Epidemiological Investigation Of Porcine Epidemic Diarrhea Virus In South China In 2014-2016 And Isolation Of PEDV SC-14 Strain

Porcine epidemic diarrhea is one of the current popular pig disease in China,it is a breeding herd intestinal infectious diseases.It can cause various clinical pigs day age of vomiting,diarrhea,dehydration and other symptoms,particularly 7 day age lactation piglet disease mortality is higher.PEDV was popular in China since 2010 and it was continues to sweep all region in our country.Dealing a hard blow to the global pig industry So the prevalence of PEDV as well as variations,and grope the virus propagation conditions have great significance.We had a epidemiological investigation and S genes evolution characteristic of porcine epidemic diarrhea strain,we established a nested RT-PCR method for differentiation between variant PEDV and classical strains,isolation and identification of Porcine epidemic diarrhea virus SC-2014 strain.The major studies include:1.Epidemiological investigation and S genes evolution characteristic of porcine epidemic diarrhea strainDuring 2014 to 2016 years,the 500 clinic samples from four provinces of south of China were detected,the result indicated JiangXi region positive rate of PEDV up to 74%,GuangXi region Positive rate lowest was 45%,four provinces of PEDV average positive rate was58%.Randomly selected 9 portions positive PEDV epidemic materials for PEDV S gene sequencing,the result indicated nine strains were variant strains,the 9 strains of pandemic strain S genetic gene homology in the 98.5%to 99.6%.Compare with the vaccine strains,variation was highly similar,and have common two insertions and one deletion,and independent of the vaccine strains to form a separate branch of the evolutionary tree G2group.2.Development of a Nested RT-PCR method for differentiation between variant PEDV and classical strainsThrough PEDV S genetic analysis that the current popular PEDV strain variation in the S gene of 58-62 aa has four amino acid insertion.According to the characteristics,this experimental design synthesis of peripheral amplification within two of specificity primers,by optimizing the amplification conditions,specificity and sensitivity of series of experiments,and complete the specificity,sensitivity test and the clinical samples test.Nested-PCR detection method to establish distinguish for variation strain and classical strain.The results show that the method can detect the nested-PCR were identified Classical PEDV strains and variant strains.Classic PEDV only 719bp band in the first round of PCR products,the second round has no band.The variation of PEDV in the first round of the PCR product only 730 band,and only 514bp of the second round.The detection method has a high specificity and sensitivity by simple operation.It offers technological means for reference to the rapid diagnosis of porcine epidemic diarrhea and epidemiology survey.3.Isolation and identification of Porcine epidemic diarrhea virus SC-14 strainPorcine epidemic diarrhea virus SC-14 strain inoculated into vero cells for blind subculture,the CPE was first present at about the 8 subculture,The cell shrinkage,turn round,refraction stronger typical cytopathic and so on.And the characteristic CPE was stability after the 20 generation.Respectively by setting different trysin concentration,different adsorption time,different PAPN concentration into training system,the isolated virus continue to propagate 10 passages,and the TCID₅₀ of the virus under different conditions were determined.The result showed that when the trysin concentration was 15?g/m L,adsorption time was 1.5 h,the concentration of PAPN added to5?g/mL,the virus titer of PEDV SC-14 strain was 10^5.23 which reached the maximum.To a certain extent,breeding conditions have been optimized.And sequenced and analyzed the whole gene sequence of PEDV SC-14 strain,The whole gene sequence of SC-14 strain was compared with that of vaccine strain CV777.SC-14 strain was divided into G1b group.There were 5 regions of gene deletion,and 3 bases were absent on S gene,and there were28 bases and 21 base deletions on the ORF1a and ORF1b genes,respectively,and on the ORF3 gene,49 bases were absent.The strain was identified as a classical wild strain.