From Deer-to-Deer: SARS-CoV-2 is efficiently transmitted and presents broad tissue tropism and replication sites in white-tailed deer.

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.

Functional evaluation of the P681H mutation on the proteolytic activation of the SARS-CoV-2 variant B.1.1.7 (Alpha) spike.

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.

Age-Related Susceptibility of Ferrets to SARS-CoV-2 Infection.

Susceptibility to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the outcome of coronavirus disease 2019 (COVID-19) have been linked to underlying health conditions and the age of affected individuals. Here, we assessed the effect of age on SARS-CoV-2 infection using a ferret model. For this, young (6-month-old) and aged (18- to 39-month-old) ferrets were inoculated intranasally with various doses of SARS-CoV-2. By using infectious virus shedding in respiratory secretions and seroconversion, we estimated that the infectious dose of SARS-CoV-2 in aged animals is ∼32 PFU per animal, while in young animals it was estimated to be ∼100 PFU. We showed that viral replication in the upper respiratory tract and shedding in respiratory secretions is enhanced in aged ferrets compared to young animals. Similar to observations in humans, this was associated with higher transcription levels of two key viral entry factors, ACE2 and TMPRSS2, in the upper respiratory tract of aged ferrets. IMPORTANCE In humans, ACE2 and TMPRSS2 are expressed in various cells and tissues, and differential expression has been described in young and old people, with a higher level of expressing cells being detected in the nasal brushing of older people than young individuals. We described the same pattern occurring in ferrets, and we demonstrated that age affects susceptibility of ferrets to SARS-CoV-2. Aged animals were more likely to get infected when exposed to lower infectious dose of the virus than young animals, and the viral replication in the upper respiratory tract and shedding are enhanced in aged ferrets. Together, these results suggest that the higher infectivity and enhanced ability of SARS-CoV-2 to replicate in aged individuals is associated, at least in part, with transcription levels of ACE2 and TMPRSS2 at the sites of virus entry. The young and aged ferret model developed here may represent a great platform to assess age-related differences in SARS-CoV-2 infection dynamics and replication.

A virulent and pathogenic infectious clone of Senecavirus A.

Senecavirus A (SVA) is a picornavirus that circulates in swine populations worldwide causing vesicular disease (VD) in affected animals. Here we developed a reverse genetics system for SVA based on the well-characterized wild-type SVA strain SD15-26 (wt SVA SD15-26). The full-length cDNA genome of SVA was cloned into a plasmid under a T7 RNA polymerase promoter. Following in vitro transcription, the genomic viral RNA was transfected into BHK-21 cells and rescue of infectious virus (rSVA SD15-26) was shown by inoculation of highly susceptible H1299 cells. In vitro characterization of the rSVA SD15-26 showed similar replication properties and protein expression levels as the wt SVA SD15-26. A pathogenesis study was conducted in 15-week-old finishing pigs to evaluate the pathogenicity and infection dynamics of the rSVA SD15-26 virus in comparison to the wt SVA SD15-26. Animals from both rSVA- and wt SVA SD15-26-inoculated groups presented characteristic SVA clinical signs (lethargy and lameness) followed by the development of vesicular lesions on the snout and/or feet. The clinical outcome of infection, including disease onset, severity and duration was similar in rSVA- and the wt SVA SD15-26-inoculated animals. All animals inoculated with rSVA or with wt SVA SD15-26 presented a short-term viremia, and animals from both groups shed similar amounts of virus in oral and nasal secretion, and faeces. Our data demonstrates that the rSVA SD5-26 clone is fully virulent and pathogenic in pigs, presenting comparable pathogenesis and infection dynamics to the wt SVA SD15-26 strain. The infectious clone generated here is a useful platform to study virulence determinants of SVA, and to dissect other aspects of SVA infection biology, pathogenesis and persistence.

Functional evaluation of the P681H mutation on the proteolytic activation the SARS-CoV-2 variant B.1.1.7 (Alpha) spike.

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 (VOC) first identified in the UK 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 while the B.1.1.7 P681H mutation may slightly increase S1/S2 cleavage this does not significantly impact viral entry or cell-cell spread.

A Novel Live Attenuated Vaccine Candidate Protects Against Heterologous Senecavirus A Challenge.

