Regulatory T Cell-like Response to SARS-CoV-2 in Jamaican Fruit Bats Transduced with Human ACE2 (preprint)

Insectivorous Old World horseshoe bats (Rhinolophus spp.) are the likely source of the ancestral SARS-CoV-2 prior to its spillover into humans and causing the COVID-19 pandemic. Natural coronavirus infections of bats appear to be principally confined to the intestines, suggesting fecal-oral transmission; however, little is known about the biology of SARS-related coronaviruses in bats. Previous experimental challenges of Egyptian fruit bats (Rousettus aegyptiacus) resulted in limited infection restricted to the respiratory tract, whereas insectivorous North American big brown bats (Eptesicus fuscus) showed no evidence of infection. In the present study, we challenged Jamaican fruit bats (Artibeus jamaicensis) with SARS-CoV-2 to determine their susceptibility. Infection was confined to the intestine for only a few days with prominent viral nucleocapsid antigen in epithelial cells, and mononuclear cells of the lamina propria and Peyers patches, but with no evidence of infection of other tissues; none of the bats showed visible signs of disease or seroconverted. Expression levels of ACE2 were low in the lungs, which may account for the lack of pulmonary infection. Bats were then intranasally inoculated with a replication-defective adenovirus encoding human ACE2 and 5 days later challenged with SARS-CoV-2. Viral antigen was prominent in lungs for up to 14 days, with loss of pulmonary cellularity during this time; however, the bats did not exhibit weight loss or visible signs of disease. From day 7, bats had low to moderate IgG antibody titers to spike protein by ELISA, and one bat on day 10 had low-titer neutralizing antibodies by a VSV pseudotyped virus assay. CD4+ helper T cells became activated upon ex vivo recall stimulation with SARS-CoV-2 nucleocapsid peptides and exhibited elevated mRNA expression of the regulatory T cell cytokines interleukin-10 and transforming growth factor beta, which may have limited inflammatory pathology. Collectively, these data show that Jamaican fruit bats are of low susceptibility to SARS-CoV-2 but that expression of human ACE2 in their lungs leads to robust infection and an adaptive immune response with low-titer antibodies and a regulatory T cell-like response that may explain the lack of prominent inflammation in the lungs. This model will allow for insights of how SARS-CoV-2 infects bats and how bat innate and adaptive immune responses engage the virus without overt clinical disease.

Ancestral lineage of SARS-CoV-2 is more stable in human biological fluids than Alpha, Beta and Omicron variants of concern (preprint)

SARS-CoV-2 is a zoonotic virus which was first identified in 2019, and has quickly spread worldwide. The virus is primarily transmitted through respiratory droplets from infected persons; however, the virus-laden excretions can contaminate surfaces which can serve as a potential source of infection. Since the beginning of the pandemic, SARS-CoV-2 has continued to evolve and accumulate mutations throughout its genome leading to the emergence of variants of concern (VOCs) which exhibit increased fitness, transmissibility, and/or virulence. However, the stability of SARS-CoV-2 VOCs in biological fluids has not been thoroughly investigated so far. The aim of this study was to determine and compare the stability of different SARS-CoV-2 strains in human biological fluids. Here, we demonstrate that the ancestral strain of Wuhan-like lineage A was more stable than the Alpha VOC B.1.1.7, and the Beta VOC B.1.351 strains in human liquid nasal mucus and sputum. In contrast, there was no difference in stability among the three strains in dried biological fluids. Furthermore, we also show that the Omicron VOC B.1.1.529 strain was less stable than the ancestral Wuhan-like strain in liquid nasal mucus. These studies provide insight into the effect of the molecular evolution of SARS-CoV-2 on environmental virus stability, which is important information for the development of countermeasures against SARS-CoV-2. ImportanceGenetic evolution of SARS-CoV-2 leads to the continuous emergence of novel variants, posing a significant concern to global public health. Five of these variants have been classified so far into variants of concern (VOCs); Alpha, Beta, Gamma, Delta, and Omicron. Previous studies investigated the stability of SARS-CoV-2 under various conditions, but there is a gap of knowledge on the survival of SARS-CoV-2 VOCs in human biological fluids which are clinically relevant. Here, we present evidence that Alpha, Beta, and Omicron VOCs were less stable than the ancestral Wuhan-like strain in human biological fluids. Our findings highlight the potential risk of contaminated human biological fluids in SARS-CoV-2 transmission and contribute to the development of countermeasures against SARS-CoV-2.

