Genetic Diversity Analysis Of PRRSV In Partial Areas Of China And The Contribution Of NSP9 Mutations To Viral Pathogenicity

Porcine reproductive and respiratory syndrome(PRRS),caused by porcine reproductive and respiratory syndrome virus(PRRSV),is characterized by reproductive failure in sows and respiratory disease in pigs,and it is a significant threat to global pig industry.PRRSV is an RNA virus,and its genome is relatively susceptible to mutation during replication,resulting in a PRRSV variant,accompanied by changes in the biological properties of the virus.Since PRRSV was first reported in China in 1996,a series of PRRSV variants have emerged successively.For example,the highly pathogenic PRRSV strain that broke out in 2006 and the NADC30-like PRRSV and GM-like PRRSV that have emerged in recent years.Due to continuous and rapid variation of PRRSV genes and antigens,the prevention and control of PRRS is more difficult.In order to understand the molecular epidemiology of PRRSV,we sequenced ORF5 sequences of 217 PRRSVs from clinical samples,retrieved all the available ORF5 sequences of PRRSVs isolated in China in 1996–2016(n = 2,213,covered 32 provinces)from GenBank,and systematically analyzed corresponding epidemiological data.The results showed that NA-type PRRSVs in China were classified into five lineages: lineage 1,lineage 3,lineage 5,lineage 8,and lineage 9.The difference at the amino acid level among lineages was above 10%.Most strains in China belonged to lineage 8(85.6%).Importantly,the emerging lineage 1 and lineage 3 strains spread rapidly,and their proportions among circulating PRRSVs have significantly increased in recent years.Because the overwhelming majority of strains were classified as belonging to lineage 8,they were further divided into seven sublineages,designated as sublineages 8.1–8.7.Furthermore,the geographical distribution of different PRRSV lineages in each province was analyzed.On the basis of the collected data,analyzed phylogenies,geographic distribution,and circulation of pigs in swine trade,possible transmission routes were drawn.All Chinese EU-type PRRSVs belonged to lineage 1.In our study,we found two consistent amino acid mutations that differed between HP-PRRSV and classical PRRSV and were located at positions 519 and 544 in non-structural protein 9.Next,a series of mutant viruses with either single or double amino acid replacements were generated based on HP-PRRSV HuN4 and classical PRRSV CH-1a infectious cDNA clones.Deletion of either of the amino acids led to a complete loss in virus viability.In both Marc-145 and porcine alveolar macrophages,the replicative efficiencies of mutant viruses based on HuN4 were reduced compared to the parent,whereas the replication level of CH-1a-derived mutant viruses was increased.Plaque growth assays showed differences between mutant and parental viruses.In infected piglets,the pathogenicity of HuN4-derived mutant viruses,assessed through clinical symptoms,viral load in sera,histopathology examination,and thymus atrophy,was reduced.Our results indicated that the amino acids at positions 519 and 544 in NSP9 were involved in the replication efficiency of HP-PRRSV and contributed to enhance pathogenicity.In this study,dominant lineages were classified further into sub-lineages and this taxonomy may form a standard for the classification of future PRRSV strains for different researchers.These results may help to understand genetic evolution of PRRSVs and provide a basis for the prevention of PRRS in China.At the same time,this study is the first to identify specific amino acids involved in PRRSV replication or pathogenicity.These findings will contribute to understanding the molecular mechanisms of PRRSV replication and pathogenicity.