| An attenuated PRRSV strain, JXA1-96, was obtained by passaging JXA1-86 on Marc-145 for 10 passages. Nine pairs of specific primers which amplified region was overlapping were designed and synthesized according to PRRSV complete genome sequence published in GenBank to amplify the whole genome fragments of JXA1-86 and JXA1-96. Finally these fragments were successfully cloned to pEasy-T1 vector and sequenced. The sequence results were stitched with DNAStar software, then the complete genome sequences of JXA1-86 and JXA1-96 were obtained.Comparison and analysis of JXA1 and JXA1-86 strain showed that amino acids encoded by Nsp5, Nsp6, Nsp8, Nsp12, M, N proteins did not change, suggesting that virulence attenuated could not be relevant to those proteins. Compared and analyzed the genomic sequences of JXA1/JXA1-86, together with those of other two virulent parental/attenuated vaccine strains, finding that there was higher amino acid mutation rate in structural proteins among the three pairs of strains, the results showed that PRRSV JXA1 strain virulence changes were more related to structure proteins variations. Compared and analyzed the genomic sequences of each generation PRRSV sequence, the variations among A928V, I1155M, E1629D in ORF1(A928V, I1155M in Nsp2, E1629D in Nsp3), I118V in GP2, H79N in GP3, I124V in GP4 and K59N in GP5 between 70th and 86th generations were found, which turned out that virulence attenuated may be associate with those variations.Nsp2, Nsp3, GP2, GP3, GP4 and GP5 protein of JXA1 and JXA1-86 were analyzed by bio informatics to explore the impact of mutations on protein, speculate the possible reasons of attenuation and provide a theoretical basis for the development of highly pathogenic PRRSV attenuated vaccine. |
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