Construction And Biological Characteristics Of Porcine Reproductive And Respiratory Syndrome Virus Mutants With Deletions In Non-structural Protein 2 Region

Porcine reproductive and respiratory syndrome?PRRS?is an important viral disease that affects the pig industry.The disease is characterized by the clinical signs of reproductive disorders in sows and respiratory difficulties in piglets and adult pigs.Since the outbreak of highly pathogenic PRRS virus?HP-PRRSV?in China in 2006,it has caused huge economic losses to the pig industry.PRRSV is highly variable,and there are many attenuated vaccine strains approved for use in China,which leads to frequent recombination between field and vaccine strain viruses.New variant strains are constantly appearing,which brings a great challenge for prevention and control of PRRSV prevalence.Although attenuated vaccines can provide protective immunity,there are still many problems in inducing cross-proection and distinguishing between vaccinated and natural infected animals.Studying on attenuated PRRSV marker strain by gene deletion may provide clues to solve the above problems.Non-structural protein 2?NSP2?is a highly variable protein encoded by the PRRSV genome,and can endure mutations,insertions and large fragment deletions in the hypervariable regions of NSP2.There are increasing evidences shows that NSP2 is one of the determinants of the virus pathogenicity.Therefore,NSP2 is an important candidate region to develop the attenuated marker virus by deletions in this gene fragments.In the previous study,one mutant virus with deletion in the amino acids sites519-565 of NSP2?named D5?was rescued based on PRRSV/GSWW/2015 infectious molecular clone.On this basis,two Full-length molecular clones with amino acid deletions at positions 467-570?pGA-AB-D13?and 628-747?pGS-AB-D14?of NSP2 were constructed separately refering to the natural deletion region of TJM vaccine strains of PRRSV reported in recent years.The full-length plasmids were linearized and transcribed in vitro to obtain whole length virus RNA genome that were applied to transfect Marc-145 cells to rescue the virus.The results showed that the construction with D13 deletion failed to get the live virus,and the construction with D14 deletion rescued the virus,indicating that the amino acids deletion at positions 628-747 of NSP2 did not affect the replication of the virus.The D14 virus was continuously passaged in Marc-145 cells for 40 generations,and the deletion region was confirmed to be stable by RT-PCR and sequencing.The replication ability of D5,D14 and parental viruses were compared in Marc145 cells by measure the TCID₅₀ and replication kinetic curves.The results showed that the rescued viruses have the similar growth characteristics to the parental virus.Compared with D5 and the parental virus,the replication level of D14 virus was slightly decreased,but without significant difference among them.In order to determine the effect of different deletions in NSP2 region on the inflammatory cytokines expression,the parental virus,D5 and D14 deletion strains were applied respectively to infected porcine alveolar macrophages?PAM?,and the mRNA levels of these cytokines were detected at different time points.The results showed that the mRNA levels of IL-6,TNF-?,IL-8 and IL-1?in D5-infected cells were significantly increased at 24 h compared with the parental virus and D14 deletion virus?P<0.05?.The mRNA levels of IL-10 and HMGB1 infected by D5 and D14 viruses were significantly decreased?P<0.05?at 12h point after infection,and the decrease of D14 was even more significant,suggesting that deletion at 519-565 amino acids of NSP2 promoted early inflammatory response.The double deletion at 519-565 and 628-747 of NSP2 significantly decreased the mRNA levels of IL-10 inhibitory cytokines and late inflammatory factor HMGB1?P<0.05?.These results indicate that NSP2 play certain roles in regulating inflammatory response and immunosuppression.