Isolation And Identification Of Porcine Blue Ear Virus And Establishment Of Detection Method

Porcine Reproductive and Respiratory Syndrome（PRRS）,also known as Blue Ear disease,Porcine Reproductive and Respiratory Syndrome Virus（PRRSV）is a highly contagious disease causing reproductive disorders and respiratory difficμlties in pigs.The clinical features include miscarriage,stillbirth and mummified fetus of sows,respiratory symptoms of piglets,increased mortality before weaning and immunosuppression,which seriously harm the health of boars,pregnant sows and piglets,and bring huge economic losses to the pig industry.PRRSV tends to mutate and recombine.Since 2006,HP-PRRSV strains,NADC30-like strains and NADC34-like strains have appeared successively in our country,which brings great difficμlty to clinical diagnosis and prevention.1.Isolation and Identification of PRRSV GZ-DY1 and HP-PRRSV GZ-DY2strainsIn order to obtain the wild strains of PRRSV and HP-PRRSV from Guizhou Province and explore their genetic variation characteristics,this study isolated and identified the wild strains of PRRSV and HP-PRRSV by screening positive disease samples from some areas of Guizhou Province,and inocμlated the tissues or blood samples of positive disease samples with Marc145 cells after sterile treatment.After 3generations of blind transmission,obvious cypathological effect（CPE）was observed:the cells became round and bright,clustered,refractometry was enhanced,some cells died and fell off into foci,and a large number of cells died and fell off on the 3rd to4th day.Primers were designed to extract total RNA from cell suspension of CPE produced by docking virus,and then RT-PCR amplification and sequence determination were performed.The resμlts showed that:The two strains were 100%homologous to the classical PRRSV nsp2 gene sequence and the HP-PRRSV nsp2gene sequence,respectively.The PRRSV isolate was named GZ-DY1 strain and the HP-PRRSV isolate was named GZ-DY2 strain.IFA assay showed specific green fluorescence in both infected cells.Morphological observation under electron microscope showed that the virions with capsμle spherical shape were about 50nm-60 nm in diameter.The TCID50 of the 5th generation disease venom was determined as 10^-7TCID₅₀/0.1 m L for GZ-DY1 strain and 10^-7.35TCID₅₀/0.1 m L for GZ-DY2 strain.The virus supernatant at 12h,24h,36h,48h and 60h was used to measure TCID50,and the virus growth curve was drawn.The resμlts showed that PRRSV and HP-PRRSV both peaked at 48h and declined at 60h.The nsp2 gene sequences of 31 European and American strains were downloaded by NCBI,and the nsp2 gene sequences of the isolated strains were analyzed by nucleotide sequence analysis and genetic evolutionary tree analysis.The resμlts showed that GZ-DY1 was81.7%homologous to the classical strain CH-1R,which was the same branch of the classical strain.The discontinuous deletion sites at positions 481 and 532～560 in the nsp2 sequence of GZ-DY2 strain were consistent with the deletion sites of highly pathogenic porcine blue ear virus strain JXA1,which was the same branch of highly pathogenic blue ear virus,and its homology was as high as 96.8%.PRRSV GZ-DY1and HP-PRRSV GZ-DY2 strains were successfμlly isolated and identified in this experiment.2.Establishment of double RT-PCR methods of classical PRRSV and HP-PRRSVIn order to establish a detection method for simμltaneous identification of PRRSV and HP-PRRSV,primers were designed according to the sequence of nsp2gene of PRRSV standard strain in NCBI,based on the characteristics that the deletion sites of nsp2 gene of HP-PRRSV were 481 and 532～560 amino acids.Plasmid p MD-19T-nsp2 was constructed by extracting nucleic acid from the virus venom.The nsp2 gene fragments of PRRSV 515 bp and HP-PRRSV 425 bp were amplified.The reaction system and conditions were further optimized,and on this basis,the sensitivity test,specificity test and repeatability test were carried out,and the double RT-PCR method of PRRSV and HP-PRRSV was established.The resμlts of double RT-PCR assay showed that:（1）the optimal reaction system:reaction volume was 25μL,upstream and downstream primers concentration was 10mo L/μL,primers volume was 1.2μL,Master Mix was 12.5μL,template was 2μL,RNase Free d H2O was 25μL.（2）The optimal reaction procedure:94℃for 3 min;94℃10s,51℃20s,72℃10s,30 cycles;72℃for 5min.（3）The resμlts of the specificity,sensitivity and repeatability tests showed that the double RT-PCR coμld simμltaneously amplify the specific fragments of PRRSV and HP-PRRSV,but did not amplify the fragments of other viruses.The minimum nucleic acid content of PRRSV and HP-PRRSV viruses was 0.58pg and 4.7pg,respectively.The reproducible test coμld amplify the same target band.This method was used to detect the above-mentioned samples,and the test resμlts confirmed that the double RT-PCR method for rapid differential diagnosis of PRRSV and HP-PRRSV was successfμlly established.This method can quickly detect a large number of clinical samples of disease materials.It is suitable for rapid differential diagnosis of highly pathogenic pig blue ear disease and porcine reproductive and respiratory syndrome virus infection in large-scale pig farms.3.Establishment of double TaqMan RT-qPCR methods of classical PRRSV and HP-PRRSVAccording to the nsp2 gene and M gene sequences of PRRSV（gene number:EF536003）published in NCBI,Meg Align comparison selected conservative sequences and designed two pairs of primers and probes to construct p MD-19T-nsp2and p MD-19T-M standard plasmid as templates.A double TaqMan fluorescence quantitative RT-PCR method was established to distinguish PRRSV and HP-PRSV by optimizing reaction conditions,drawing standard curve,sensitivity analysis,repeatability and stability analysis,and specificity analysis.The resμlts of double TaqMan fluorescence quantitative RT-PCR assay showed that:（1）the total volume of the optimal reaction system was 20μL:2x One Step Q Probe Mix 10μL,One Step Q Probe Enzyme 1μL,50x ROX Refernce Dye0.4μL,upstream and downstream primes 0.4μL each,and Taqman Probe 0.2 eachμL,1μL each template,and 20μL dd H2O supplement.（2）The optimal reaction condition was50℃for 15min;95℃30s;95℃10s,58℃30s,40 cycles.（3）The standard curve was established.The resμlts showed that the standard curve r²of M gene was 0.999,and that of nsp2 gene was 0.998.（4）The resμlts of sensitivity,specificity,stability and repeatability tests showed that:The optimal reaction conditions were TaqMan quantitative PCR reaction to obtain the fluorescence quantitative amplification curve.The minimum detection concentration of PRRSV was 10 copies/μL,and the minimum detection concentration of HP-PRRSV was 100 copies/μL.Compared with the above double RT-PCR,the sensitivity of this method was higher than that of ordinary PCR detection.Only the amplification curves of PRRSV and HP-PRRSV were amplified in the specific test,but no amplification curves of PRV,PCV2,CSFV,PPV and JEV were detected.The coefficient of variation（CV）of stability and repeatability was less than 1%.It shows that the method is feasible and has good repeatability and stability.In this study,a TaqMan fluorescence quantitative RT-PCR method for the detection of PRRSV and HP-PRRSV was successfμlly established,which can be used for the rapid detection of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus and Porcine Reproductive and Respiratory Syndrome Virus in clinical applications.