Reproduction de : Thèse d'exercice : Médecine vétérinaire : Toulouse 3 : 2008. / Titre provenant de l'écran titre. Bibliogr. p. 81-86.

Nipah virus (NiV) is a highly pathogenic and zoonotic paramyxovirus in the subfamily Paramyxovirinae, genus Henipavirus. The virus causes outbreaks of severe febrile encephalitis with a high mortality rate in humans, and of encephalitic and respiratory disease but with a low mortality rate in pigs. The innate immune response has a critical role in limiting viral infection by activating antiviral state and adaptive immune response. As pigs are able to overcome the infection with NiV, the working hypothesis was that IFN induced signaling pathways are not completely inhibited by NiV in infected porcine cells enabling an antiviral state to be established. Indeed, there was no block of eIF2α phosphorylation in porcine fibroblast (ST) and monocytic-like (IPAM) cells, and human fibroblast (MRC5) cells. To address the potential activation of an alternative IFN induced pathway, the MAPK signaling pathways were examined. The findings revealed that NiV infection triggers different kinetics of phosphorylation of ERK and p38 MAPK in the selected cell types. The data also indicates that p38 MAPK to be indispensable for NiV replication in vitro especially in immune cells. As the involvement of immune cells in viral spread and in immune modulation of porcine adaptive immune response were reported. The next hypothesis stated that NiV infects immune cells and affects the population frequencies of PBMC in pigs. In vitro, productive viral replication was detected in monocytes, CD6+CD8+ T lymphocytes and NK cells, by recovery of infectious virus, anti-genomic RNA and detection of structural N and non-structural C proteins. B lymphocytes, CD4-CD8-, as well as CD4+CD8- T lymphocytes were not permissive to NiV. In NiV infected piglets, the expansion of the CD4+CD8- T cells early post infection was consistent with a functional humoral response. In contrast, significant drop in CD4+CD8- T cell frequency was observed in piglets which succumbed to the experimental infection, supporting vaccine studies that antibody development is a critical component of protective immune response. Thus, both aspects of innate and adaptive immune response are affected and contribute to NiV pathogenesis. These findings will help researchers to design and establish vaccination programs that would be more effective in pigs.

Two types of porcine circovirus (PCV) exist, referred to as PCV1 and PCV2. PCV2 has been associated with disease syndromes in pigs, including that designated postweaning multisystemic wasting syndrome (PMWS), which has been identified in all regions of the world bar Australia (Hamel et al., 1998; Allan et al., 1999a; Onuki et al., 1999; Martelli et al., 2000; Kyriakis et al., 2000; Wellenberg et al., 2000; Done et al., 2001; Trujano et al., 2001; Saradell et al., 2004; Castro et al., 2004; Jemersic et al., 2004; Maldonado et al., 2004; Wang et al., 2004; Motovski and Segales, 2004; Garkavenko et al., 2005). PMWS affects young weaner pigs and results in weight loss, tachypnea, dyspnea, enlarged lymph nodes and jaundice (Harding, 1998). PCV2 may also cause or contribute to other swine diseases such as congential tremors (CT) (Stevenson et al., 1999), porcine dermatitis and nephropathy syndrome (PDNS) (Rosell et al., 2000), reproductive failure (Meehan et al., 2001) and several other emerging disease syndromes. PCV1 is currently considered to be non-pathogenic. Although PMWS has not been reported in Australia, information on the distribution, variation and further characterisation of PCV in Australian pigs was necessary as it might provide insights into why there is no PCV-associated disease in this country. The results reported in this thesis involved the detection and further study of porcine circovirus in Australia. This chapter provides an outline of this thesis and the work undertaken, while Chapter 2 is a review of the relevant literature with particular reference to circoviral diseases. Chapter 3 describes the detection of both PCV1 and PCV2 in the Australian pig herd, using a multiplex PCR designed to differentiate between the two viral types. The association of Australian PCV with two disease outbreaks was also investigated. Following the detection of both viruses, it was important to genetically compare Australian PCV with overseas strains known to cause disease, and this was achieved with a sequencing and phylogenetic study as described in Chapter 4. Possible reasons for the genetic groupings and distribution of different PCV2 strains worldwide are also discussed in this chapter. As PMWS is as yet unidentified in Australian pigs, the importation of pig meat into Australia from countries with the disease requires careful monitoring. Current protocols for the cooking of imported pig meat were designed to inactivate porcine reproductive and respiratory disease virus (PRRSV), and as such may not be effective against PCV. In this study (Chapter 5), Australian PCV2 was successfully infected into cell culture, and detected using a variety of techniques. Subsequently, thermal stability experiments were performed using a newly-developed immunoperoxidase (IPMA) test. It was anticipated that this study would determine whether current importation protocols require revision, and the results would suggest that this is the case, with PCV2 unaffected by treatment comparable with current cooking protocols. While no animal experiments were undertaken in this study, it may become necessary to infect pigs with Australian PCV to determine viral pathogenicity. Cell culture inoculums have been used in the past overseas, but problems with contamination and viral titre have been encountered (Fenaux et al., 2001). Viral infectious clones can be used to overcome these problems, so an infectious clone of Australian PCV2 was constructed, as described in Chapter 6. While time constraints prevented the clone from being infected into culture, it is anticipated that the construct would be infectious as it is based on a previously published method (Hattermann et al., 2004). Chapter 7 is a general discussion of the results and conclusions from this study. The detection and characterisation of Australian PCV as described in this study has provided further information on the status of PCV in the Australian pig herd, and also developed diagnostic tests to assist in future research. These tools will be important when assessing and managing the risk of Australia experiencing PCV-associated diseases.

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Sex pre-selection in livestock offspring to produce dairy cows and specific male and females lines for heterosis in swine are important economic goals in animal husbandry. The long-term goal of the study is to separate porcine and bovine chromosome X- or Y-bearing sperms using sex-specific antibodies. H-Y antigen encoded by jumonji, AT-rich interactive domain 1d (Rbp2-like) (Jarid1d) is defined as a male histocompatibility antigen that causes rejection of male skin grafts by female recipients of the same inbred strain of rodents. Porcine and bovine JARID1D genes are estimated between 3.5 kb and 4.5 kb, however there was only 457 bp and 99 bp respectively found in the database. In this study, the porcine and bovine sex-specific genes, JARID1D were cloned using in silico cloning, PCR and cDNA library screening. We used human JARID1D gene as a probe or template to search the ESTs of porcine and bovine in NCBI and TIGR databases and combined the results from two databases and assembled the ESTs using CAP3 program. We also constructed porcine and bovine testis cDNA libraries, and performed porcine testis cDNA libraries screening. To study this model in the mouse, Jarid1d H-Y epitope TENSGKDI was synthesized and used to immune the rabbits. Dot blotting analysis revealed the specificity of the immuned antibodies to mouse Jarid1d H-Y epitope TENSGKDI, but western blotting analysis did not confirm the results. Immunofluorescence analysis of mouse sperms reacted with anti-mouse Jarid1d H-Y epitope TENSGKDI-specific antibody didn¡¦t show a 1:1 ratio of stained sperms to unstained sperms. The specificity of this antibody needed to be improved. The data shows partial JARID1D gene of pig was cloned, and the anti-mouse Jarid1d H-Y epitope TENSGKDI-specific antibodies were prepared, however the specificity of anti-mouse Jarid1d H-Y epitope TENSGKDI-specific antibodies to surface protein expression on mouse sperms needs more confirmations.

Reproduction de : Thèse d'exercice : Médecine vétérinaire : Toulouse 3 : 2008. / Titre provenant de l'écran titre. Bibliogr. p.114-115.

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