GII.4 human norovirus and G8P[1] bovine-like rotavirus in oysters (Crassostrea gigas) from Argentina.

Bivalve mollusks have been widely recognized as an important source of foodborne virus. The aim of this work was to determine the presence of norovirus (NoV) and rotavirus (RVA) in Pacific cupped oyster (Crassostrea gigas) from Buenos Aires, Argentina. A total of 88 oyster were processed. 7% of pooled samples resulted positive for NoV GII by RT-qPCR. The nucleotide analysis showed that it was closely related to GII.4/Sydney. Regarding RVA, 21% were positive by RT-qPCR targeting the NSP3 gene. RVA from one pool was isolated in cell culture and infective viral particles were evidenced by immunofluorescence. The genotype constellation of RVA/Oyster-wt/Crassostrea gigas_BA/2015/G8P[1] isolated strain was G8-P[1]-I2-R2-C2-M2-A3-N2-T6-E2-H3, which has a bovine-like genome backbone. Notably, RVA possesses an E2 genotype which is different from the characteristic E12 genotype of RVA circulating in animal species from South America. Our findings evidence not only the presence of enteric viruses in oysters from Argentina, but most important the viability of RVA. This result pose the need to implement surveillance programs to prevent potential foodborne viral outbreaks due to the consumption of contaminated shellfish.

Identification of the gamma irradiation dose applied to ground beef that reduces Shiga toxin producing Escherichia coli but has no impact on consumer acceptance.

The aims of the present study were: a) to estimate the minimal dose of gamma irradiation required to reduce 5 log CFU/g of native O157 and non-O157 Shiga toxin-producing Escherichia coli population in ground beef samples inoculated with high inoculum; b) to assess its effectiveness in samples with low inoculum and 3) to evaluate consumer acceptance. Based on the results, 1 kGy was estimated as the minimal dose of gamma irradiation required to reduce 5 log CFU/g of STEC in ground beef. However, when samples with low inoculum level were subjected to 1 kGy, 3.9% of the samples were positive for stx and eae genes after an enrichment step. Consumer acceptance analysis was carried out with samples subjected to 2.5 kGy and no significant differences were found between irradiated and control samples. Therefore, 2.5 kGy was identified as the gama irradiation dose that reduces STEC but has no impact on consumer acceptance of ground beef.

Quantitation of cytokine gene expression by real time PCR in bovine milk and colostrum cells from cows immunized with a bovine rotavirus VP6 experimental vaccine.

In a previous work, VP6 recombinant protein was produced using baculovirus system and it was evaluated in a colostrum-deprived calf model. This vaccine was able to protect calves against viral challenge without inducing neutralizing antibodies (NAb), suggesting that another immunological effectors were involved in the protection observed. In this work, groups of cows (n=4) were immunized in the last third of gestation with a bovine rotavirus (BRV) experimental vaccine and with a VP6 subunit vaccine. At birth, colostrums from vaccinated and non-vaccinated cows were processed and viable colostral mononuclear cells were obtained. With the purpose of determining the cytokine patterns generated by cells from immune secretions (colostrums and milk), a relative quantification by real time PCR was standardized. Quantitative real time PCR (qPCR) was used to determine transcript levels of IL-4, IL-6, IL-10, IL-12, IFN-γ and IFN-α from these cells. Colostral and milk mononuclear cells expressed a different cytokine transcript expression pattern regarding the vaccine used. These results demonstrated that the colostral cellular population was active and could exert its action influencing the final immune response.

VP8* antigen produced in tobacco transplastomic plants confers protection against bovine rotavirus infection in a suckling mouse model.

Group A rotavirus is a major leading cause of diarrhea in mammalian species worldwide. In Argentina, bovine rotavirus (BRV) is the main cause of neonatal diarrhea in calves. VP4, one of the outermost capsid proteins, is involved in various virus functions. Rotavirus infectivity requires proteolytic cleavage of VP4, giving an N-terminal non-glycosilated sialic acid-recognizing domain (VP8*), and a C-terminal fragment (VP5*) that remains associated with the virion. VP8* subunit is the major determinant of the viral infectivity and one of the neutralizing antigens. In this work, the C486 BRV VP8* protein was produced in tobacco chloroplasts. Transplastomic plants were obtained and characterized by Southern blot, northern blot and western blot. VP8* was highly stable in the transplastomic leaves, and formed insoluble aggregates that were partially solubilized by sonication. The recombinant protein yield was 600 µg/g of fresh tissue (FT). Both the soluble and insoluble fractions of the VP8* plant extracts were able to induce a strong immune response in female mice as measured by ELISA and virus neutralization test. Most important, suckling mice born to immunized dams were protected against oral challenge with virulent rotavirus. Results presented here contribute to demonstrate the feasibility of using antigens expressed in transplastomic plants for the development of subunit vaccines.

Passive protection to bovine rotavirus (BRV) infection induced by a BRV VP8* produced in plants using a TMV-based vector.

We have previously reported on the use of a tobacco mosaic virus (TMV) vector TMV-30B to express foreign viral antigens for use as experimental immunogens. Here we describe the development of an improved TMV-30B vector that adds a sequence of 7 histidine residues to the C-terminus of recombinant proteins expressed in the vector. We used this TMV-30B-HISc vector to express the VP8* fragment of the VP4 protein from bovine rotavirus (BRV) strain C-486 in plants. Recombinant VP8* protein was purified from N. benthamiana leaves at 7 days post-inoculation by immobilized metal affinity chromatography. The plant-produced VP8* was initially detected using anti-His tag mAb and its antigenic nature was confirmed using both monoclonal and polyclonal specific antisera directed against BRV. Adult female mice, inoculated by the intraperinoteal route with an immunogen containing 4 microg of recombinant VP8*, developed a specific and sustained response to the native VP8* from the homologous BRV. Eighty five percent of suckling mice from immunized dams that were challenged with the homologous virus at the fifth day of age were protected from virus as compared to 35% of the pups from mothers immunized with a control protein. These results demonstrate that the plant-produced VP8* was able to induce passive protection in the new born through the immunization of dams. This suggests that the technology presented here provides a simple method for using plants as an inexpensive alternative source for production of recombinant anti-rotavirus antigens.