Isolation And Identification Of A PRRSV Strain From Xinjiang And Its Effect On Abundance Of Swine FcRn Protein

Porcine reproductive and respiratory syndrome(PRRS),caused by porcine reproductive and respiratory syndrome virus(PRRSV),is an acute contact infectious diseases leading to severe respiratory diseases.Highly pathogenic porcine reproductive and respiratory syndrome(HP-PRRS)has occurred in Jiangxi,Henan,Hunan and other provinces since 2006,which has brought severe economic losses to the pig industry in China.It is of great significance to explore the genetic evolution and pathogenic mechanism of PRRSV.As one of the important components of the immune system,mucosal immunity mainly plays a role in mediating the transfer of Ig A to the mucosal surface through the polymeric immunoglobulin receptor(p Ig R).In recent years,studies have found that a certain amount of Ig G is also present in mucosal secretions.Whose transport to the mucosal surface is mainly mediated by neonatal Fc receptor(Fc Rn),Ig G protects the body from pathogens playing an important role in the invasion process.Via the uterine vaginal mucosa or the respiratory mucosa,Body’s mucosal immunity can be strongly disrupted by the infection of PRRSV,which can induce strong immune suppression.So Fc Rn may also play an important role in PRRSV infection.At present,a large amount of research on Fc Rn is mainly focused on humans and mice,and there is little research on porcine Fc Rn and its function in virus-infected organisms.Based on the research of h Fc Rn function and regulatory mechanism of p Fc Rn exposed to pathogenic,the study demonstrates the affection of p Fc Rn and regulation principle in PAMs exposed to PRRSV.This study concentrates on the effects of PRRSV infection on the expression of Fc Rn,deeply analyzing the molecular mechanism of PRRSV affecting Fc Rn expression,with a view to providing clues to the molecular mechanism of PRRSV immune escape and persistent infection.The main results of studies were as follows: 1.Isolation and identification of HP-PRRSV and analysis of genome-wide genetic evolutionA PRRSV strain was successfully isolated from the blood of suspected PRRSV infected pigs in Xinjiang Uygur Autonomous Region in 2018,and named as XJ-China-2018.In this study,the Marc-145 cell line was used to isolate and culture the clinical material.The isolate was subjected to examinations by RT-PCR,plaque test,IFA identification.After plaque purification,the complete genome of the isolate was amplified by RT-PCR,spliced by Seq Man software,and genetically evolved with the PRRSV whole genome,Nsp2 and ORF5 gene nucleotide sequences published on the NCBI.The results showed that clinical material was positive of nucleic acid of PRRSV.The CPE of Marc-145 cells were appeared after 72 h of post-infection.Antigenicity positive were proved by IFA tests.The isolated strain appertained to the highly pathogenic PRRSV subgroup of China according to its whole genome,Nsp2 and ORF5 nucleotide sequence.2.Effect of PRRSV infection on pulmonary alveolar macrophages on Fc Rn expression and molecular mechanismFirst of all,automatic cell counter and indirect immunofluorescence was used to exclude the effect of cell death induced by PRRSV infection on detecting Fc Rn expression of PRRSV-affected PAMs.It was found that the level of Fc Rn protein can significantly down-regulate in PRRSV-affected PAMs by Western blot,whether by avirulent or virulent strains.What’s more,this down-regulation trend was dose-dependent and may be related to virus replication.In order to explore the molecular mechanism of PRRSV down-regulating Fc Rn in PAMs,protease inhibitor MG132 and apoptosis inhibitor Z-VAD-FMK were used to treat PAMs cells after PRRSV infection,and only MG132 was found to significantly inhibit the trend of Fc Rn’s downregulation in PAMs infected by PRRSV.The above results indicate that PRRSV infection might induce the degradation of Fc Rn via the proteasome pathway,which leads to the downregulation of Fc Rn protein levels in PAMs.In summary,this experiment successfully isolated a strain of PRRSV from clinical samples,and studies have shown that PRRSV might reduce the level of Fc Rn protein through the ubiquitinated proteasome pathway.This study provided a theoretical basis for the molecular mechanism of mucosal immunity suppressed by PRRSV infection,and offered clues for the study of PRRSV immune escape and persistent infection.