Analyzing genetic mutations of porcine reproductive and respiratory syndrome virus strains by serial passages in cell line from field wild strain

Abstract

In this study, we compared the genetic mutation and virulence of the attenuated PRRSV strains obtained by 95 serial passages in Marc-145 cells with the parental virulent strain (designated as BG81) isolated in Vietnam. Results showed that there were marked changes in virulence: pigs inoculated with BG81 exhibited high fever ( 41◦C), which lasted for 12 days, and presented typical clinical symptoms of PRRSV; otherwise, pigs inoculated with BG895 (from passage 95), maintained mean rectal temperature from 39,5oC to 39,9oC, did not develop any significant clinical symptoms. Whole genomes of the attenuated strains were significantly different, but their sequence lengths were conserved, i.e., 15,321 nucleotides. The attenuated strain from passage 95 (BG895) contained 38 nucleotide substitutions that resulted in 14 amino acid changes. Most of these changes (about 65%) occurred before passage 50. The 14 amino acid changes were distributed in Nsp1, Nsp4, Nsp9, Nsp10, GP2, E, GP3, GP4, GP5 and N. Specially, there were two single substitutes within a codon in ORF3, corresponding to parallel mutation at position F143L. However, structural protein (M) and eight non-structural proteins (Nsp2, Nsp3, Nsp5, Nsp6, Nsp7, Nsp8, Nsp11 and Nsp12) among the 19 PRRSV proteins, remained conserved, without any mutations and supposed for consideration as irrelative to the attenuation process. It is interesting that in the gene coding for the smallest structural protein (E protein), there was the highest mutation rate among all of the structural genes analyzed, and genetically, seemed to be a highly variable region. These changes may provide the molecular bases for the observation of the attenuated phenotype in pigs. Thus, our variation results obtained between the attenuated BG895 and the parental virulent BG81 strains provide appropriate molecular data for potential use to test and control the masterseed strain in production of a PRRSV vaccine in Vietnam.