Genetic Evolution Analysis Of Porcine Reproductive And Respiratory Syndrome Virus Genome In Shandong Province And Surrounding Areas From 2013 To 2018

Porcine Reproductive and Respiratory Syndrome?PRRS?is a viral infectious disease caused by the Porcine Reproductive and Respiratory Syndrome Virus?PRRSV?.It can cause abortion of sows,premature births,stillbirths,deformed babies and piglets,and the respiratory tract of fattening pigs and other symptoms.It has been 25 years since PRRS was introduced to China in 1995.Especially after 2001,the disease began to occur frequently.In recent years,due to the frequent mutations of PRRSV,the types of strains PRRSV in China are diverse and complicated,which makes the prevention and control of PRRS extremely difficult.In order to understand the prevalence and mutation of Porcine Reproductive and Respiratory Syndrome Virus in Shandong and surrounding areas,this study tested and identified positive samples of 2000 suspected positive pathogens stored in our laboratory since 2013 via RT-PCR.After virus isolation and identification,twenty strains PRRSV were isolated.The twenty strains isolated PRRSV were subjected to RNA extraction and reverse transcribed into cDNA.And the designed full-length 12 pairs of primers were used for whole genome amplification,sequencing and sequence splicing.The spliced gene sequences represent those in GenBank domestic and abroad.The PRRSV reference strains were compared for genetic sequence homology,phylogenetic tree,the important amino acid deduced sites of some NSP2 and ORF5 genes,and analysis of some recombinant strains.The analysis results show that the twenty strains PRRSV isolated from the study form three large branches on the gene phylogenetic tree,namely lineage8/sublineage8.1?CH-1a-like?,lineage8/sublineage 8.7?JXA1-like?and lineage5/sublineage 5.1?VR-2332-like?.Among them,six strains PRRSV have the highest homology with VR-2332 in the reference strains.They are located in the same branch as VR-2332.They belong to sublineage 5.1?VR-2332-like?,a PRRSV strain which has the highest homology with the classic strain CH-1a.The nucleotide and amino acid homology are 99.3%and 98.7%,respectively.They are in the same branch as CH-1a,belonging to sublineage 8.1?CH-1a-like?.There are thirteen strains PRRSV nucleotide homology between them.It is 95.5%-99.9%,amino acid homology is 90.8%-98.1%,and belongs to the same large branch as JXA1,HUN4,etc.and belongs to sublineage 8.7?JXA1-like?.Among them,two strains PRRSV independently formed a small branch in the NSP2 genetic evolutionary tree,and a PRRSV strain independently formed a small branch in the genome-wide genetic evolutionary tree.They were between Lineage 5 and8,which suggested a possible gene recombination phenomenon.According to the amino acid derived from the NSP2 gene,there are thirteen strains PRRSV belonging to the sublineage 8.7.And the highly pathogenic representative JXA1isolated in this study have the same 30 discontinuous amino acid deletion characteristics,in which there are four strains PRRSV?SDGM-7-3-2016,SDJY-2017,SDLC-23-2015,SDRZ-10-12-2015?and five consistenting mutations?422aa,465aa,502aa,590aa,605aa?.A PRRSV strain belong to sublineage 8.1.Compared with CH-1a,has four amino acid mutations?396aa,461aa,482aa,514aa?and a amino acid deletion?632aa?occurred.HBHD-2017 is similar with VR-2332,and SDWF-p8-3-2014 has amino acid mutations and deletions?503aa,595-597aa?.The remaining four had highly pathogenic thirty discontinuous amino acid deletion characteristics.Genetic recombination occurred in the NSP2 ares.The ORF5 gene deduced amino acids found that all isolates mainly showed amino acid mutations.Among them,the virus virulence-related sites were located in the sublineage 5.1isolates.Except for HBHD-2017 from R¹5151 to I¹⁵¹,the remaining isolates were from R¹33 to Q¹³,R¹5151 to K¹⁵¹.The sublineage 8.1 and sublineage 8.7 were characterized by strong strains R¹³and R¹⁵¹.Among the 137aa difference between vaccine strains and wild strains,a total of six strains PRRSV of sublineage 5.1 were identical to the VR-2332 and RespPRRS MLV vaccine strains.The results indicate that they are most likely to return from attenuated vaccine strains to virulent strains.In addition,among the 39aa is important for neutralizing antibody binding,sublineage 5.1 is L³⁹,sublineage 8.1is F³⁹,and sublineage 8.7 is I³⁹.These amino acid changes may make the virus immune escape.Through the analysis of N-glycosylation sites,all isolates are still highly conserved at44-46aa,51-53aa N-glycosylation sites.Compared with VR-2332,isolates in sublineage 5.1had different loss and increase of N-glycosylation sites in 33-35aa and 34-36aa,a isolate PRRSV in sublineage 8.1 did not change compared with CH-1a.Compared with JXA1,Located in sublineage8.7 isolates PRRSV two have increased glycosylation sites at 33-35aa and 34-36aa,lost glycosylation sites at 35-37aa,and three strains lost at 30-32aa.In view of the glycosylation sites,the number of these N-glycosylation sites may affect the virus virulence.From the time of the entire sublineage 8.7 isolates PRRSV,the N-glycosylation sites have gradually neutralized the epitope the tendency to move,which in turn masks the neutralizing epitope,leads to vaccine immunization failure.Genetic recombination analysis showed that there are seven strains isolates exist genetic recombination in all PRRSV isolates.One of the recombination patterns consisted of VR-2332,JXA1,and CH-1a.The remaining six strains PRRSV consisted of VR-2332 and JXA1.Comparing the recombinant strains PRRSV isolated in this research with recombination strains isolated by other research teams between 2013 and 2018,it was found that recombination breakpoints are distributed throughout the genome without specific area.It is high-frequency distribution area is located in the NSP2,NSP9,ORF2,ORF4,and ORF7regions,and occasionally occurs in other ares.The results show that the HP-PRRSV epidemic in Shandong province and surrounding areas are still severe from 2013 to 2018.Besides,classic strains PRRSV are still existing,new recombinant strains PRRSV are constantly emerging.By analysis of the NSP2 and GP5 genes PRRSV are constantly emerging new mutations gene mutations and deletions on especially.The analysis of recombinant strains found,because of current vaccines unreasonable selection and non-standard use,it leads to increase the probability of strain variation.Thus,it is particularly important to further understand and investigate the genetic variation and molecular epidemiology of PRRSV epidemic strains.This study not only help us understand to genetic variation of PRRSV in Shandong Province and surrounding ares,but also provide a data reference for effective prevention and control strategies.