Senecavirus A (SVA) is an emerging picornavirus causing vesicular disease (VD) clinically indistinguishable from foot-and-mouth disease (FMD) in pigs. Currently there are no vaccines currently available for SVA. Here we developed a recombinant SVA strain (rSVAm SacII) using reverse genetics and assessed its immunogenicity and protective efficacy in pigs. In vivo characterization of the rSVAm SacII strain demonstrated that the virus is attenuated, as evidenced by absence of lesions, decreased viremia and virus shedding in inoculated animals. Notably, while attenuated, rSVA mSacII virus retained its immunogenicity as high neutralizing antibody (NA) responses were detected in inoculated animals. To assess the immunogenicity and protective efficacy of rSVA mSacII, 4-week-old piglets were sham-immunized or immunized with inactivated or live rSVA mSacII virus-based formulations. A single immunization with live rSVA mSacII virus via the intramuscular (IM) and intranasal (IN) routes resulted in robust NA responses with antibodies being detected between days 3-7 pi. Neutralizing antibody responses in animals immunized with the inactivated virus via the IM route were delayed and only detected after a booster on day 21 pi. Immunization with live virus resulted in recall T cell proliferation (CD4+, CD8+, and CD4+/CD8+ T cells), demonstrating efficient stimulation of cellular immunity. Notably, a single dose of the live attenuated vaccine candidate resulted in protection against heterologous SVA challenge, as demonstrated by absence of overt disease and reduced viremia, virus shedding and viral load in tissues. The live attenuated vaccine candidate developed here represents a promising alternative to prevent and control SVA in swine.

Persistent Infection and Transmission of Senecavirus A from Carrier Sows to Contact Piglets.

Senecavirus A (SVA) is a picornavirus that causes acute vesicular disease (VD), that is clinically indistinguishable from foot-and-mouth disease (FMD), in pigs. Notably, SVA RNA has been detected in lymphoid tissues of infected animals several weeks following resolution of the clinical disease, suggesting that the virus may persist in select host tissues. Here, we investigated the occurrence of persistent SVA infection and the contribution of stressors (transportation, immunosuppression, or parturition) to acute disease and recrudescence from persistent SVA infection. Our results show that transportation stress leads to a slight increase in disease severity following infection. During persistence, transportation, immunosuppression, and parturition stressors did not lead to overt/recrudescent clinical disease, but intermittent viremia and virus shedding were detected up to day 60 postinfection (p.i.) in all treatment groups following stress stimulation. Notably, real-time PCR and in situ hybridization (ISH) assays confirmed that the tonsil harbors SVA RNA during the persistent phase of infection. Immunofluorescence assays (IFA) specific for double-stranded RNA (dsRNA) demonstrated the presence of double-stranded viral RNA in tonsillar cells. Most importantly, infectious SVA was isolated from the tonsil of two animals on day 60 p.i., confirming the occurrence of carrier animals following SVA infection. These findings were supported by the fact that contact piglets (11/44) born to persistently infected sows were infected by SVA, demonstrating successful transmission of the virus from carrier sows to contact piglets. Results here confirm the establishment of persistent infection by SVA and demonstrate successful transmission of the virus from persistently infected animals.IMPORTANCE Persistent viral infections have significant implications for disease control strategies. Previous studies demonstrated the persistence of SVA RNA in the tonsil of experimentally or naturally infected animals long after resolution of the clinical disease. Here, we showed that SVA establishes persistent infection in SVA-infected animals, with the tonsil serving as one of the sites of virus persistence. Importantly, persistently infected carrier animals shedding SVA in oral and nasal secretions or feces can serve as sources of infection to other susceptible animals, as evidenced by successful transmission of SVA from persistently infected sows to contact piglets. These findings unveil an important aspect of SVA infection biology, suggesting that persistently infected pigs may function as reservoirs for SVA.

Senecavirus A 3C Protease Mediates Host Cell Apoptosis Late in Infection.

Senecavirus A (SVA), an oncolytic picornavirus used for cancer treatment in humans, has recently emerged as a vesicular disease (VD)-causing agent in swine worldwide. Notably, SVA-induced VD is indistinguishable from foot-and-mouth disease (FMD) and other high-consequence VDs of pigs. Here we investigated the role of apoptosis on infection and replication of SVA. Given the critical role of the nuclear factor-kappa B (NF-κB) signaling pathway on modulation of cell death, we first assessed activation of NF-κB during SVA infection. Results here show that while early during infection SVA induces activation of NF-κB, as evidenced by nuclear translocation of NF-κB-p65 and NF-κB-mediated transcription, late in infection a cleaved product corresponding to the C-terminus of NF-κB-p65 is detected in infected cells, resulting in lower NF-κB transcriptional activity. Additionally, we assessed the potential role of SVA 3C protease (3Cpro) in SVA-induced host-cell apoptosis and cleavage of NF-κB-p65. Transient expression of SVA 3Cpro was associated with cleavage of NF-κB-p65 and Poly (ADP-ribose) polymerase (PARP), suggesting its involvement in virus-induced apoptosis. Most importantly, we showed that while cleavage of NF-κB-p65 is secondary to caspase activation, the proteolytic activity of SVA 3Cpro is essential for induction of apoptosis. Experiments using the pan-caspase inhibitor Z-VAD-FMK confirmed the relevance of late apoptosis for SVA infection, indicating that SVA induces apoptosis, presumably, as a mechanism to facilitate virus release and/or spread from infected cells. Together, these results suggest an important role of apoptosis for SVA infection biology.

