ferrets may help in the race for finding the best therapeutic drugs and vaccines against SARS-CoV-2. A recent study that investigated the efficacy of different antivirals agents using infected ferrets, has revealed a reduction in the overall clinical scores following treatment with three antiviral agents; lopinavir-ritonavir, hydroxychloroquine sulphate, and emtricitabine-tenofovir 18 (Table S2).

| Non-human primates
NHPs are considered as one of the best animal models to investigate potential antiviral drugs, therapeutic agents, and to gain a better understanding of the aspects related to pathology in different infectious diseases. 103

| Other animals
Other animals that have been used for vaccine studies in Covid-19 research include pigs and rabbits (Table S2). However, there is a lack YOUNES ET AL. of studies on these animals, and thus, further investigation on the usefulness of these animals as models is needed. hamsters and ferrets may serve as a useful model for studying the virus transmission and the efficacy of antiviral drugs to limit the spread of the infection. Most importantly, the NHPs were demonstrated to be the most relevant animal model to assess the antiviral agents and vaccine effectiveness before rapid deployment to clinical trials on humans. Although there are more than 180 vaccine candidates and more than 23 well-known pharmaceutical companies that are trying to produce an effective vaccine against SARS-CoV-2, until now, no vaccines were finally approved for human use. However, the current literature data so far proposed that safe vaccines could be available within the coming few months. For more information about vaccine development, the readers are advised to refer back to this comprehensive review and website. 106,107 It is clear now that among all virus proteins, the most antigenic target for successful active or passive vaccination against all coronavirus, including SARS-CoV-2, is the S1 subunit of the spike S proteins; more specifically, the RBDs of the S1 subunit. That is, if a vaccinated host produces NAbs to the RBD, it will block virus binding and, consequently, viral replication. In fact, it was shown that passive vaccination with a cocktail of humanized monoclonal antibodies targeting the RBD has shown effectiveness in the treatment of Covid-19 patients. 108 The RBD viral antigen could be injected in a form of self-replicating mRNA or a recombinant protein. Different delivery systems are currently being used in clinical trials for delivering the virus RNA or the recombinant protein such as the lipid nanoparticles or the non-pathogenic adenovirus associated vector. Moreover, other companies are also trying to produce live attenuated (a genetically weakened version of the virus) or chemically inactivated whole virus vaccine. 106 Finally, our systematic search revealed that viral shedding from pets is not sufficient to infect other members of the family or other animals. However, susceptible animals could serve as reservoirs of the virus, necessitating careful ongoing animal management and surveillance.

ACKNOWLEDGMENTS
Open access funding provided by the Qatar National Library. This The statements made herein are solely the responsibility of the authors.