Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19) in humans, has a broad host range, and is able to infect domestic and wild ...animal species. Notably, white-tailed deer (WTD, Odocoileus virginianus), the most widely distributed cervid species in the Americas, were shown to be highly susceptible to SARS-CoV-2 in challenge studies and reported natural infection/exposure rates approaching 30-40% in free-ranging WTD in the U.S. Thus, understanding the infection and transmission dynamics of SARS-CoV-2 in WTD is critical to prevent future zoonotic transmission to humans, at the human-WTD interface during hunting or venison farming, and for implementation of effective disease control measures. Here, we demonstrated that following intranasal inoculation with SARS-CoV-2 B.1 lineage, WTD fawns (~8-month-old) shed infectious virus up to day 5 post-inoculation (pi), with high viral loads shed in nasal and oral secretions. This resulted in efficient deer-to-deer transmission on day 3 pi. Consistent a with lack of infectious SARS-CoV-2 shedding after day 5 pi, no transmission was observed to contact animals added on days 6 and 9 pi. We have also investigated the tropism and sites of SARS-CoV-2 replication in adult WTD (3-4 years of age). Infectious virus was detected up to day 6 pi in nasal secretions, and from various respiratory-, lymphoid-, and central nervous system tissues, indicating broad tissue tropism and multiple sites of virus replication. The study provides important insights on the infection and transmission dynamics of SARS-CoV-2 in WTD, a wild animal species that is highly susceptible to infection and with the potential to become a reservoir for the virus in the field.
The origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing the global coronavirus disease 19 (COVID-19) pandemic, remains a mystery. Current evidence suggests a ...likely spillover into humans from an animal reservoir. Understanding the host range and identifying animal species that are susceptible to SARS-CoV-2 infection may help to elucidate the origin of the virus and the mechanisms underlying cross-species transmission to humans. Here we demonstrated that white-tailed deer (
), an animal species in which the angiotensin converting enzyme 2 (ACE2) - the SARS-CoV-2 receptor - shares a high degree of similarity to humans, are highly susceptible to infection. Intranasal inoculation of deer fawns with SARS-CoV-2 resulted in established subclinical viral infection and shedding of infectious virus in nasal secretions. Notably, infected animals transmitted the virus to non-inoculated contact deer. Viral RNA was detected in multiple tissues 21 days post-inoculation (pi). All inoculated and indirect contact animals seroconverted and developed neutralizing antibodies as early as day 7 pi. The work provides important insights into the animal host range of SARS-CoV-2 and identifies white-tailed deer as a susceptible wild animal species to the virus.
Given the presumed zoonotic origin of SARS-CoV-2, the human-animal-environment interface of COVID-19 pandemic is an area of great scientific and public- and animal-health interest. Identification of animal species that are susceptible to infection by SARS-CoV-2 may help to elucidate the potential origin of the virus, identify potential reservoirs or intermediate hosts, and define the mechanisms underlying cross-species transmission to humans. Additionally, it may also provide information and help to prevent potential reverse zoonosis that could lead to the establishment of a new wildlife hosts. Our data show that upon intranasal inoculation, white-tailed deer became subclinically infected and shed infectious SARS-CoV-2 in nasal secretions and feces. Importantly, indirect contact animals were infected and shed infectious virus, indicating efficient SARS-CoV-2 transmission from inoculated animals. These findings support the inclusion of wild cervid species in investigations conducted to assess potential reservoirs or sources of SARS-CoV-2 of infection.
