Anthropogenic Infection of Domestic Cats With SARS-CoV-2 Alpha Variant B.1.1.7 Lineage in Buenos Aires.

SARS-CoV-2 reverse zoonosis, particularly to domestic animals, and the potential role of infected animals in perpetuating the spread of the virus is an issue of increasing concern. In this case report, we identified the natural infection of two cats by SARS-CoV-2, in Argentina, whose owner had been previously infected by SARS-CoV-2. Viral genetic material was detected in feline oropharyngeal (OP) and rectal (R) swab by RT-qPCR, and sequence analysis revealed that the virus infecting the owner and one cat were genetically similar. The alpha variant (B.1.1.7 lineage) was identified with a unique additional mutation, strongly suggesting human-to-cat route of transmission. This study reinforces the One Health concept and the importance of integrating human, animal, and environmental perspectives to promptly address relevant health issues.

Efficacy of the spray-drying treatment to inactivate the bovine leukemia virus in bovine colostrum.

Previous studies have shown the presence of bovine leukemia virus (BLV) in colostrum and milk of naturally infected cows. The relationship between virus or provirus and specific antibodies in these secretions is particular to each infected cow and will probably determine whether the consumption of colostrum or milk from these naturally infected dams provides an infective or a protective effect in recipient calves. Our recent findings suggest that this issue is a key point in BLV transmission in very young calves. Based on this, the aim of the present study was to determine the effect of the spray-drying treatment of colostrum on BLV infectivity. The treatment was done on scale-down conditions, using fresh colostrum from BLV-negative cows spiked with infective BLV. Residual infectivity was tested in susceptible lambs. Lambs inoculated with colostrum spiked with BLV-infected cells or cell-free BLV showed evidence of infection 60 d after inoculation, whereas none of the lambs inoculated with spray-dried colostrum showed evidence of infection 60 d after inoculation. These results provide direct evidence that the experimental spray-drying process used in this study was effective in inactivating infectious BLV in colostrum. These findings suggest that the risk for BLV transmission could be reduced if milk and colostrum were treated by spray-drying prior to consumption in dairy facilities. The effect of spray-drying on the functional properties and stability of the antibodies present in colostrum under long-term storage should be further investigated.

Immune cells transferred by colostrum do not influence the immune responses to foot-and-mouth disease primary vaccination.

Little is known about the influence of maternal antibodies and immune cells transferred through colostrum on the immune responses of calves to the currently used foot-and-mouth disease (FMD) vaccines. Here we evaluated the humoral and cellular immune responses induced by vaccination of colostrum-deprived calves and calves that received equivalent amounts of colostrum preparations that differed in the presence or absence of maternal immune cells but contained the same quantity and quality of anti-foot-and-mouth disease virus (FMDV) antibodies. Three groups of 32-d-old calves (n = 3 per group) were deprived of colostrum and fed either whole immune colostrum or a cell-free colostrum preparation containing only anti-FMDV antibodies. All groups were immunized with 1 dose of an oil-adjuvanted commercial vaccine. Blood samples were collected periodically before vaccination and weekly after vaccination. Immune responses specific to FMDV were assessed based on T-cell proliferation, IFN-γ production, total and neutralizing serum antibodies, and isotype profile. All vaccinated calves developed IFN-γ and lymphoproliferative responses, irrespective of the colostrum received. Colostrum-deprived animals responded to vaccination with a primary IgM response followed by an increase of IgG1 titers. Conversely, antibody titers decreased in all colostrum-fed calves after vaccination. This study demonstrates for the first time that maternal immune cells transferred to the calves through colostrum do not modify immune responses to FMD vaccine, and it confirms the interference of maternal antibodies in the induction of humoral but not cell-mediated immune responses.

In Vitro and In Vivo Characterization of a Typical and a High Pathogenic Bovine Viral Diarrhea Virus Type II Strains.

