Identification And Pathogenicity Of Novel PRRSV Variants

Porcine Reproductive and Respiratory Syndrome(PRRS)is an important animal disease that seriously endangers the global pig industry and causes significantly economic losses.Porcine Reproductive and Respiratory Syndrome Virus(PRRSV)is one of the most rapdily evolving RNA viruses.Since it was detected in China in 1995,multiple genotypes and subtypes of PRRSV isolates have been found in Chinese swine herds,which causes huge prevention and control pressure to combat PRRS.Aim at the above scientific issues,in this dissertation,firstly,universal and quadruplex PRRSV real-time RT-PCR assay were established for simultaneously detection and differentiation of distinct PRRSV isolates.Secondly,we isolated two highly pathogenic PRRSV(HP-PRRSV)isolates with high genomic homology but from two farms showing distinct clinical manifestation.Further animal challenge study confirmed that they have different pathogenicity and whole genome sequence analysis identified the differential amino acid residues;Thirdly,we designed,constructed and rescued the infectious clones of these two isolates,which provides a platform for investigating their distinct pathogenicity.1.Establishment of universal and quadruplex PRRSV real-time RT-PCR assayIn this study,two pairs of primers and a TaqMan-MGB probe were designed for the detection of PRRSV 1 and PRRSV2;four pairs of primers and four TaqMan-MGB probes were designed for simultaneous detection and differentiation of four major groups of PRRSV isolates.The developed universal and quadruplex real-time RT-PCR assay exhibited good specificity,sensitivity,repeatability and reproducibility by optimizing the detection system and procedure.In addition,the newly developed real-time RT-PCR assays were further validated by comparing with a universal PRRSV conventional RT-PCR assay on the detection of 664 clinical samples collected from 2016 to 2019 in China.Based on the clinical performance,the agreements between the universal and quadruplex real-time RT-PCR assays and the conventional RT-PCR assay were 99.55%and 99.40%,respectively.Two samples were determined to be co-infected with NL-PRRSV2 and HP-PRRSV2 isolates by the quadruplex real-time RT-PCR assay,demonstrating that this assay has the ability to determine co-infection.This study provides promising alternative tools for simultaneous detection and differentiation of PRRSV circulating in Chinese swine herds.2.Identification and pathogenicity analysis of novel highly pathogenic PRRSV variants XJ17-5 and JSTZ712-12During the detection process of routine epidemiological investigation by quadruplex real-time RT-PCR assay in 2017,two PRRSV variants were identified from a severe abortion farm and a clinically healthy farm,respectively.The viruses were isolated and denominated as XJ17-5 and JSTZ1712-12.Genomic sequencing indicated that their genomes are both 14,960 bp in length sharing 99.45%nucleotide identity.Sequence alignments identified a discontinuous 30-amino-acid deletion and a continuous 120-amino-acid deletion in nsp2 of both isolates.Genome-based phylogenetic analysis confirmed that XJ17-5 and JSTZ1712-12 belong to the HP-PRRSV subtype but form a new branch with other isolates containing the same 150-amino-acid deletion in nsp2.Pathogenic analysis showed that XJ17-5 is highly virulent causing 60%mortality,while JSTZ1712-12 is avirulent for piglets.Furthermore,fragment comparisons identified 34-amino-acid differences between XJ17-5 and JSTZ1712-12 that might be associated with the distinct virulence.The identification of highly homologous HP-PRRSV variants with new genetic feature and distinct virulence contributes to further analyze the pathogenesis and evolution of PRRSV in the field3.Construction and rescue of XJ17-5 and JSTZ1712-12 infectious clonesTo explore the amino acids associated with the distinct pathogenicity of XJ17-5 and JSTZ1712-12 isolates,XJ17-5 and JSTZ1712-12 infectious clones were designed,constructed and resuced.Based on the rXJ17-5 and rJXTZ1712-12 infectious clones,rXJ-EGFP and rJS-RFP were constructed and rescued to analyze the replication ability of these two rescued viruses.In addition,rXJ-120AA and rJS-120AA were also constructed and rescued to study the influence of nsp2 120AA deletion on virus replication and virulence.Furthermore,chimeric infectious clones of XJ17-5 and JSTZ1712-12 were constructed and rescued to investigate the key regions associated with their pathogenicity.The established reverse genetic systems provide a technical platform to elucidate amino acids associating with their distinct pathogenicity.