Detection of SARS-CoV-2 Omicron variant (B.1.1.529) infection of white-tailed deer (preprint)

White-tailed deer (Odocoileus virginianus) are highly susceptible to infection by SARS-CoV-2, with multiple reports of widespread spillover of virus from humans to free-living deer. While the recently emerged SARS-CoV-2 B.1.1.529 Omicron variant of concern (VoC) has been shown to be notably more transmissible amongst humans, its ability to cause infection and spillover to non-human animals remains a challenge of concern. We found that 19 of the 131 (14.5%; 95% CI: 0.10-0.22) white-tailed deer opportunistically sampled on Staten Island, New York, between December 12, 2021, and January 31, 2022, were positive for SARS-CoV-2 specific serum antibodies using a surrogate virus neutralization assay, indicating prior exposure. The results also revealed strong evidence of age-dependence in antibody prevalence. A significantly ({chi}2, p < 0.001) greater proportion of yearling deer possessed neutralizing antibodies as compared with fawns (OR=12.7; 95% CI 4-37.5). Importantly, SARS-CoV-2 nucleic acid was detected in nasal swabs from seven of 68 (10.29%; 95% CI: 0.0-0.20) of the sampled deer, and whole-genome sequencing identified the SARS-CoV-2 Omicron VoC (B.1.1.529) is circulating amongst the white-tailed deer on Staten Island. Phylogenetic analyses revealed the deer Omicron sequences clustered closely with other, recently reported Omicron sequences recovered from infected humans in New York City and elsewhere, consistent with human to deer spillover. Interestingly, one individual deer was positive for viral RNA and had a high level of neutralizing antibodies, suggesting either rapid serological conversion during an ongoing infection or a breakthrough infection in a previously exposed animal. Together, our findings show that the SARS-CoV-2 B.1.1.529 Omicron VoC can infect white-tailed deer and highlights an urgent need for comprehensive surveillance of susceptible animal species to identify ecological transmission networks and better assess the potential risks of spillback to humans.

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

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 naive sheep was not highly efficient; hovewer, 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.

Effects of Spike Mutations in SARS-CoV-2 Variants of Concern on Human or Animal ACE2-Mediated Virus Entry and Neutralization (preprint)

SARS-CoV-2 is a zoonotic agent capable of infecting humans and a wide range of animal species. Over the duration of the pandemic, mutations in the SARS-CoV-2 Spike protein (S) have arisen in circulating viral populations, culminating in the spread of several variants of concern (VOC) with varying degrees of altered virulence, transmissibility, and neutralizing antibody escape. In this study, we employed lentivirus-based pseudotyped viruses that express specific SARS-CoV-2 S protein substitutions and cell lines that stably express ACE2 from nine different animal species to gain insights into the effects of VOC mutations on viral entry and antibody neutralization capability. All animal ACE2 receptors tested, except mink, support viral cell entry for pseudoviruses expressing the parental (prototype Wuhan-1) S at levels comparable to human ACE2. Most single S substitutions (e.g., 452R, 478K, 501Y) did not significantly change virus entry, although 614G and 484K resulted in a decreased efficiency in viral entry. Conversely, combinatorial VOC substitutions in the S protein were associated with significantly increased entry capacity of pseudotyped viruses compared to that of the parental Wuhan-1 pseudotyped virus. Similarly, infection studies using live ancestral (USA-WA1/2020), Alpha, and Beta SARS-CoV-2 viruses in hamsters revealed a higher replication potential for the Beta variant compared to the ancestral prototype virus. Moreover, neutralizing titers in sera from various animal species, including humans, were significantly reduced by single substitutions of 484K or 452R, double substitutions of 501Y-484K, 452R-484K and 452R-478K and the triple substitution of 501Y-484K-417N, suggesting that 484K and 452R are particularly important for evading neutralizing antibodies in human, cat, and rabbit sera. Cumulatively, this study reveals important insights into the host range of SARS-CoV-2 and the effect of recently emergent S protein substitutions on viral entry, virus replication and antibody-mediated viral neutralization. Author summaryCells stably expressing ACE2 from various animals and a lentivirus-based SARS-CoV-2 pseudotyped virus assay were established to study SARS-CoV-2 cell entry. The results demonstrated that ACE2 from a wide range of animal species facilitate S-mediated virus entry into cells, which is supported by in silico data as well as natural and experimental infection studies. Pseudotyped viruses containing mutations in the RBD of S representative of the Alpha, Gamma, and especially Beta, variants of concern demonstrated that certain mutations are associated with increased viral entry compared to the parental S. The Beta variant was also observed to have a replicative advantage in vitro and in vivo compared to the prototype virus. Pseudotyped viruses containing combinatorial substitutions of 501Y-484K-417K, 614G-501Y-484K and 614G-501Y-484K-417N increased viral entry via ACE2 across multiple species. The 501Y or 478K single substitution did not significantly affect neutralizing capacity of immune sera compared to the prototype strain, but the addition of 484K or 452R substitutions significantly reduced the neutralizing titers.