Non-cytopathic (ncp) type 2 bovine viral diarrhea virus (BVDV-2) is widely prevalent in Argentina causing high mortality rates in cattle herds. In this study, we characterized an Argentinean ncp BVDV-2 field isolate (98-124) compared to a high-virulence reference strain (NY-93), using in silico analysis, in vitro assays, and in vivo infections of colostrum-deprived calves (CDC) to compare pathogenic characters and virulence. In vitro infection of bovine peripheral blood mononuclear cells (PBMC) with BVDV 98-124 induced necrosis shortly after infection while NY-93 strain increased the apoptotic rate in infected cells. Experimental infection of CDC (n = 4 each) with these strains caused an enteric syndrome. High pyrexia was detected in both groups. Viremia and shedding were more prolonged in the CDC infected with the NY-93 strain. In addition, NY-93 infection elicited a severe lymphopenia that lasted for 14 days, whereas 98-124 strain reduced the leukocyte counts for 5 days. All infected animals had a diminished lymphoproliferation activity in response to a mitogen. Neutralizing and anti-NS3 antibodies were detected 3 weeks after infection in all infected calves. Virulence was associated with a more severe clinical score, prolonged immune-suppression, and a greater window for transmission. Studies of apoptosis/necrosis performed after in vitro PBMC infection also revealed differences between both strains that might be correlated to the in vivo pathogenesis. Our results identified 98-124 as a low-virulence strain.

Serologic evidence of HoBi-like virus circulation in Argentinean water buffalo.

HoBi-like pestiviruses (also known as bovine viral diarrhea virus 3) have been sporadically reported from naturally infected cattle in Brazil, Asia, and Europe. Although HoBi-like viruses seem to be endemic in Brazilian cattle and buffalo, they have not been studied in the other countries of South America to our knowledge. Herein we report serologic results of buffalo from 12 large farms in Argentina located near the Brazilian border. These buffalo were not vaccinated against pestiviruses. Our results indicate that HoBi-like virus may be circulating in the northeastern region of Argentina given that half of the analyzed animals showed high levels of neutralizing antibodies against the pestivirus. The HoBi-like seropositive animals were also checked for neutralizing antibodies against BVDV-1a, BVDV-1b, and BVDV-2, and in most cases these animals had low levels or no detectable antibodies against these other pestiviruses. Our study suggests a need for continued pestivirus surveillance in Argentinean cattle and buffalo.

Bovine Viral Diarrhea Virus Infects Monocyte-Derived Bovine Dendritic Cells by an E2-Glycoprotein-Mediated Mechanism and Transiently Impairs Antigen Presentation.

Infection of professional antigen presenting cells by viruses can have a marked effect on these cells and important consequences for the generation of subsequent immune responses. In this study, we demonstrate that different strains of bovine viral diarrhea virus (BVDV) infect bovine dendritic cells differentiated from nonadherent peripheral monocytes (moDCs). BVDV did not cause apoptosis in these cells. Infection of moDC was prevented by incubating the virus with anti-E2 antibodies or by pretreating the cells with recombinant E2 protein before BVDV contact, suggesting that BVDV infects moDC through an E2-mediated mechanism. Virus entry was not reduced by incubating moDC with Mannan or ethylenediaminetetraacetic acid (EDTA) before infection, suggesting that Ca(2+) and mannose receptor-dependent pathways are not mediating BVDV entry to moDC. Infected moDC did not completely upregulate maturation surface markers. Infection, but not treatment with inactivated virus, prevented moDC to present a third-party antigen to primed CD4(+) T cells within the first 24 hours postinfection (hpi). Antigen-presenting capacity was recovered when viral replication diminished at 48 hpi, suggesting that active infection may interfere with moDC maturation. Altogether, our results suggest an important role of infected DCs in BVDV-induced immunopathogenesis.

Desarrollo de una vacuna mejorada de la subunidad del virus de la diarrea viral bovina basada en la glicoproteína E2 fusionada con un anticuerpo de cadena única que se dirige a las células presentadoras de antígenos / Development of an enhanced bovine viral diarrhea virus subunit vaccine based on E2 glycoprotein fused to a single chain antibody which targets to antigen-presenting cells

Bovine viral diarrhea virus (BVDV) is an important cause of economic losses worldwide. E2 is an immunodominant protein and a promising candidate to develop subunit vaccines. To improve its immunogenicity, a truncated E2 (tE2) was fused to a single chain antibody named APCH, which targets to antigen-presenting cells. APCH-tE2 and tE2 proteins were expressed in the baculovirus system and their immunogenicity was firstly compared in guinea pigs. APCH-tE2 vaccine was the best one to evoke a humoral response, and for this reason, it was selected for a cattle vaccination experiment. All the bovines immunized with 1.5Ag of APCH-tE2 developed high levels of neutralizing antibodies against BVDV up to a year post-immunization, demonstrating its significant potential as a subunit vaccine. This novel vaccine is undergoing scale-up and was transferred to the private sector. Nowadays, it is being evaluated for registration as the first Argentinean subunit vaccine for cattle

