Effects Of Nsp2 Deletions On PRRSV Genome And Replication Efficiency

Abstract

Summary: The effects of various nsp2 deletion mutants on their genome stabilities and virus replication efficiencies were examined in this study. Four different nsp2 variants were identified in NA type PRRSV isolated from Hong Kong pig farms. Among them, three possess 1 (aa482, AFCD04), 50 (aa470-519, AFCD17) and 131 (aa328-453, 483-487, AFCD34) amino acid deletion respectively, and one with no deletion (AFCD37) in nsp2 gene. After 10 successive passages in MARC-145 cells, the nucleotide sequences of ORF1a, ORF5 and ORF7 of the progenies were compared with that of their parental strains. AFCD37 showed the highest genome stability and no nucleotide substitution was observed in ORF1a, ORF5 and ORF7. AFCD04 showed 4 nucleotide substitutions (3 out of 5 were mis-sense mutation) in ORF1a gene after 10 passages but none in ORF5 and ORF7. AFCD17 accumulated 26 nucleotide substitutions and among them 9 substitutions contributed to the alternation in amino acid residues. AFCD34 showed the highest number of substitution among 4 nsp2 variants. A total of 44 nucleotides were substituted and 19 of them led into mis-sense mutation. Besides, the result also shows that substitutions in ORF1a may affect the stability of structural genes. AFCD17 strain ac-cumulated 8 mis-sense mutations within nsp4-6 genes in passage 8, while nucleotide substitution in ORF5 was observed in passage 9. Apart from genome stability, deletion in nsp2 may also affect the replication efficiency of the virus. Replication kinetics profiles reveal that AFCD37 and AFCD04 strains showed no significant difference between parental viruses and progenies. While the progenies of AFCD17 and AFCD34 showed 6.8 fold and 4.7 fold increase in virus titers respectively. To conclude, deletions in nsp2 may contribute to the variations in genome stability of PRRSV. Large segment of deletion leads into an increase in number of nucleotide substitutions in the genome. Furthermore, substitutions in nsp4-7 eventually affect the stability of ORF5 structural gene. Accumulation of substitutions in ORF1a also increases the replication efficiency of PRRSV in MARC-145 cells. Further investigation on the role of nsp2 deletion will be performed by reverse genetic approach.