Infection and transmission of SARS-CoV-2 and its alpha variant in pregnant white-tailed deer (preprint)

SARS-CoV-2, a novel Betacoronavirus, was first reported circulating in human populations in December 2019 and has since become a global pandemic. Recent history involving SARS-like coronavirus outbreaks (SARS-CoV and MERS-CoV) have demonstrated the significant role of intermediate and reservoir hosts in viral maintenance and transmission cycles. Evidence of SARS-CoV-2 natural infection and experimental infections of a wide variety of animal species has been demonstrated, and in silico and in vitro studies have indicated that deer are susceptible to SARS-CoV-2 infection. White-tailed deer (Odocoileus virginianus) are amongst the most abundant, densely populated, and geographically widespread wild ruminant species in the United States. Human interaction with white-tailed deer has resulted in the occurrence of disease in human populations in the past. Recently, white-tailed deer fawns were shown to be susceptible to SARS-CoV-2. In the present study, we investigated the susceptibility and transmission of SARS-CoV-2 in adult white-tailed deer. In addition, we examined the competition of two SARS-CoV-2 isolates, representatives of the ancestral lineage A (SARS-CoV-2/human/USA/WA1/2020) and the alpha variant of concern (VOC) B.1.1.7 (SARS-CoV-2/human/USA/CA_CDC_5574/2020), through co-infection of white-tailed deer. Next-generation sequencing was used to determine the presence and transmission of each strain in the co-infected and contact sentinel animals. Our results demonstrate that adult white-tailed deer are highly susceptible to SARS-CoV-2 infection and can transmit the virus through direct contact as well as vertically from doe to fetus. Additionally, we determined that the alpha VOC B.1.1.7 isolate of SARS-CoV-2 outcompetes the ancestral lineage A isolate in white-tailed deer, as demonstrated by the genome of the virus shed from nasal and oral cavities from principal infected and contact animals, and from virus present in tissues of principal infected deer, fetuses and contact animals.

SARS-CoV-2 variants of concern have acquired mutations associated with an increased spike cleavage (preprint)

For efficient cell entry and membrane fusion, SARS-CoV-2 spike (S) protein needs to be cleaved at two different sites, S1/S2 and S2 by different cellular proteases such as furin and TMPRSS2. Polymorphisms in the S protein can affect cleavage, viral transmission, and pathogenesis. Here, we investigated the role of arising S polymorphisms in vitro and in vivo to understand the emergence of SARS-CoV-2 variants. First, we showed that the S:655Y is selected after in vivo replication in the mink model. This mutation is present in the Gamma Variant Of Concern (VOC) but it also occurred sporadically in early SARS-CoV-2 human isolates. To better understand the impact of this polymorphism, we analyzed the in vitro properties of a panel of SARS-CoV-2 isolates containing S:655Y in different lineage backgrounds. Results demonstrated that this mutation enhances viral replication and spike protein cleavage. Viral competition experiments using hamsters infected with WA1 and WA1-655Y isolates showed that the variant with 655Y became dominant in both direct infected and direct contact animals. Finally, we investigated the cleavage efficiency and fusogenic properties of the spike protein of selected VOCs containing different mutations in their spike proteins. Results showed that all VOCs have evolved to acquire an increased spike cleavage and fusogenic capacity despite having different sets of mutations in the S protein. Our study demonstrates that the S:655Y is an important adaptative mutation that increases viral cell entry, transmission, and host susceptibility. Moreover, SARS-COV-2 VOCs showed a convergent evolution that promotes the S protein processing.