The global distribution and constant evolution are challenges for the control of porcine reproductive and respiratory syndrome virus (PRRSV), one of the most important viruses affecting swine ...worldwide. Effective control of PRRSV benefits from genotyping, which currently relies on Sanger sequencing. Here we developed and optimized procedures for real-time genotyping and whole genome sequencing of PRRSV directly from clinical samples based on targeted amplicon- and long amplicon tiling sequencing using the MinION Oxford Nanopore platform. Procedures were developed and tested on 154 clinical samples (including lung, serum, oral fluid and processing fluid) with RT-PCR Ct values ranging from 15 to 35. The targeted amplicon sequencing (TAS) approach was developed to obtain sequences of the complete ORF5 (main target gene for PRRSV genotyping) and partial ORF4 and ORF6 sequences of both PRRSV-1 and PRRSV-2 species. After only 5 min of sequencing, PRRSV consensus sequences with identities to reference sequences above 99% were obtained, enabling rapid identification and genotyping of clinical PRRSV samples into lineages 1, 5 and 8. The long amplicon tiling sequencing (LATS) approach targets type 2 PRRSV, the most prevalent viral species in the U.S. and China. Complete PRRSV genomes were obtained within the first hour of sequencing for samples with Ct values below 24.9. Ninety-two whole genome sequences were obtained using the LATS procedure. Fifty out of 60 sera (83.3%) and 18 out of 20 lung samples (90%) had at least 80% of genome covered at a minimum of 20X sequence depth per position. The procedures developed and optimized in this study here are valuable tools with potential for field application during PRRSV elimination programs.
The COVID-19 pandemic caused by the SARS-CoV-2 virus remains a global public health crisis. Although widespread vaccination campaigns are underway, their efficacy is reduced owing to emerging ...variants of concern
. Development of host-directed therapeutics and prophylactics could limit such resistance and offer urgently needed protection against variants of concern
. Attractive pharmacological targets to impede viral entry include type-II transmembrane serine proteases (TTSPs) such as TMPRSS2; these proteases cleave the viral spike protein to expose the fusion peptide for cell entry, and thus have an essential role in the virus lifecycle
. Here we identify and characterize a small-molecule compound, N-0385, which exhibits low nanomolar potency and a selectivity index of higher than 10
in inhibiting SARS-CoV-2 infection in human lung cells and in donor-derived colonoids
. In Calu-3 cells it inhibits the entry of the SARS-CoV-2 variants of concern B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta). Notably, in the K18-human ACE2 transgenic mouse model of severe COVID-19, we found that N-0385 affords a high level of prophylactic and therapeutic benefit after multiple administrations or even after a single administration. Together, our findings show that TTSP-mediated proteolytic maturation of the spike protein is critical for SARS-CoV-2 infection in vivo, and suggest that N-0385 provides an effective early treatment option against COVID-19 and emerging SARS-CoV-2 variants of concern.
The goal of this study was to evaluate survival of important viral pathogens of livestock in animal feed ingredients imported daily into the United States under simulated transboundary conditions. ...Eleven viruses were selected based on global significance and impact to the livestock industry, including Foot and Mouth Disease Virus (FMDV), Classical Swine Fever Virus (CSFV), African Swine Fever Virus (ASFV), Influenza A Virus of Swine (IAV-S), Pseudorabies virus (PRV), Nipah Virus (NiV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Swine Vesicular Disease Virus (SVDV), Vesicular Stomatitis Virus (VSV), Porcine Circovirus Type 2 (PCV2) and Vesicular Exanthema of Swine Virus (VESV). Surrogate viruses with similar genetic and physical properties were used for 6 viruses. Surrogates belonged to the same virus families as target pathogens, and included Senecavirus A (SVA) for FMDV, Bovine Viral Diarrhea Virus (BVDV) for CSFV, Bovine Herpesvirus Type 1 (BHV-1) for PRV, Canine Distemper Virus (CDV) for NiV, Porcine Sapelovirus (PSV) for SVDV and Feline Calicivirus (FCV) for VESV. For the remaining target viruses, actual pathogens were used. Virus survival was evaluated using Trans-Pacific or Trans-Atlantic transboundary models involving representative feed ingredients, transport times and environmental conditions, with samples tested by PCR, VI and/or swine bioassay. SVA (representing FMDV), FCV (representing VESV), BHV-1 (representing PRV), PRRSV, PSV (representing SVDV), ASFV and PCV2 maintained infectivity during transport, while BVDV (representing CSFV), VSV, CDV (representing NiV) and IAV-S did not. Notably, more viruses survived in conventional soybean meal, lysine hydrochloride, choline chloride, vitamin D and pork sausage casings. These results support published data on transboundary risk of PEDV in feed, demonstrate survival of certain viruses in specific feed ingredients ("high-risk combinations") under conditions simulating transport between continents and provide further evidence that contaminated feed ingredients may represent a risk for transport of pathogens at domestic and global levels.
