| In order to effectively control and prevent the porcine reproductive and respiratory syndrome (PRRS), so as to accelerate the development of pig-breeding industry, three directions of investigation on PRRS has been performed, which were scientific evaluation on the safety and protective efficacy of attenuated strains of PRRSV, exploration of molecular mechanism of attenuation of PRRSV in vitro, and the phylogenic analysis and molecular characterization of PRRSV in Northern China, Central China and Southern China.Firstly, with the purpose of scitific evaluation on the safety and protective efficacy of BJ-F80, BJ-F150, GD-F80and GDr180, which are attenuated strains of HP-PRRSV BJ and GD, were injected into piglets, and then the piglets inoculated with different PRRSV were evaluated through clinical signs, weight changes, viremia and antibody level till28days post inoculation, subsequently all piglets were challenged with virulent PRRSV, and periodic autopsies for the observation of gross lesion, meanwhile, lungs were collected for the tissue sections, which were then applied for H.E. staining and immunohistochemistry staining (IHC), flurescent quantitative PCR (FQ-PCR) was performed to detect the nucleic acid of PRRSV in lungs and sera, and antibody level of different time were tested with ELISA. Results displayed varying degrees of lesions in piglets inoculated with BJ-F80, BJ-F150and GD-F80at28days post inoculation, while no damage appeared in piglets inoculated with GDr180and piglets of control group. Results of IHC and FQ-PCR showed faster propagation of BJ-F80, BJ-F150and GD-F80in vivo than GDr180, and the antibody level of piglets inoculated with GDr180were higher than that in other groups. To sum up, the safety and immunizing protection of GDr180achieved ideal standard yet BJ-F80, BJ-F150and GD-F80were attenuated not well enough.Secondly, to explore the molecular mechanism of attenuation of PRRSV in vitro, seven and eight strains of different generations of PRRSV BJ and GD were applied for comparative analysis respectively.68and50mutations of nucleotides were found in BJ→BJ-F150and GD→Dr180respectively, and caused37and33variation of amino acids each, notably, several variation sites were found variated repeatedly from one amino acid to another and then change it back in BJ→BJ-F150, same scene was not observed in GD→GDr180, suggested the risk of returning virulence might be associated with the instability of variation. In addition, BJ/BJ-F150, GD/GDr180combined with VR-2332/RespPRRS MLV, JA142/Ingelvac ATP, CH-la/CH-1R and JXA1/JXA1-R were applied for the genomic sequences of comparative analysis,74mutation sites were summarized and suggested the connection with molecular mechanism of attenuation in vitro. Besides that, phylogenic analysis of former-mentioned virulent/attenuated PRRSVs and epidemic PRRSVs of China from1996to2014revealed that, two main branches of epidemic PRRSVs were closely related with the attuated live vaccines, reminded widespread and frequent evolvement of PRRSV might be associated with the over-usage of attenuate live vaccines, and which should be brought to the forefront in clinical production. Lastly, three strains of PRRSV were isolated from clinical samples of Northern China, Central China and Southern China respectively, which were all belonged to the HP-PRRSV of NA-type, and possessed the deletion of1+29amino acids in NSP2. One amino insertion was remarkablely found in GP5of GD-2011, which was never seen in any other reports. Comparative analysis and phylogenic analysis of three newly isolated PRRSV and other PRRSVs of Northern China, Central China and Southern China from1996to2014were performed, results indicated that the genetic diversity of PRRSV in Northern China and Southern China were much more complicated than that in Central China, and several mutations exhibited same variation pattern with the GD→GDr180and JXA1→JXA1-R, which alerted the attention should be paid into the rational usage of attenuated live vaccine. |
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