| Porcine Reproductive and Respiratory Syndrome(PRRS),caused by Porcine Reproductive and Respiratory Syndrome Virus(PRRSV),is mainly characterized by sow reproductive disorders and piglet respiratory diseases.It is an acute and highly contagious viral infectious disease.PRRSV is the National Medium and Long-Term Animal Disease Prevention and Control Plan(2012-2020)epidemic prevention disease.Effective prevention and control of PRRS is of great significance to the healthy development of the pig breeding industry.This paper investigates the status of PRRSV in pigs from slaughterhouses in 21 cities in Guangdong Province,and make a further analyzes of the genetic variation of viral genes to provide some date for scientifically formulating PRRS prevention and control measures.1.PRRSV universal real-time fluorescent RT-PCR detection kit comparison experimentIn order to more accurately reflect the actual infection status of pigs in the slaughterhouse,a more suitable kit was selected.This study compared the PRRSV universal real-time fluorescent RT-PCR detection kits produced by 6 manufacturers experiment.In this study,PRRSV nucleic acid standards and wild strains were used as comparison samples to compare the detection sensitivity,specificity,and repeatability of each kit.After statistical analysis and comprehensive evaluation,the selected kits of F company had a Kappa value of 0.97,a coefficient of variation within 1%,and a Youden index of0.91.The kit was used in subsequent epidemiological investigation experiments.2.The 2019-2020 PRRSV epidemiological survey of slaughterhouses in Guangdong ProvinceDuring the period of 2019-2020,at least 2 slaughterhouses were selected from 21 prefectures and municipalities in Guangdong Province to carry out investigations,involving 133 slaughterhouses,10-25 healthy pigs to select at random from each slaughterhouse with a total of 2560 collected sample of swine lymph nodes was tested for viral nucleic acid of the PRRSV-NA and PRRSV-EU by using real-time fluorescent RT-PCR detection method.In response to the large number of PRRSV-NA,the positive samples of PRRSV-NA were subjected to PRRSV-NA(Nsp21594 ～ 1680 mutation Strain),NADC30-like and JXA1-R real-time fluorescence RT-PCR detection.The test results showed that the PRRSV positive rate was 13.91%(356/2560),of which the PRRSV-NA was 12.03%(308/2560)and the PRRSV-EU was 2.58%(66/2560).In addition,the positive rate of PRRSV-NA(Nsp2 1594-1680 variants)was 21.43%(66/308);the positive rate of vaccine strain JXA1-R was 10.06%(31/308),and the positive rate of NADC30-like was 0.65%(2 /308).It is worth noting that the positive samples of JXAI-R are all derived from PRRSV-NA(Nsp21594～1680 mutant strain).The copy number of PRRSV nucleic acid test samples in pigs in pig slaughterhouses in Guangdong Province is not high,and repeated testing is required.The rate of latent infection is high.It is mainly carried by PRRSV-NA,and had a small percentage by PRRSV-EU.The lymph node samples Ct/Cp value of the slaughterhouse is mostly low,which needs to be detected repeatedly,indicating that the virus is in a state of low-level infection.3.PRRSV ORF5 gene sequencing analysis.The results of ORF5 gene sequence analysis of 15 successfully obtained viruses showed: 3 Strains are PRRSV-EU;12 are PRRSV-NA: including Lineage8(Sublineage8.7)5 strains,Lineage3 3 strains,Lineage1 2 strains,Lineage5(Sublineage5.1)2 strains.In terms of genetic similarity,the PRRSV-EU is 90.2%～90.4%,the PRRSV-NA is 87.5%～99.6%,the detected nucleotide homology between the three European strains and the LV strain ORF5 gene is 86.9%～88.2%,and the genetic variation is large.Analysis of the amino acids encoded by the ORF5 gene of the PRRSV-NA revealed that there were many amino acid variations in the protein signal peptide,neutralizing epitope,and three cell epitope regions.Among them: among the neutralizing epitopes,5 strains had L39 I mutation,2 strains had L39 S mutation,and 1 strain had L39 T mutation.These mutations may have a certain impact on the immunogenicity of GP5 protein,thereby affecting the immune effect of the vaccine.In addition,among the 13 th and 151 st amino acid sites related to virulence,9 strains have Q13 R mutation and 4 strains have R151 K mutation,which may cause virulence enhancement and new high Pathogenic strains.In summary,this study provides a reference for the selection of PRRS pathogenic diagnostic reagents and the verification of other kits through comparative evaluation of PRRSV detection kits;through PRRSV molecular epidemiological investigations of pigs in slaughterhouses in Guangdong Province The analysis aims to understand the recessive transmission of PRRSV in asymptomatic pigs in Guangdong province,the epidemic status of the strains and the degree of virus variation,and provide a basis for scientifically formulating PRRS prevention and control measures. |
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