African swine fever virus is transmissible through animal consumption of contaminated feed. To determine virus survival during transoceanic shipping, we calculated the half-life of the virus in 9 ...feed ingredients exposed to 30-day shipment conditions. Half-lives ranged from 9.6 to 14.2 days, indicating that the feed matrix environment promotes virus stability.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent causing the COVID-19 pandemic. SARS-CoV-2 B.1.1.7 (Alpha), a WHO variant of concern first identified in the United Kingdom in ...late 2020, contains several mutations including P681H in the spike S1/S2 cleavage site, which is predicted to increase cleavage by furin, potentially impacting the viral cell entry. Here, we studied the role of the P681H mutation in B.1.1.7 cell entry. We performed assays using fluorogenic peptides mimicking the Wuhan-Hu-1 and B.1.1.7 S1/S2 sequence and observed no significant difference in furin cleavage. Functional assays using pseudoparticles harboring SARS-CoV-2 spikes and cell-to-cell fusion assays demonstrated no differences between Wuhan-Hu-1, B.1.1.7, or a P681H point mutant. Likewise, we observed no differences in viral growth between USA-WA1/2020 and a B.1.1.7 isolate in cell culture. Our findings suggest that, although the B.1.1.7 P681H mutation may slightly increase S1/S2 cleavage, this does not significantly impact viral entry or cell-cell spread.
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•SARS-CoV-2 B.1.1.7 P681H mutation in the spike is predicted to enhance viral infection•P681H does not significantly impact furin cleavage, viral entry, or cell-cell spread•Other mutations in the SARS-CoV-2 B.1.1.7 VOC may account for increased infection rates
Virology
The porcine epidemic diarrhea virus (PEDV) spike (S) protein is the major target of neutralizing antibodies against PEDV. Here immunodominant neutralizing epitopes of PEDV were identified using a ...panel of S-specific monoclonal antibodies (mAbs). Ten of eleven S-specific mAbs successfully neutralized PEDV infectivity in vitro. Notably, epitope mapping by peptide ELISAs revealed that nine of these mAbs recognized linear neutralizing epitopes located in the N-terminus of the S2 glycoprotein subunit (amino acids aa 744–759, 747–774 and/or 756–771). Additionally, one mAb recognized a neutralizing epitope located in the C-terminus of S2 (aa 1371–1377), while only one neutralizing mAb reacted against a region of the S1 glycoprotein subunit (aa 499–600). Notably, mAbs that recognized epitopes within the S2 subunit presented the highest neutralizing activity against PEDV. Together these results indicate that the S2 glycoprotein subunit contains major antigenic determinants and, perhaps, the immunodominant neutralizing epitopes of PEDV.
•A panel of PEDV S-specific neutralizing antibodies were developed and characterized.•S-specific neutralizing mAbs presented potent neutralizing activity against PEDV in vitro.•Epitope mapping revealed that most neutralizing mAbs target the N-terminus of the S2 protein subunit.•S2 subunit seems to contain immunodominant neutralizing epitopes of PEDV.
Orf virus (ORFV) has been used as a vaccine delivery vector for multiple animal species. Several strategies are being used to improve the immunogenicity and efficacy of ORFV vectors, including the ...use of poxviral promoter(s) with strong early and late activity capable of driving the expression of the heterologous genes for a prolonged time and eliciting a potent immune response. Here, we used RNA-sequencing (RNA-Seq) approach to analyze the transcriptome of ORFV during infection in primary ovine cells. Based on the transcriptional profile of individual ORFV genes, we identified ORFV promoters with strong early and late activity and have shown that they can be used to express heterologous genes in ORFV vectors. Our results show that the intergenic regulatory sequence containing core promoter sequences present upstream of ORF112 (p112) and ORF116 (p116) lead to markedly higher transgene expression than conventional poxviral promoters. Thus, these promoters are valuable alternatives to express transgenes in poxviral vectors.
•Complete overview of the temporal gene expression cascade of Orf virus.•Four gene classes were identified: immediate-early, early, intermediate, and late.•Promoters that result in robust heterologous gene expression by poxviral vectors were identified.•Parapoxvirus p116 induces robust heterologous gene expression by ORFV-based vectors.
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