Susceptibility of sheep to experimental co-infection with the ancestral lineage of SARS-CoV-2 and its alpha variant

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for a global pandemic that has had significant impacts on human health and economies worldwide. SARS-CoV-2 is highly transmissible and the cause of coronavirus disease 2019 (COVID-19) in humans. A wide range of animal species have also been shown to be susceptible to SARS-CoV-2 infection by experimental and/or natural infections. Domestic and large cats, mink, ferrets, hamsters, deer mice, white-tailed deer, and non-human primates have been shown to be highly susceptible, whereas other species such as mice, dogs, pigs, and cattle appear to be refractory to infection or have very limited susceptibility. Sheep (Ovis aries) are a commonly farmed domestic ruminant that have not previously been thoroughly investigated for their susceptibility to SARS-CoV-2. Therefore, we performed in vitro and in vivo studies which consisted of infection of ruminant-derived cell cultures and experimental challenge of sheep to investigate their susceptibility to SARS-CoV-2. Our results showed that sheep-derived cell cultures support SARS-CoV-2 replication. Furthermore, experimental challenge of sheep demonstrated limited infection with viral RNA shed in nasal and oral swabs primarily at 1-day post challenge (DPC), and also detected in the respiratory tract and lymphoid tissues at 4 and 8 DPC. Sero-reactivity was also observed in some of the principal infected sheep but not the contact sentinels, indicating that transmission to co-mingled naïve sheep was not highly efficient; however, viral RNA was detected in some of the respiratory tract tissues of sentinel animals at 21 DPC. Furthermore, we used challenge inoculum consisting of a mixture of two SARS-CoV-2 isolates, representatives of the ancestral lineage A and the B.1.1.7-like alpha variant of concern (VOC), to study competition of the two virus strains. Our results indicate that sheep show low susceptibility to SARS-CoV-2 infection, and that the alpha VOC outcompeted the ancestral lineage A strain.

First serological evidence of SARS-CoV-2 natural infection in small ruminants

“…They found no infectious virus shedding and no viral RNA was detectable by nasal-oral swabs in sheep, while 2 of 3 goats showed positive results by RT-PCR. Moreover, challenged small ruminants show anti-SARS-CoV-2 detectable antibodies but for a short time after infection and low titers of neutralizing antibodies (Gaudreault et al 2021;Bosco-Lauth et al 2021). Altogether, these observations have led to the conclusion that sheep and goats have low susceptibility to SARS-CoV-2 and natural infection is unlikely to occur (Gaudreault et al 2021;Villanueva-Saz et al, 2021;Bosco-Lauth et al, 2021).…”

“…Thus, the absence or limited clinical signs may lead to underdiagnosed cases of SARS-CoV-2 infection in animal species. Furthermore, it has been demonstrated by experimental and natural infections, that positive animals show low amounts of viral shedding, during a very short time (Murphy and Ly, 2021;Ulrich et al, 2020;Falkenberg et al, 2021;Gaudreault et al 2021;Cardillo et al, 2022;Fiorito et al, 2022). Therefore, serological examinations, revealing a previous viral infection, are a useful tool to investigate SARS-CoV-2 host range and prevalence in animal species, and, in particular, virus serum neutralization assay (VSN), that is species-independent and is based on the principle of in vitro interaction between specific neutralizing antibodies and the investigated virus (Wernike et al, 2020).…”

“…The susceptibility of domestic ruminants to SARS-CoV-2 is currently under investigated, albeit these species are of particular interest since, as for other species, cattle and sheep ACE2 shows affinity to SARS-CoV-2 RBD (Zhai et al, 2020: Di Teodoro et al, 2021 and, in particular, livestock are often in close contact with farmworkers and, during grazing, with potential known SARS-CoV-2 wildlife reservoirs (Gaudreault et al 2021;Fiorito et al, 2022), such as white-tailed deer in North America (Hale et al, 2022), the recently reported Asian small-clawed and Eurasian otters, (Padilla-Blanco et al, 2022) and/or still unknown susceptible feral species (WOAH, 2022c). Particular concerns have also been highlighted by Lam and colleagues (2020), who, in order to predict infection risks, analyzed a modelling S-protein:ACE2 complex from sheep, by calculating energetic changes of the complex due to species-specific mutations, as well as by examining chemical-physical affinity due to specific groups.…”

“…Interestingly, it has been demonstrated that ACE2 orthologs of some small ruminants allow cell entry of SARS-CoV-2, indicating that sheep and goats may harbor as well as spread the virus (Li et al, 2020). Furthermore, an in silico study evaluating the interaction between ACE2 receptor and the S protein, has evidenced the susceptibility of sheep (Damas et al, 2020) to SARS-CoV-2 infection and, later, by ex vivo organ cultures (EVOCs) and in vitro experiments, it has been observed that the ovine ACE2-expressing cells of the respiratory tract are able to sustain viral replication (Gaudreault et al 2021;Di Teodoro et al, 2021). To date, no natural infection in sheep and goats has been observed.…”

Serological screening in wild ruminants in Germany, 2021/22: No evidence of SARS-CoV-2, bluetongue virus or pestivirus spread but high seroprevalences against Schmallenberg virus

“…Natural SARS-CoV-2 infections linked to human exposure have been reported in American mink, ferrets, felines, canines and primates [34]. For domestic ruminants such as cattle, goat and sheep a very low susceptibly was demonstrated during experimental infection studies [35][36][37], and natural infections of cattle also seem to be a rare event [38]. However, SARS-CoV-2 or specific antibodies were detected frequently in freeranging white-tailed deer (Odocoileus virginianus) in North America [29][30][31], but it remains unclear how these deer acquired the virus from humans.…”