El virus de la diarrea viral bovina (BVDV) es causante de importantes pérdidas económicas a nivel mundial. La proteína E2 es la inmunodominante del virus y es la candidata para desarrollar vacunas de subunidad. Para mejorar su inmunogenicidad, una versión truncada de la E2 (tE2) se fusionó a un anticuerpo de cadena simple (APCH), que se dirige a las células presentadoras de antígeno. Se expresaron las proteínas APCH-tE2 y tE2 en el sistema de baculovirus y su inmunogenicidad fue evaluada y comparada en cobayos; la proteína APCH-tE2 fue la que indujo la mejor respuesta humoral. Por dicha razón se la evaluó en bovinos utilizando 1,5µg de antígeno. Los animales presentaron altos títulos de anticuerpos neutralizantes contra BVDV hasta un año posinmunización. Esta nueva vacuna está en proceso de escalado y se transfirió al sector privado. Actualmente se está evaluando para su registro como la primera vacuna argentina de subunidad para bovinos

Development of an enhanced bovine viral diarrhea virus subunit vaccine based on E2 glycoprotein fused to a single chain antibody which targets to antigen-presenting cells.

Bovine viral diarrhea virus (BVDV) is an important cause of economic losses worldwide. E2 is an immunodominant protein and a promising candidate to develop subunit vaccines. To improve its immunogenicity, a truncated E2 (tE2) was fused to a single chain antibody named APCH, which targets to antigen-presenting cells. APCH-tE2 and tE2 proteins were expressed in the baculovirus system and their immunogenicity was firstly compared in guinea pigs. APCH-tE2 vaccine was the best one to evoke a humoral response, and for this reason, it was selected for a cattle vaccination experiment. All the bovines immunized with 1.5 µg of APCH-tE2 developed high levels of neutralizing antibodies against BVDV up to a year post-immunization, demonstrating its significant potential as a subunit vaccine. This novel vaccine is undergoing scale-up and was transferred to the private sector. Nowadays, it is being evaluated for registration as the first Argentinean subunit vaccine for cattle.

Validation of an indirect ELISA to detect antibodies against BoHV-1 in bovine and guinea-pig serum samples using ISO/IEC 17025 standards.

Two ELISAs to quantify antibodies to BoHV-1 in the sera of cattle and immunized guinea pigs were developed and validated using ISO/IEC 17025 standards. The cut-off value of the assay was established at 20% positivity of a high positive control for screening of cattle. Using this threshold, the assay properly classified the OIE bovine reference sera EU1, EU2 and EU3. For vaccine potency testing, a cut-off of 40% was selected for both species. The reliability of the assays, given by their diagnostic sensitivity and specificity, using the threshold of 40% was 89.7% and 100%, respectively, for bovines and 94.9% and 100% for guinea pigs, respectively. There was almost perfect agreement between the ELISA and virus neutralization results. In addition, after vaccination, there was a good correlation between the neutralizing and ELISA antibody titers of the serum from the same bovine or guinea pig, sampled at 60 and 30 days post-vaccination, respectively (R(bovine)=0.88, R(guinea pig)=0.92; p<0.0001). A similar correlation was observed when analyzing the mean antibody titers of groups of vaccinated animals (R(bovine)=0.95 and R(guinea pig)=0.97; p<0.0001), indicating the relevance of the ELISAs for batch to batch vaccine potency testing in the target species and in the laboratory animal model. The intermediate precision of the assays expressed as the relative coefficient of variation (CV) of the positive control assayed over a 3-year period in the same laboratory was 22.2% for bovines and 23.1% for guinea pigs. The reproducibility of both techniques obtained in inter-laboratory assays was CV=12.4% for bovines and CV approximately 0 for guinea pigs, which met the requirements of the OIE (CV<30%). The validated ELISAs represent important methods for vaccine potency testing and for controlling BoHV-1 infections.