| Porcine reproductive and respiratory syndrome virus(PRRSV)is one of the most important pathogenes.It can cause severe reproductive failure in sows,poor viability of piglets,and respiratory disease with secondary infection in growing pigs,resulting in huge economically loss in the world pig industry.The commerical vaccines can not provide completely protection against this disease.PRRSV belongs to RNA virus,which can be varied easily.It has been introduced to China and broke out for several times.Highly pathogenic PRRSV(HP-PRRSV)emerged in China in 2006.And the vaccines have been wildly used for controlling this disease.However,the epidemic situation is not stable,and the predominant circulating strains exerted genetic diversity.The recombination,mutation or revert virulence of the vaccine virus strains has not been completely understood.In this study,a total of 293 clinical samples were collected from pigs from different swine herds that experienced diseases in 16 provinces of China in 2015.The epidemic PRRSV strains were detected by RT-PCR,and the whole genome sequencing of 28 PRRSV isolates were detected and analyzed together with the sequences of 623 PRRSV strains in GenBank data base.It was found that four subgenotypes of PRRSV North American and PRRSV European currently circulate in China,clarifying the epidemic of highly pathogenic PRRSV JXA1-R vaccine strain in China and its genetic characteristics.It provides a theoretical basis for the prevention and control of the disease.The thesis contents were as following:1.Virus surveillance and clinical epidemic diagnosis of PRRSA total of 293 clinical lung samples were collected from pigs from 72 swine herds in 16 provinces of China in 2015.The viral detection was based on polymerase chain reaction(PCR)and RT-PCR with primers specific to PRRSV,porcine circovirus type 2(PCV-2),classical swine fever virus(CSFV),porcine parvovirus(PPV),and porcine pseudorabies virus(PRV),respectively.The positive rate of PRRSV was 39.93%.Moreover,we found 8 cases of PRRSV European.A total of 24 representative pig farms with severe respiratory disease and positive to PRRSV were selected.Clinical and pathological findings showed that high and continuous fever,labored breathing,blue ears,blue nose,erythematous blanching rashes,pimples in the ears or on the back,and loss of appetite.Pathological examination results showed hyperplasia,edema,and hemorrhage in the lungs,and severe hemorrhagic spots in the lymph nodes.IHC results indicated obvious PRRSV antigen in the lungs of diseased piglets,and it was diagnosed as PRRS.These results reveal that PRRS was still prevalent in China in 2015.It provides useful materials for the study of genetic variation of predominant circulating strains of PRRS.2.The genetic variation of PRRSV and molecular characteristics of vaccine JXAl-R-like strains.A total of 28 infected samples collected from 24 pig farms in nine provinces were further selected for whole genome sequencing of PRRSV.Phylogenetic analysis and recombination screening were performed with the full genome sequences of 28 strains and other 623 reference sequences of PRRSV.Phylogenetic results showed that the 28 strains were nested into sublineage 10.5(classic highly pathogenic[HP]-PRRSV),sublineage 10.6(HP-PRRSV-like strains and related recombinants),sublineage 10.7(potential vaccine JXA1-R-like strains),and lineage 9(NADC30-like strains and recombinants of NADC30-like strains),respectively,suggesting that multiple subgenotypes of PRRSV currently circulate in China.It was found that 44 JXA1-R-like strains,which included the 10 potential JXA1-R-like strains in this study and 34 Chinese strains in the GenBank database,shared the highest nucleotide identities with JXA1-R strains(>99%).The geographical distribution results revealed that those potential JXA1-R-like strains were detected in all 16 provinces or autonomous cities(regions)with a wide geographical range in China.Recombination analyses showed that 9 of 28 isolates and one isolate from other laboratory were potential complicated recombinants between the vaccine JXA1-R-like strains and predominant circulating strains,which being by three kinds of inner-lineage,inter-lineage,and both inner-lineage and inter-lineage.The strains 15GD2,15GD3,15HUN1,15HUN2,and 10FS1-GD were the products of inner-lineage recombination events between 2009-2010 HP-PRRSV-like strains(sublineage 10.6)and vaccine JXA1-R strains(sublineage 10.7).15JX1,15HEN1,and 15SC3 were inter-lineage recombination events between strains NADC30(lineage 9)and JXA1-R.Third,15HEB1,and 15LN3 were mosaic recombination events between strains NADC30,2009-2010 HP-PRRSV,and JXA1-R.The complicated recombination events exist in Nsp2 or Nsp9 genes.One PRRSV strain 15HUN3,collected from pig herds suffered severe disease,were selected for further analysis in genomic sequence level.Compared with vaccine JXA1 P80,15HUN3 had 70 mutation in nt level and 30 mutation of aa level.Among them,Nsp8 or ORF5 exhibited the high mutation rate in nt level,and GP5 showed the high mutation rate in aa level.These mutations may relate to the reverting of 15HUN3.3.Genetic variation of European type PRRSV in ChinaPRRSV isolate 15HEN1_EU was selected from the 8 PRRSV European positive samples for the whole genome sequence and sequence analysis.Phylogenetic results indicated that 15HEN1_EU of 15092 bp is closest to BJEU06-1,which was predominant circulating strains in China,and belong to subgroup 1.3.Compared with other PRRSV European isolates,many deletion,insertion,and mutation characteristics were observed in the genomic sequence of 15HEN1_EU.Such as,a 5-aa deletion and 1-aa insertion in Nsp2,1-aa deletion in GP3 or GP4,and mutation sites in UTR?Nsp2?GP3?GP4?GP5 and N were located.Although 15HEN1_EU proved not be the result of recombination,five strains(NVDC-NM1-2011,NVDC-FJ,NVDC-NM2,NVDC-NM3,HLJB1)isolated in China were potential recombinants,which enriched the epidemiological data of PRRSV. |
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