Antigenic and immunogenic properties of the canine distemper virus nucleocapsid protein expressed in Escherichia coli employing codon optimized synthetic gene / Propriedades antigênicas e imunogênicas da proteína do nucleocapsídeo do virus da cinomose canina expressa em Escherichia coli empregando gene sintético com codons otimizados

Despite common occurrence and importance of canine distemper disease the majority of tests currently available for diagnosis are hampered by either low sensitivity or specificity. In this study it was evaluated antigenic and immunogenic characteristics of a conserved region of nucleocapsid protein of canine distemper virus (rCDV NP) expressed in Escherichia coli employing a codon optimized synthetic gene. The expression of rCDVNP in Star strain (mean 300μg/mL, purified) was confirmed by SDS-PAGE and Western blot analysis by using His-Tag monoclonal antibodies. Western blot and ELISA, employing positive and negative control dog sera, demonstrated the rCDVNP antigenicity. The rCDVNP was inoculated in hens and immunoglobulin Y (IgY) was purified from the egg yolk. The mean yield of IgY was 28.55mg/mL. IgY reacted with the recombinant protein as demonstrated by Western blot and ELISA assays. In summary, our findings demonstrated that rCDVNP is antigenic since CDV positive dog sera recognized the protein in vitro. Additionally, the rCDVNP proved to be immunogenic in hens being possible to isolate a high concentration of specific IgY antibodies from the egg yolk. Taken together, these results indicate that the rCDVNP along with the specific IgY could be useful tools for development of the canine distemper immunodiagnostic assays.(AU)

Apesar da ocorrência comum e importância da cinomose canina, a maioria dos testes atualmente disponíveis para diagnóstico são prejudicados pela baixa sensibilidade ou especificidade. Neste estudo foram avaliadas características antigênicas e imunogênicas de uma região conservada da proteína do nucleocapsídeo do virus da cinomose canina (rCDV NP) expressa em Escherichia coli empregando um gene sintético e codons otimizados. A expressão na cepa Star (média de 300μg/mL, purificada) foi confirmada por SDS-PAGE e Western blot utilizando anticorpos monoclonais anti-His-Tag. A antigenicidade da rCDVNP foi demonstrada por western blot e ELISA empregando soros de cães positivos e negativos. A rCDVNP foi inoculada em galinhas e imunoglobulina Y (gY) foi obtida e purificada a partir da gema. A produção média de IgY foi 28.55mg/mL. Anticorpos IgY reagiram com a proteína recombinante, quando analisados por Western blot e ELISA. Em resumo, nossos achados demonstram que a rCDVNP produzida é antigênica, uma vez que os anticorpos de soro de cães positivos para CDV reconheceram a proteína in vitro. Além disso, a rCDVNP foi imunogênica em galinhas, sendo possível isolar anticorpos IgY específicos a partir da gema do ovo em altas concentrações. Tomados em conjunto, estes resultados indicam que a rCDVNP juntamente com a IgY específica podem ser ferramentas úteis para elaborar ensaios de imunodiagnóstico de cinomose canina.(AU)

Pathogenicity and cross-reactive immune responses of a historical and a contemporary Senecavirus A strains in pigs.

The goals of this study were to compare the pathogenicity and infection dynamics of a historical and a contemporary SVA strains (SVV 001 and SD15-26) and to assess cross-neutralizing and cross-reactive T cell responses following experimental infection in pigs. Both SVA strains successfully infected all inoculated animals, resulting in viremia and robust antibody and cellular immune responses. SVA SD15-26 infection resulted in characteristic clinical signs and vesicular lesions, however, SVA SVV 001 did not cause overt clinical disease with inoculated animals remaining clinically normal during the experiment. Notably, neutralization- and -recall IFN-γ expression-assays revealed marked cross-neutralizing antibody and cross-reactive T cell responses between the two viral strains. Together these results demonstrate that the historical SVA SVV 001 strain presents low virulence in pigs when compared to the contemporary SVA SD15-26 strain. Additionally, immunological assays indicate that SVA SVV 001 and SD15-26 are antigenically related and share conserved antigenic determinants.