Seasonal stability of SARS-CoV-2 in biological fluids (preprint)

Transmission of SARS-CoV-2 occurs by close contact with infected persons through droplets, the inhalation of infectious aerosols and the exposure to contaminated surface. Previously, we determined the virus stability on different types of surfaces under indoor and seasonal climatic conditions. SARS-CoV-2 survived the longest on surfaces under winter conditions, followed by spring/fall and summer conditions, suggesting the seasonal pattern of stability on surfaces. However, under natural conditions, the virus is secreted in various biological fluids from infected humans. In this respect, it remains unclear how long the virus survives in various types of biological fluids. This study explored the SARS-CoV-2 stability in human biological fluids under different environmental conditions and estimated the half-life. The virus was stable for up to 21 days in nasal mucus, sputum, saliva, tear, urine, blood, and semen; it remained infectious significantly longer under winter and spring/fall conditions than under summer conditions. In contrast, the virus was only stable up to 24 hours in feces and breast milk. These findings demonstrate the potential risk of infectious biological fluids in SARS-CoV-2 transmission and have implications for its seasonality.

The N501Y mutation in SARS-CoV-2 spike leads to morbidity in obese and aged mice and is neutralized by convalescent and post-vaccination human sera (preprint)

The current COVID-19 (coronavirus disease 19) pandemic, caused by SARS-CoV-2, disproportionally affects the elderly and people with comorbidities like obesity and associated type 2 diabetes mellitus. Small animal models are crucial for the successful development and validation of antiviral vaccines, therapies and to study the role that comorbidities have on the outcome of viral infections. The initially available SARS-CoV-2 isolates require adaptation in order to use the mouse angiotensin converting enzyme 2 (mACE-2) entry receptor and to productively infect the cells of the murine respiratory tract. We have mouse-adapted SARS-CoV-2 by serial passaging a clinical virus isolate in the lungs of mice. We then used low doses of this virus in mouse models for advanced age, diabetes and obesity. Similar to SARS-CoV-2 infection in humans, the outcome of infection with mouse-adapted SARS-CoV-2 resulted in enhanced morbidity in aged and diabetic obese mice. Mutations associated with mouse adaptation occurred in the S, M, N and ORF8 genes. Interestingly, one mutation in the receptor binding domain of the S protein results in the change of an asparagine to tyrosine residue at position 501 (N501Y). This mutation is also present in the newly emerging SARS-CoV-2 variant viruses reported in the U.K. (20B/501Y.V1, B1.1.7 lineage) that is epidemiologically associated with high human to human transmission. We show that human convalescent and post vaccination sera can neutralize the newly emerging N501Y virus variant with similar efficiency as that of the reference USA-WA1/2020 virus, suggesting that current SARS-CoV-2 vaccines will protect against the 20B/501Y.V1 strain.

A trans-complementation system for SARS-CoV-2 (preprint)

The biosafety level-3 (BSL-3) requirement to culture severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a bottleneck for research and countermeasure development. Here we report a trans-complementation system that produces single-round infectious SARS-CoV-2 that recapitulates authentic viral replication. We demonstrate that the single-round infectious SARS-CoV-2 can be used at BSL-2 laboratories for high-throughput neutralization and antiviral testing. The trans-complementation system consists of two components: a genomic viral RNA containing a deletion of ORF3 and envelope gene, and a producer cell line expressing the two deleted genes. Trans-complementation of the two components generates virions that can infect naive cells for only one round, but does not produce wild-type SARS-CoV-2. Hamsters and K18-hACE2 transgenic mice inoculated with the complementation-derived virions exhibited no detectable disease, even after intracranial inoculation with the highest possible dose. The results suggest that the trans-complementation platform can be safely used at BSL-2 laboratories for research and countermeasure development.

