Analysis Of The Correlation Between Porcine Reproductive And Respiratory Syndrome Virus Replication And Its Pathogenicity And Superinfection

Porcine reproductive and respiratory syndrome virus(PRRSV)is an important pathogen impacting economically global swine industry.The emerging highly pathogenic PRRSV(HP-PRRSV)and its epidemic brought about incalculable losses to the Chinese pig production.Compared with classical PRRSV strains,HP-PRRSV shares strong replication efficiency and fatal virulence for pigs,causing atypical PRRS which is characterized by high fever,high morbidity and high mortality.To date,the molecular mechanisms of the correlation between PRRSV replication and its pathogenicity are not understood fully.This study focuses on the critical amino acids in nsp9 and nsp 10 which are responsible for the replication ability and virulence of HP-PRRSV,and meanwhile dissects the relationship between the replication and superinfection resistance of PRRSV.The aim of our present work is to provide valuable evidence for better understanding the molecular mechanism of the correlation between PRRSV replication and its pathogenicity.Previous studies have demonstrated that both nsp9 and nsp 10 contribute the fatal virulence of the Chinese HP-PRRSV.To futher analysze how nsp9 and nsp 10 affect PRRSV distribution and viral loads in vivo,six-weeks healthy piglets were intranasal ly inoculated with the chimeric viruses with the swapped nsp9-and nsp 10-coding regions(RvJHn9n10 and RvHJn9n10)between their parental viruses(RvJXwn and RvHB-1/3.9)and the virus loads in different tissues were detected on 7 d and 14 d post-inoculation.Compared with RvJXwn,RvJHn9n10-inoculated pigs presented less lung lesions and significantly reduced viral loads in lung and inguinal lymph nodes(ILNs);the ORF7 gene copy numbers and PRRSV antigen in lung,thymus,tonsil,spleen,ILNs,submandibular lymph nodes(SLNs),mesenteric lymph nodes(MLNs),and hilar lymph nodes(HLNs)decreased significantly,so did the positive rates of ORF7 gene in other tissues(liver and stomach etc.).On the contrary,compared with RvHB-1/3.9,RvHJn9n10-inoculated pigs displayed more severe inflammatory cellular infiltration,interstitial expansion and hemorrhage in lung,accompanied with higher virus loads in these tissues,and higher ORF7 gene positive rates in other tissues(liver and stomach etc.).The results indicated that nsp9 and nsp10 of HP-PRRSV contributed to the severity of lung lesions,and virus distribution and loads in vivo,adding the data in relation to nsp9 and nsp 10 affecting PRRSV replication efficiency and pathogenicity.The potential amino acid sites in nsp9 and nsp10 affecting the replication efficiency and pathogenicity of PRRSV were identified in this study.We used the chimeric full-length cDNA clones pWSK-RvJHn9n10 and pWSK-RvHJn9nlO to construct a serial of full-length cDNA clones with mutated differential amino acid sites in nsp9 and nsp10 between JXwn06 and HB-1/3.9.The replication efficiencies of these mutated viruses in PAMs were examined and compared by drawing growth curves,together with the parental viruses(RvJXwn,RvHB-1/3.9,RvJHn9n10 and RvHJn9n10).The results showed that the amino acid in the position 586 and 592 of nsp9 effected the virus replication efficiency.To further confirm the influence of these two residues on PRRSV replication,the full-length cDNA clone pWSK-RvJXwn and pWSK-RvJHn10 were used to construct a serial of full-length cDNA clones with mutated differential amino acid sites in nsp9 and the mutant viruses were rescued.The results indicated that the mutations of the residues 586 and 592 in nsp9 decreased the virus titers and viral RNA synthesis efficiency of HP-PRRSV in PAMs,while the mutations of the residues 427 and 609 in nsp9 had no effect.Then the mutant viruses based on RvJXwn,RvJHn10 and RvHJn10 were used to analyze the effect of these two residues on viral pathogenicity.Our data revealed that the mutation of Thr to Ala in 586 and Ser to Thr in 592 of HP-PRRSV nsp9 alleviated the temperature reaction and lung lesions of the infected piglets,and declined the replication efficiency of HP-PRRSV in vivo and delayed the death time of the infected piglets,resulting in the decreased mortality of piglets.However,the mutations of the residues 427 and 609 displayed no changes in clincal responses,lung leisons,virus loads and mortality of the infected piglets compared with their parental viruses.On the other hand,the mutations of Ala to Thr in the position 586 and Thr to Ser in 592 in RvHJn10 could not enhance the pathogenicity of the virus.Collectively,our results suggest that the residues in the position 586 and 592 of nsp9 are critical sites contributing to the replication efficiency and fatal virulence of HP-PRRSV.The correlation between the superinfection resistance and replication of PRRSV was analyzed.The 3×Myc tag was inserted into RvJXwn,RvHB-1/3.9 and RvCHsx1401 to distinguish the primary and secondary infection of PRRSV.This insertion had no effect on virus replication.In superinfection assay,our results indicated the secondary infection of same PRRSV strain was suppressed by the initial infection in PAMs,and the superinfection resistance could be established within 2 h following primary infection of PRRSV.While the inactivated virions by UV irradiation and CHX treatment could not display superinfection resistance.The data of virus attachment and entry assay as well as vRNA and cRNA synthesis assay suggested that the superinfection resistance of PRRSV appeared at the step of viral RNA synthesis rather than the step of entry.The superinfection resistance between different strains was also investigated.The results showed that RvMyc-HB-1/3.9-.or RvMyc-CHsx 1401-infected PAMs were resistant to RvJXwn infection,RvMyc-JXwn-or RvMyc-CHsx1401-infected PAMs were resistant to RvHB-1/3.9 infection,while RvCHsx1401-infected PAMs could not be resistant to the superinfection of RvMyc-JXwn and RvMyc-HB-1/3.9,indicating that the superinfection resistance does not always occur between different strains and it depends on the strain causing primary infection.The chimeric viruses by swapping different coding regions of RvJXwn based on RvCHsx1401(SJ1a,SJ1b and SJSP)were rescued and used as primary infection strains in superinfection assay.The ORFla and ORF1b of PRRSV were shown to be key coding regions to establish superinfection resistance in PAMs.Our results suggest that the superinfection resistance might confer the protectitive mechanism of PRRSV modified live virus(MLV)vaccines.In summary,our present studies have added further data regarding the fact that nsp9 and nsp10 are closely related to the replication efficiency and pathogenicity of HP-PRRSV,and dissected that the residues in 586 and 592 of nsp9 are critical amino acid sites determining the replication efficiency and fatal virulence of HP-PRRSV,and revealed the role of virus replication in PRRSV superinfection resistance establishment.Our findings help us better understand the molecular mechanism of HP-PRRSV pathogenesis and the protective mechanism of PRRSV MLV vaccines.