Pathogenicity And Its Molecular Basis Of Genotype 1 PRRSV Strain GZ11-G1

Porcine reproductive and respiratory syndrome (PRRS) is regarded as one of the most severely infectious diseases for swine industry worldwide, and the causative agent is porcine reproductive and respiratory syndrome virus (PRRSV). According to the genetic and antigenic differences, PRRSV can be classified into genotype 1 (European type) and genotype 2 (North American type). Currently much attention has been paid to the emergence of clinical infection of genotype 1 PRRSV in China. Thus it is essential to conduct the study of genotype 1 PRRSV. In the present study, we systematically analyzed pathogenicity of a strain (designated as GZ11-G1) of genotype 1 PRRSV for piglets, and constructed the infectious cDNA clone of this virus, and investigated the molecular basis associated with this virus by means of site-mutagenesis, in an attempt to provide the necessary technology and basis for further study on pathogenesis of genotype 1 PRRSV.Six-week-old SPF piglets were inoculated with GZ11-G1 and vaccine virus (Amervac PRRS) of genotype 1 PRRSV. The body temperatures and clinical symptoms of the inoculated piglets were monitored and recorded, and virus loads in the sera, virus excretion, kinetics of antibody generation, and tissues lesions and their immunohistochemical staining were examined. The results showed that the piglets infected by GZ11-G1 displayed transient fever, respiratory disorder and severe diffuse interstitial pneumonia, indicating that GZ11-G1 is pathogenic for piglets.Based on the whole genome sequences of GZ11-G1 and vaccine virus, respective several genome fragments were amplified by using RT-PCR. One restriction enzyme digestion site was introduced by silent mutagenesis. Then the amplified fragments were inserted into the vector pWSK-29T to construct the full-length cDNA clones pWSK-GZ11-G1 and pWSK-Amervac, respectively. After linearizing and transcription in vitro, RNAs were transfected into BHK-21 cells, and the viable viruses were rescued and identified. The result indicated that the full-length cDNA clones pWSK-GZ11-G1 and pWSK-Amervac were infectious. Two viruses were successfully rescued and named as Rv-GZ11 and Rv-Amervac, which present the similar growth property in vitro to the parental virus.By genome or amino acid sequence alignment and analysis of corresponding regions (5’UTR, 3’UTR, ORF1b and ORF2-6) among 10 strains of PRRSV genotype 1 (including GZ11-G1 and Amervac PRRS), four amino acid mutations in Nsp9-and 10-coding regions and 3 amino acid mutations in GP2-coding region were found, namely the position 93 (G) in Nsp9,the position 281(P), 304 (V), and 401 (K) in Nsp10, and the position 5(H),120 (G) and 252 (S) in GP2. With the cDNA infectious clone and site-mutagenesis technology, six mutated viruses were generated, which named as Rv-G-A-GP2, Rv-A-G-GP2, Rv-A-G-GP2+Nsp9-10, Rv-G-A-GP2+Nsp9-10, Rv-G-A-Nsp9-10 and Rv-A-G-Nsp9-10. The growth curves in vitro showed that the mutated viruses with 3 amino acid sites in GP2 shared a similar kinetics to their parental rescued viruses, and while the mutated viruses with 4 amino acids in Nsp9-10 (Rv-A-G-Nsp9-10, Rv-A-G-GP2+Nsp9-10) has the increased virus titers at the early stage, and wheras the Rv-G-A-Nsp9-10 and Rv-G-A-GP2+Nsp9-10 led to 12-h and 24-h delay of virus peak titer, with lower virus titers at all time points compared with the rescued parental virus (P<0.05). The pathogenicity analysis revealed that, compared with parental virus (Rv-Amervac), Rv-A-G-Nsp9-10 displayed more severe clinical symptoms, viremia and interstitial pneumonia for piglets; in contrast, Rv-G-A-Nsp9-10 could cause short-term viremia with lower virus titer and transient fever, and slighter pathological changes than the parental virus (Rv-GZ11). The above data suggest that 4 amino acids in Nsp9-10 coding region are related to the enhanced growth ability in vitro and in vivo and pathogenicity for piglets of Rv-GZ 11.In summary, our results showed that:(ⅰ) the PRRSV GZ11-G1 belonging to genotype 1 is pathogenic for piglets; (ⅱ) the full-length cDNA infectious clone of PRRSV genotype 1 was successfully constructed; (ⅲ) four amino acids in Nsp9-10 coding region of PRRSV genotype 1 strain GZ11-G1 are related to its growth ability and pathogenicity. Our present studies provide a useful technological platform for further studying molecular mechanism of pathogenesis of PRRSV genotype 1, as well as valuable scientific evidence for illuminating its pathogenesis.