Azithromycin Plus Zinc Sulfate Rapidly and Synergistically Suppresses IκBα-Mediated In Vitro Human Airway Cell ACE2 Expression for SARS-CoV-2 Entry (preprint)

Large-scale efforts have been persistently undertaken for medical prophylaxis and treatment of COVID-19 disasters worldwide. A variety of novel viral spike protein-targeted vaccine preparations have recently been clinically distributed based on accelerated approval. We revisited the early but inconclusive clinical interest in the combination of azithromycin and zinc sulfate repurposing with safety advantages. In vitro proof of concept was provided for rapid and synergistic suppression of ACE2 expression following treatments in human airway cells, Calu-3 and H322M. The two representative ACE2-expressing human airway cells indicate the upper and lower respiratory tracts. Prophylactic and early therapeutic roles of azithromycin combined with zinc are proposed for virus cellular entry prevention potential bridging to effective antibody production.

Topoisomerase 1 inhibition therapy protects against SARS-CoV-2-induced inflammation and death in animal models. (preprint)

The ongoing pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is currently affecting millions of lives worldwide. Large retrospective studies indicate that an elevated level of inflammatory cytokines and pro-inflammatory factors are associated with both increased disease severity and mortality. Here, using multidimensional epigenetic, transcriptional, in vitro and in vivo analyses, we report that Topoisomerase 1 (Top1) inhibition suppresses lethal inflammation induced by SARS-CoV-2. Therapeutic treatment with two doses of Topotecan (TPT), a FDA-approved Top1 inhibitor, suppresses infection-induced inflammation in hamsters. TPT treatment as late as four days post-infection reduces morbidity and rescues mortality in a transgenic mouse model. These results support the potential of Top1 inhibition as an effective host-directed therapy against severe SARS-CoV-2 infection. TPT and its derivatives are inexpensive clinical-grade inhibitors available in most countries. Clinical trials are needed to evaluate the efficacy of repurposing Top1 inhibitors for COVID-19 in humans.

Designed proteins assemble antibodies into modular nanocages (preprint)

Antibodies are widely used in biology and medicine, and there has been considerable interest in multivalent antibody formats to increase binding avidity and enhance signaling pathway agonism. However, there are currently no general approaches for forming precisely oriented antibody assemblies with controlled valency. We describe the computational design of two-component nanocages that overcome this limitation by uniting form and function. One structural component is any antibody or Fc fusion and the second is a designed Fc-binding homo-oligomer that drives nanocage assembly. Structures of 8 antibody nanocages determined by electron microscopy spanning dihedral, tetrahedral, octahedral, and icosahedral architectures with 2, 6, 12, and 30 antibodies per nanocage match the corresponding computational models. Antibody nanocages targeting cell-surface receptors enhance signaling compared to free antibodies or Fc-fusions in DR5-mediated apoptosis, Tie2-mediated angiogenesis, CD40 activation, and T cell proliferation; nanocage assembly also increases SARS-CoV-2 pseudovirus neutralization by -SARS-CoV-2 monoclonal antibodies and Fc-ACE2 fusion proteins. We anticipate that the ability to assemble arbitrary antibodies without need for covalent modification into highly ordered assemblies with different geometries and valencies will have broad impact in biology and medicine.

Susceptibility of midge and mosquito vectors to SARS-CoV-2 by natural route of infection (preprint)

