Evaluation of virus decontamination techniques for porcine embryos produced in vitro.

The objective of this study was to explore approaches to decontaminate embryos either contaminated naturally or under experimental conditions with different viruses. Embryos were obtained from in vitro maturation and fertilisation of porcine oocytes. After 7 days of development, morula and blastocyst stages were exposed for 1 h to the following viruses: encephalomyocarditis virus (EMCV), porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus (PRRSV), and bovine viral diarrhea virus (BVDV) at an infectivity of 100 TCID50/mL. Embryos samples were treated with different washing procedures, which all included the following standard washing solutions: PBS+0.4% BSA (five times for 10 s), Hank's+0.25% trypsin (two times for 60-90 or 120-150 s, or one time of 5 min), Hank's+0.1 mg/mL DNase 1+20 U/mL RNase One (one time of 30 min) and PBS+0.4% BSA again (five times for 10s). Two new approaches were used to improve trypsin treatment, 0.1% hyaluronidase (one time for 5 min) instead of trypsin and a pre-incubation with oviductal cells. Therefore, in the first experiment, oocytes received standard maturation treatments and in the second, they were also co-incubated with oviductal cells for the last 3 h of maturation. The effectiveness of the different washing techniques in removing viruses was evaluated by polymerase chain reaction (PCR) analysis. In the first experiment, trypsin treatment did not eliminate PRRSV, PPV, PCV, and EMCV from contaminated embryos. Surprisingly, treatment with hyaluronidase eliminated all tested viruses. In the second experiment, all viruses tested were removed from the oocytes following the different enzymatic treatments. In conclusion, in vitro embryo decontamination was more effective following exposure to oviductal secretions and hyaluronidase eliminated more virions than trypsin in washing techniques.

Alternative codon usage of PRRS virus ORF5 gene increases eucaryotic expression of GP(5) glycoprotein and improves immune response in challenged pigs.

Pigs exposed to GP(5) protein of PRRSV by means of DNA immunization develop specific neutralizing and protecting antibodies. Herein, we report on the consequences of codon bias, and on the favorable outcome of the systematic replacement of native codons of PRRSV ORF5 gene with codons chosen to reflect more closely the codon preference of highly expressed mammalian genes. Therefore, a synthetic PRRSV ORF5 gene (synORF5) was constructed in which 134 nucleotide substitutions were made in comparison to wild-type gene (wtORF5), such that 59% (119) of wild-type codons were replaced with known preferable codons in mammalian cells. In vitro expression in mammalian cells of synORF5 was considerably increased comparatively to wtORF5, following infection with tetracycline inducible replication-defective human adenoviral vectors (hAdVs). After challenge inoculation, SPF pigs vaccinated twice with recombinant hAdV/synORF5 developed earlier and higher antibody titers, including virus neutralizing antibodies to GP(5) than pigs vaccinated with hAdV/wtORF5. Data obtained from animal inoculation studies suggest direct correlation between expression levels of immunogenic structural viral proteins and immune response.

Sequence of the 3'-terminal end (8.1 kb) of the genome of porcine haemagglutinating encephalomyelitis virus: comparison with other haemagglutinating coronaviruses.

A cytopathogenic coronavirus, serologically identified as porcine haemagglutinating encephalomyelitis virus (HEV), has recently been associated with acute outbreaks of wasting and encephalitis in nursing piglets from pig farms in southern Québec and Ontario, Canada. The 3'-terminal end of the genome of the prototype HEV-67N strain and that of the recent Québec IAF-404 field isolate, both propagated in HRT-18 cells, were sequenced. Overall, sequencing data indicated that HEV has remained antigenically and genetically stable since its first isolation in North America in 1962. Compared with the prototype strain of bovine enteropathogenic coronavirus (BCoV), HEV, as well as the human respiratory coronavirus (HCoV-OC43) showed a major deletion in their ORF4 gene. Deduced amino acid sequences for both HEV strains revealed 89/88, 80, 93/92 and 95/94% identities with the structural proteins HE, S, M and N of BCoV and HCoV-OC43, respectively. Major variations were observed in the S1 portion of the S gene of both HEV strains, with only 73/71% amino acid identities compared with those of the two other haemagglutinating coronaviruses.

Eucaryotic expression of the nucleocapsid protein gene of porcine circovirus type 2 and use of the protein in an indirect immunofluorescence assay for serological diagnosis of postweaning multisystemic wasting syndrome in pigs.

The purpose of this study was to develop a sensitive, rapid, and inexpensive immunofluorescence assay (IFA) using a recombinant porcine circovirus type 2 (PCV2) nucleocapsid protein for the serological detection of PCV2-specific antibodies in pig sera. The viral nucleocapsid protein encoded by the PCV2 ORF2 gene has recently been identified as the most immunoreactive viral protein that carries type-specific antigenic determinants. The ORF2 sequence of the IAF-2897 strain of PCV2 has been cloned into a pCEP5 eucaryotic expression vector under the control of the cytomegalovirus promoter, downstream of a polyhistidine sequence tag. The recombinant plasmid was used in transfection experiments with human epithelial kidney 293 cells that were further tested, and positive expression of the viral nucleocapsid protein was confirmed by IFA and Western blotting. Strong, specific fluorescence was observed in the nuclei of transfected cells. Test specificity to PCV2 was verified with several related infectious agents. Sensitivity was compared to that of standard IFA using PCV2-infected cells by evaluating the reactivities of 44 field serum samples from pigs on farms with a porcine population suffering from postweaning multisystemic wasting syndrome. The recombinant nucleocapsid-based test was able to detect 15 more positive-testing pigs than the PCV2-based IFA. Therefore, the relative sensitivity of the latter test was estimated at only 57.1% compared to that of the recombinant nucleocapsid-based test. The recombinant fusion protein has been purified by affinity chromatography and is being used to develop further sensitive serological tests.

Increased proteolytic activity and matrix metalloprotease expression in lungs during infection by porcine reproductive and respiratory syndrome virus.

The local increase in the secretion of extracellular proteases, allowing cleavage of the extracellular matrix and thereby facilitating the infiltration of T cells, monocytes and neutrophils, is a hallmark of chronic inflammation and autoimmunity. In pulmonary genetic diseases, such as emphysema and cystic fibrosis, proteases can also favour the development of local immunodeficiency by degrading key regulators of the immune response, such as CD4, CD8, IgG, ICAM-1 and C3b receptors. Since several infectious agents can give rise to severe pulmonary disorders associated with opportunistic infections, we sought to determine whether an increase in proteolytic activity occurred during infection with porcine reproductive and respiratory syndrome virus (PRRSV), the causative agent of a new disease in swine characterized by severe respiratory problems in young pigs. Piglets were infected with the virus and bronchoalveolar lavages were collected at various times post-infection to measure the net proteolytic activity. It was shown that PRRSV infection leads to a significant increase in proteolytic activity in pulmonary fluids. Maximal activity was found at 7 and 14 days post-infection, with a return towards normal levels at day 42. Zymographic analyses showed a significant increase in the secretion of matrix metalloproteases (MMPs) 2 and 9, two enzymes involved in tissue remodelling. Histological analyses showed a correlation between the increase in proteolytic activity and the appearance of lesions that were characterized by massive lymphomononuclear cell infiltration. These results suggest that virus infection of the lungs can lead to a transient increase in proteolytic activity that could favour opportunistic infection.