Molecular Epidemiology Of Porcine Reproductive And Respiratory Syndrome Virus And Effect Of Conserved Amino Acids In Nsp7? On PRRSV Infectivity

Porcine reproductive and respiratory syndrome(PRRS)has caused enormously economic losses since its emergence in the United States in the late 1980 s.The causative agent,PRRSV is characteristic of genetically extensive variation and recombination,leading to the widely spread of numerous mutant strains in the world.PRRSV has very unique and complex immunological characteristics.Until now,the pathogenesis has not been fully revealed yet and PRRS has not been effectively controlled by the traditional strategies.Here,we explore the prevalence of PRRSV and the effects of conserved sites in Nsp7 on the infectivity and proliferation of PRRSV,which will lay the foundation for the prevention and treatment of PRRS(1)Epidemiological investigation of PRRSVIn this study,31 ORF5 and 18 partial Nsp2 genes were amplified and analyzed to investigate the epidemic characteristics of PRRSVs in Central China since 2014.Phylogenetic tree based on ORF5 revealed that,among them,18 isolates were clustered into subgroup 1,represented by the NADC30;11 isolates were clustered into the HP-PRRSVs subgroup(subgroup 3).Homology analysis showed NADC30-like PRRSV isolates in subgroup 1 shared 91.71%-99.34% nucleotides identities with each other.And for the HP-PRRSV isolates in subgroup 3,the nucleotides identities with each other was 98.84%-99.67%.In addition,NADC30-like PRRSV isolates contained extensive amino acid(aa)substitutions in crucial motifs,such as potential neutralization epitopes and the N-glycosylation sites.Interestingly,S32 H,N33D,D34 N,S36G substitutions in GP5 would cause HNhx(NADC30-like)losing all the potential N-Glycosylation sites at aa 30-35.To better understand the characteristics of novel NADC30-like PRRSV circulating in China,we isolate a NADC30-like strain named HNhx and analyze its characteristic.HNhx contains specific mutations at aa 30-35.Full-length HNhx genome sequence was obtained byamplifying 6 overlapping fragments covering the full-length genome using reverse transcription-PCR(RT-PCR).Recombination analysis revealed that HNhx was the result of recombination between the NADC30 virus and HP-PRRSV vaccine strain JXA1-P80.The recombined region located in Nsp4(nt 5,261)to Nsp9(nt 7,911).Our data suggested that the NADC30-like PRRSV strains spread quickly and are now circulating and prevalent in Central China as well as the classical HP-PRRSV strains.HP-PRRSVs were relatively conserved in Central China.Reciprocally,the NADC30-like PRRSV strains underwent extensive mutation and recombination,leading to rapid evolution.(2)Effects of conserved sites in Nsp7 on PRRSV infectivityComparing the complete amino acid sequences of all available Genotype II PRRSV strains,we found a conservative region(aa 30-50)at Nsp7? of all the PRRSVs.In addition,one conserved amino acid site(aa 38)was found in the region of aa 30-50 by comparing the deduced amino acid sequences of Nsp7? of all the members within arterivirus.Research on the conserved region and amino acid site might be helpful to discovering the function of Nsp7? in the PRRSV replication process.So we constructed the reverse genetics system of PRRSV based on the genome of the HP-PRRSV strain HN07-1.To construct the infectious complete c DNA clone,4 fragments covering full-length HN07-1 genome were cloned into p CDNA3.1(+)vector(r HN07-1),respectively.Meanwhile,an infectious c DNA clone expressing foreign gene GFP(r HN07-1-EGFP)as a marker was construceted.The EGFP gene was inserted between the ORF1 b and ORF2 in another infectious clone.The plasmids were transfected into the susceptible Marc145 cells.After one passage,obvious cytopathic effect(CPE)of Marc145 cells was observed.The results of Western blot and immunofluorescent assay(IFA)revealed that rescued viruses were generated successfully.In addition,the growth kinetic of r HN07-1 was similar to HN07-1,which indicated the constructed infectious clone could be used for reverse genetic manipulation.Deletetion or mutation of the conserved region and aa sites in Nsp7?did not produce infectious virus,indicating that the aa 37/38 might be the key sites of Nsp7? influencing the proliferation of PRRSV.