SARS-CoV-2 is a recently emerged, highly contagious virus and the cause of the current pandemic. It is a zoonotic virus, although its animal origin is not clear yet. Person-to-person transmission occurs by inhalation of infected droplets and aerosols, or by direct contact with contaminated fomites. Arthropods transmit numerous viral, parasitic, and bacterial diseases; however, the potential role of arthropods in SARS-CoV-2 transmission is not fully understood. Thus far, a few studies have demonstrated that SARS-CoV-2 replication is not supported in cells from certain insect species nor in certain species of mosquitoes after intrathoracic inoculation. In this study, we expanded the work of SARS-CoV-2 susceptibility to biting insects after ingesting a SARS-CoV-2infected blood meal. Species tested included Culicoides sonorensis biting midges, as well as Culex tarsalis and Culex quinquefasciatus mosquitoes, all known biological vectors for numerous RNA viruses. Arthropods were allowed to feed on SARS-CoV-2 spiked blood and at various time points post infection analyzed for the presence of viral RNA and infectious virus. Additionally, cell lines derived from C. sonorensis (W8a), Ae. aegypti (C6/36), Cx. quinquefasciatus (HSU), and Cx. tarsalis (CxTrR2) were tested for SARS-CoV-2 susceptibility. Our results indicate that none of the biting insects, nor the insect cell lines support SARS-CoV-2 replication. We conclude, that biting insect do not pose a risk for transmission of SARS-CoV-2 to humans or animals following a SARS-CoV-2 infected blood meal.

Stability of SARS-CoV-2 on surfaces (preprint)

We report the stability of SARS-CoV-2 on various surfaces under indoor, summer and spring/fall conditions. The virus was more stable under the spring/fall condition with virus half-lives ranging from 17.11 to 31.82 hours, whereas under indoor and summer conditions the virus half-lives were 3.5-11.33 and 2.54-5.58 hours, respectively.

Susceptibility of swine cells and domestic pigs to SARS-CoV-2 (preprint)

The emergence of SARS-CoV-2 has resulted in an ongoing global pandemic with significant morbidity, mortality, and economic consequences. The susceptibility of different animal species to SARS-CoV-2 is of concern due to the potential for interspecies transmission, and the requirement for pre-clinical animal models to develop effective countermeasures. In the current study, we determined the ability of SARS-CoV-2 to (i) replicate in porcine cell lines, (ii) establish infection in domestic pigs via experimental oral/intranasal/intratracheal inoculation, and (iii) transmit to co-housed naive sentinel pigs. SARS-CoV-2 was able to replicate in two different porcine cell lines with cytopathic effects. Interestingly, none of the SARS-CoV-2-inoculated pigs showed evidence of clinical signs, viral replication or SARS-CoV-2-specific antibody responses. Moreover, none of the sentinel pigs displayed markers of SARS-CoV-2 infection. These data indicate that although different porcine cell lines are permissive to SARS-CoV-2, five-week old pigs are not susceptible to infection via oral/intranasal/intratracheal challenge. Pigs are therefore unlikely to be significant carriers of SARS-CoV-2 and are not a suitable pre-clinical animal model to study SARS-CoV-2 pathogenesis or efficacy of respective vaccines or therapeutics.

SARS-CoV-2 infection, disease and transmission in domestic cats (preprint)

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the cause of Coronavirus Disease 2019 (COVID-19) and responsible for the current pandemic. Recent SARS-CoV-2 susceptibility and transmission studies in cats show that the virus can replicate in these companion animals and transmit to other cats. Here, we present an in-depth study of SARS-CoV-2 infection, associated disease and transmission dynamics in domestic cats. Six 4- to 5-month-old cats were challenged with SARS-CoV-2 via intranasal and oral routes simultaneously. One day post challenge (DPC), two sentinel contact cats were co-mingled with the principal infected animals. Animals were monitored for clinical signs, clinicopathological abnormalities and viral shedding throughout the 21 DPC observation period. Postmortem examinations were performed at 4, 7 and 21 DPC to investigate disease progression. Viral RNA was not detected in blood but transiently in nasal, oropharyngeal and rectal swabs and bronchoalveolar lavage fluid as well as various tissues. Tracheobronchoadenitis of submucosal glands with the presence of viral RNA and antigen was observed in airways of the infected cats on 4 and 7 DPC. Serology showed that both, principal and sentinel cats, developed SARS-CoV-2-specific and neutralizing antibodies to SARS-CoV-2 detectable at 7 DPC or 10 DPC, respectively. All animals were clinically asymptomatic during the course of the study and capable of transmitting SARS-CoV-2 to sentinels within 2 days of comingling. The results of this study are critical for our understanding of the clinical course of SARS-CoV-2 in a naturally susceptible host species, and for risk assessment of the maintenance of SARS-CoV-2 in felines and transmission to other animals and humans.