Passive immunity to porcine epidemic diarrhea virus following immunization of pregnant gilts with a recombinant orf virus vector expressing the spike protein.

Passive immunity is critical for protection of neonatal piglets against porcine epidemic diarrhea virus (PEDV). Here, we investigated the immunogenicity of an orf virus (ORFV) vector expressing the full-length spike (S) protein of PEDV (ORFV-PEDV-S) in pregnant gilts and its ability to confer passive immunity and protection in piglets. Three doses of ORFV-PEDV-S were given to two groups of PEDV-negative pregnant gilts, with the last dose being administered two weeks prior to farrowing. One of the two groups immunized with the ORFV-PEDV-S recombinant virus was also exposed to live PEDV orally on day 31 post-immunization (pi). Antibody responses were assessed in serum, colostrum and milk of immunized gilts, and passive transfer of antibodies was evaluated in piglet sera. The protective efficacy of ORFV-PEDV-S was evaluated after challenge of the piglets with PEDV. PEDV-specific IgG, IgA and neutralizing antibody (NA) responses were detected in ORFV-PEDV-S-immunized and ORFV-PEDV-S-immunized/PEDV-exposed gilts. PEDV NA, IgG and IgA were detected in the serum of piglets born to immunized gilts, demonstrating the transfer of antibodies through colostrum and milk. Piglets born to immunized gilts showed reduced morbidity and a marked reduction in mortality after PEDV challenge in comparison to control piglets. Piglets born to gilts that received ORFV-PEDV-S and were exposed to live PEDV showed stronger NA responses and lower clinical scores when compared to piglets born to gilts immunized with ORFV-PEDV-S alone. These results demonstrate the potential of ORFV as a vaccine delivery platform capable of eliciting passive immunity against PEDV.

Pathogenesis of Senecavirus A infection in finishing pigs.

Senecavirus A (SVA) is an emerging picornavirus that has been associated with vesicular disease and neonatal mortality in swine. Many aspects of SVA infection biology and pathogenesis, however, remain unknown. Here the pathogenesis of SVA was investigated in finishing pigs. Animals were inoculated via the oronasal route with SVA strain SD15-26 and monitored for clinical signs and lesions associated with SVA infection. Viraemia was assessed in serum and virus shedding monitored in oral and nasal secretions and faeces by real-time reverse transcriptase quantitative PCR (RT-qPCR) and/or virus isolation. Additionally, viral load and tissue distribution were assessed during acute infection and following convalescence from disease. Clinical signs characterized by lethargy and lameness were first observed on day 4 post-inoculation (pi) and persisted for approximately 2-10 days. Vesicular lesions were first observed on day 4 pi on the snout and/or feet, affecting the coronary bands, dewclaws, interdigital space and heel/sole of SVA-infected animals. A short-term viraemia was observed between days 3 and 10 pi, whereas virus shedding was detected between days 1 and 28 pi in oral and nasal secretions and faeces. Notably, RT-qPCR and in situ hybridization (ISH) performed on tissues collected on day 38 pi revealed the presence of SVA RNA in the tonsils of all SVA-infected animals. Serological responses to SVA were characterized by early neutralizing antibody responses (day 5 pi), which coincided with decreased levels of viraemia, virus shedding and viral load in tissues. This study provides significant insights into the pathogenesis and infectious dynamics of SVA in swine.

Synthesis, antifungal and cytotoxic activities of 2-aryl-3-((piperidin-1-yl)ethyl)thiazolidinones.

A series of sixteen novel thiazolidinone derivatives were synthesized from the efficient one-pot reaction of 2-(piperidin-1-yl)ethylamine, arenealdehydes and mercaptoacetic acid in good yields. Identification and characterization of products were achieved by NMR and GC-MS techniques. The in vitro antifungal activities of all synthesized compounds were evaluated against seven fungi: Candida albicans, Candida parapsilosis, Candida guilliermondii, Cryptococcus laurentii, Geotrichum sp, Trichosporon asahii and Rhodotorula sp. The results are expressed as the Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) and the best results were found against the Rhodotorula sp yeast. Two thiazolidinones (4h and 4l), MIC and MFC (16.5 µg/mL) proved to be 1.6 times more active than fluconazole and four of them (4b, 4e, 4g and 4k (MIC and MFC 25 µg/mL)) showed similar activity of standard drug to Rhodotorula sp. In addition, the cytotoxicity of thiazolidinones 4a-p was evaluated on cultured Vero cells and most of them displayed low toxicity (above 98 µg/mL). These preliminary and important results could be considered a starting point for the development of new antifungal agents.