Stability of SARS-CoV-2 in Biological Fluids of Animals.

Since its first emergence in 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued to evolve genetically, jump species barriers, and expand its host range. There is growing evidence of interspecies transmission including infection of domestic animals and widespread circulation in wildlife. However, knowledge of SARS-CoV-2 stability in animal biological fluids and their role in transmission is still limited as previous studies focused on human biological fluids. Therefore, this study aimed to determine the SARS-CoV-2 stability in biological fluids from three animal species, cats, sheep and white-tailed deer (WTD). Saliva, feces, 10% fecal suspensions, and urine of cats, sheep, and WTD were mixed with a known concentration of virus and incubated under indoor and three different climatic conditions. Our results show that the virus was stable for up to 1 day in the saliva of cats, sheep, and WTD regardless of the environmental conditions. The virus remained infectious for up to 6 days in feces and 15 days in fecal suspension of WTD, whereas the virus was rather unstable in cat and sheep feces and fecal suspensions. We found the longest survival of SARS-CoV-2 in the urine of cats, sheep, and WTD. Furthermore, side-by-side comparison with different SARS-CoV-2 strains showed that the Alpha, Delta, and Omicron variants of concern were less stable than the ancestral Wuhan-like strain in WTD fecal suspension. The results of our study provide valuable information for assessing the potential role of various animal biological fluids in SARS-CoV-2 transmission.

Ancestral Lineage of SARS-CoV-2 Is More Stable in Human Biological Fluids than Alpha, Beta, and Omicron Variants of Concern.

SARS-CoV-2 is a zoonotic virus 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. 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 the 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. IMPORTANCE Genetic evolution of SARS-CoV-2 leads to the continuous emergence of novel virus variants, posing a significant concern to global public health. Five of these variants have been classified to date 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.

Characterization of SARS-CoV-2 Spike mutations important for infection of mice and escape from human immune sera.

Due to differences in human and murine angiotensin converting enzyme 2 (ACE-2) receptor, initially available SARS-CoV-2 isolates could not infect mice. Here we show that serial passaging of USA-WA1/2020 strain in mouse lungs results in "mouse-adapted" SARS-CoV-2 (MA-SARS-CoV-2) with mutations in S, M, and N genes, and a twelve-nucleotide insertion in the S gene. MA-SARS-CoV-2 infection causes mild disease, with more pronounced morbidity depending on genetic background and in aged and obese mice. Two mutations in the S gene associated with mouse adaptation (N501Y, H655Y) are present in SARS-CoV-2 variants of concern (VoCs). N501Y in the receptor binding domain of viruses of the B.1.1.7, B.1.351, P.1 and B.1.1.529 lineages (Alpha, Beta, Gamma and Omicron variants) is associated with high transmissibility and allows VoCs to infect wild type mice. We further show that S protein mutations of MA-SARS-CoV-2 do not affect neutralization efficiency by human convalescent and post vaccination sera.

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

Severe acute respiratory syndrome coronavirus 2 (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 (S) protein have arisen, culminating in the spread of several variants of concern (VOCs) with various degrees of altered virulence, transmissibility, and neutralizing antibody escape. In this study, we used pseudoviruses that express specific SARS-CoV-2 S protein substitutions and cell lines that express angiotensin-converting enzyme 2 (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 ancestral prototype S at levels comparable to human ACE2. Most single S substitutions did not significantly change virus entry, although 614G and 484K resulted in a decreased efficiency. Conversely, combinatorial VOC substitutions in the S protein were associated with increased entry of pseudoviruses. Neutralizing titers in sera from various animal species were significantly reduced against pseudoviruses expressing the S proteins of Beta, Delta, or Omicron VOCs compared to the parental S protein. Especially, substitutions in the S protein of the Omicron variant significantly reduced the neutralizing titers of the sera. 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. IMPORTANCE The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to have devastating impacts on global health and socioeconomics. The recent emergence of SARS-CoV-2 variants of concern, which contain mutations that can affect the virulence, transmission, and effectiveness of licensed vaccines and therapeutic antibodies, are currently becoming the common strains circulating in humans worldwide. In addition, SARS-CoV-2 has been shown to infect a wide variety of animal species, which could result in additional mutations of the SARS-CoV-2 virus. In this study, we investigate the effect of mutations present in SARS-CoV-2 variants of concern and determine the effects of these mutations on cell entry, virulence, and antibody neutralization activity in humans and a variety of animals that might be susceptible to SARS-CoV-2 infection. This information is essential to understand the effects of important SARS-CoV-2 mutations and to inform public policy to create better strategies to control the COVID-19 pandemic.

