Molecular Diagnosis And Etiology Investigation Of Porcine Reproductive And Respiratory Disorder Syndrome

“Blue ear Disease” is also known as Porcine reproductive and respiratory syndrome(PRRS),Porcine reproductive and respiratory syndrome(PRRSV)caused reproductive dysfunction of sows and respiratory diseases of piglets.It is an acute and highly infectious viral infectious disease.PRRSV is most susceptible to pregnant sows and piglets 0-30 days of age and can be transmitted through a variety of routes.The disease is not only a single infection in production and breeding,but also easy to be mixed with a variety of reproductive disorders,and has a synergistic effect,causing great harm to the pig industry.In this study,two detection methods were established: universal fluorescence quantitative PCR and multiple PCR.The two methods have strong specificity and high sensitivity.In this study,a systematic sampling evaluation scheme was designed.During the period September 2020-December 2022,4445 samples were collected from pig farms in Hubei,Hunan,Sichuan,Chongqing and Guangdong provinces,which were suspected to be infected with PRRSV.The molecular diagnostic method established in this study was applied to systematically evaluate and monitor the samples.The etiological background of PRRS in 5 provinces was preliminarily identified,and the prevalence and transmission route of blue ear disease in large-scale pig farms in different provinces were revealed,as well as the genetic changes of different strains of the same pathogen.These data will provide us with scientific information for the early warning and forecast of blue ear disease in large-scale pig farms as well as the connection with clinical practice,which is of great significance for the development of scientific and precise prevention and control strategies that vary from field to field,and for the purification and phased prevention and control measures of blue ear disease.I.Establishment of molecular detection methods for porcine reproductive and respiratory disorder syndrome:In this paper,one set of probe primers(2 Primers + Probe)and one pair of merge primers(2 Primers)were designed with reference to the PRRSV NSP2 gene sequence registered in Gen Bank to screen highly conserved fragments.Among them,the probe primer is a universal fluorescent probe PCR method for detecting PRRSV,and the conditions of its primer,probe,reverse transcriptase concentration and annealing temperature are optimized.Finally,the sensitivity of the universal real-time PCR detection method was determined to be 1.36×100 copies/μL.Other pathogens(porcine epidemic diarrhea virus(PEDV),porcine parvovirus(PPV),porcine circovirus type 2(PCV2),etc.)preserved in this laboratory were amplified by this method,and the nucleic acids of Escherichia coli and Staphylococcus aureus were amplified,and no amplification curves appeared.Repeatability experiments were carried out: the coefficient of variation between batches was divided into 0.40%～1.02%;The coefficient of variation in the batch is 0.36%～0.60%;All were less than 1.5%,which proved that the method had good stability and repeatability.The combination primer was used as multiplex RT-PCR detection method to identify three PRRSV strains,and the amplification fragments were classic strain 1029 bp,highly pathogenic variant 939 bp,and NADC30 like strain 636 bp,which could effectively amplify and identify PPRSV-positive samples with CT value <34,and the lower limit of detection was 1.36×101 copies/μL.Amplification of these pathogens did not reveal specific bands.Both methods are important for early warning of PRRS and for developing more effective early warning measures.II.Molecular epidemiological investigation of porcine reproductive and respiratory disorders syndrome in some parts of China:From September 2020-December 2022,4445 samples were detected by PRRSV from five provinces,including Hubei Province,Hunan Province,Sichuan Province,Chongqing Region and Guangdong Province,and the universal real-time PCR method and multiplex RTPCR method were used for detection.The overall positivity rate was 13.94%(620/4445),indicating that PRRS is still widely spread.The high infection rates in Chongqing,Guangdong,Hunan,Sichuan and Hubei were 17.74%(66/372),15.81%(62/392),14.04%(125/890),13.60%(51/375)and 12.83%(310/2416),respectively.In different breeding periods,the infection rate of nursery pigs was the highest 19.31%,followed by 13.82%,13.18%,12.93% and 12.68% of breeding pigs,0-30 days old piglets,farrowing sows and primary sows,respectively,and the infection rate of breeding boars was 6.98%.PRRSV can be detected in semen,colostrum,placenta,nasal swabs,vulva swabs,and blood.The results of PRRSV typing and identification: 94 classic strains,186 highly pathogenic variants and334 NADC30 like strains were identified.It shows that NADC30 like strain is still the dominant epidemic strain;Classical strains and highly pathogenic variants still exist and circulate clinically,and cause certain harm.III.Analysis of genetic variation of NSP2 gene in porcine reproductive and respiratory syndrome virus:After sequencing 15 positive samples,the nucleotide homology and derived amino acid homology between classic strain sequences were 78.7%～99.0%,73.3%～98.9%,respectively.The nucleotide homology and derived amino acid homology between sequences of highly pathogenic variants were 97.3%～99.9% and 97.0%～99.7%,respectively,and the nucleotide homology and derived amino acid homology between sequences of NADC30-like strains identified by sequencing were 88.3%～98.4%,81.6%～96.2%,respectively.The phylogenetic tree of PRRSV NSP2 gene was constructed and analyzed and found that the PRRSV strain was divided into European(PRRSV 1)and American(PRRSV 2),and the American strain could be divided into 4 lineages: lineage 1,3,5,8(Lineage 1,3,5,8),and a total of 15 PRRSV strains from five provinces identified in this experiment were all American(PRRSV2),of which 5 belonged to subline 1.9(NADC30 like)in lineage 1;5 belong to subline 8.7 in lineage 8(JXA1 like);2 belong to Lineage 8.1(CH-1a like)in lineage 8;3 belong to Lineage5.1(VR-2332 like)in lineage 5.Through the analysis of the amino acid mutation site of PRRSV NSP2,it was found that the classical strain and high pathogenic variant sequenced in 5 provinces had little difference in amino acids from its reference strain,and the NADC30 like strain sequenced in 5 province had great amino acid variation compared with its reference strain,and the amino acid variation was relatively easy to occur at NSP2 amino acid sites 309,373,375,375,381,390,416,425,429,432,437,445,474,477,485 and 500.