Mutations in SARS-CoV-2 variants of concern link to increased spike cleavage and virus transmission.

SARS-CoV-2 lineages have diverged into highly prevalent variants termed "variants of concern" (VOCs). Here, we characterized emerging SARS-CoV-2 spike polymorphisms in vitro and in vivo to understand their impact on transmissibility and virus pathogenicity and fitness. We demonstrate that the substitution S:655Y, represented in the gamma and omicron VOCs, enhances viral replication and spike protein cleavage. The S:655Y substitution was transmitted more efficiently than its ancestor S:655H in the hamster infection model and was able to outcompete S:655H in the hamster model and in a human primary airway system. Finally, we analyzed a set of emerging SARS-CoV-2 variants to investigate how different sets of mutations may impact spike processing. All VOCs tested exhibited increased spike cleavage and fusogenic capacity. Taken together, our study demonstrates that the spike mutations present in VOCs that become epidemiologically prevalent in humans are linked to an increase in spike processing and virus transmission.

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

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 in humans. A wide range of animal species have also been shown to be susceptible to SARS-CoV-2 by experimental and/or natural infections. Sheep are a commonly farmed domestic ruminant that have not 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 cells and experimental challenge of sheep to investigate their susceptibility to SARS-CoV-2. Our results showed that sheep-derived kidney cells support SARS-CoV-2 replication. Furthermore, the experimental challenge of sheep demonstrated limited infection with viral RNA shed in nasal and oral swabs at 1 and 3-days post challenge (DPC); viral RNA was also detected in the respiratory tract and lymphoid tissues at 4 and 8 DPC. Sero-reactivity was 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 respiratory tract tissues of sentinel animals at 21 DPC. Furthermore, we used a 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, 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 variant outcompeted the lineage A strain.

Infection and transmission of ancestral SARS-CoV-2 and its alpha variant in pregnant white-tailed deer.

ABSTRACTSARS-CoV-2 was first reported circulating in human populations in December 2019 and has since become a global pandemic. Recent history involving SARS-like coronavirus outbreaks have demonstrated the significant role of intermediate hosts in viral maintenance and transmission. 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 (WTD) are amongst the most abundant and geographically widespread wild ruminant species in the US. Recently, WTD 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 WTD. In addition, we examined the competition of two SARS-CoV-2 isolates, representatives of the ancestral lineage A and the alpha variant of concern (VOC) B.1.1.7 through co-infection of WTD. 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 WTD 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 WTD, as demonstrated by the genome of the virus shed from nasal and oral cavities from principal infected and contact animals, and from the genome of virus present in tissues of principal infected deer, fetuses and contact animals.

Experimental re-infected cats do not transmit SARS-CoV-2.

SARS-CoV-2 is the causative agent of COVID-19 and responsible for the current global pandemic. We and others have previously demonstrated that cats are susceptible to SARS-CoV-2 infection and can efficiently transmit the virus to naïve cats. Here, we address whether cats previously exposed to SARS-CoV-2 can be re-infected with SARS-CoV-2. In two independent studies, SARS-CoV-2-infected cats were re-challenged with SARS-CoV-2 at 21 days post primary challenge (DPC) and necropsies performed at 4, 7 and 14 days post-secondary challenge (DP2C). Sentinels were co-mingled with the re-challenged cats at 1 DP2C. Clinical signs were recorded, and nasal, oropharyngeal, and rectal swabs, blood, and serum were collected and tissues examined for histologic lesions. Viral RNA was transiently shed via the nasal, oropharyngeal and rectal cavities of the re-challenged cats. Viral RNA was detected in various tissues of re-challenged cats euthanized at 4 DP2C, mainly in the upper respiratory tract and lymphoid tissues, but less frequently and at lower levels in the lower respiratory tract when compared to primary SARS-CoV-2 challenged cats at 4 DPC. Viral RNA and antigen detected in the respiratory tract of the primary SARS-CoV-2 infected cats at early DPCs were absent in the re-challenged cats. Naïve sentinels co-housed with the re-challenged cats did not shed virus or seroconvert. Together, our results indicate that cats previously infected with SARS-CoV-2 can be experimentally re-infected with SARS-CoV-2; however, the levels of virus shed was insufficient for transmission to co-housed naïve sentinels. We conclude that SARS-CoV-2 infection in cats induces immune responses that provide partial, non-sterilizing immune protection against re-infection.