Isolation And Identification Of Porcine Epidemic Diarrhea Virus And Genetic Variation Analysis Of The ORF3 Gene,M Gene And N Gene From Anhui In 2013

Porcine Epidemic Diarrhea(PED)is caused by Porcine Epidemic Diarrhea Virus(PEDV),which characterized by loss of appetite,vomi.PED causes huge economic losses to the pig industry.In PEDV strain ORF3 gene is very important when it encodes an ion channel protei;M gene encodes a transmembrane protein,and it is important for ssembling and budding in virus;N gene encodes the nucleocapsid protein gene,and it has the basic significance of viral RNA.To understand the epidemiological characteristics and changes of PEDV in Anhui province,the author isolated 6 PEDV strains from different parts of Anhui,clonied and sequenced of ORF3 gene,M gene and N gene.In this study,we used Trizol method to have RNA,and TAKARA reverse transcription kit to reverse transcrib CDNA which stored at-20 ℃.We used Oligo7.0 to design the whole sequence of all the genes and they ware identified CV777(AF353511.1).The PCR product was subjected to agarose gel electrophoresis,observed or photographed in the gel imaging system.Since then used Vero cells to take a blind passing,and then SYBR Green I real-time PCR.SYBR Green I real-time PCR results showed the established PEDV standard curve correlation coefficient is 0.999 and amplification efficiency is 103%.It mean there are 6 PEDV strains,and we named them AH1301,AH1302,AH1303,AH1304,AH1305,AH1306.The full-length of ORF3 gene,M gene and N gene,were cloned,sequenced and analysised.Gene sequence analysis showed that: the length of six ORF3 gene are 675 bp,containing a complete open reading frame encoding 224 amino acids;the length of six M gene are 681 bp,containing a complete open reading frame encodes 226 amino acids;the length of six N gene are 1326 bp,encoding 441 amino acids.The percentage of identity at the nucleotide level of them among the 6 strains were98.8%-100%,97.5%-100%,96.2%-99.7%;while amino acid sequences shared99.6%-100%,97.8%-100%,97.3%-100%.Compared with the standard CV777 strain,6ORF3 emerged 2 new amino acids;there were 5 M genes have a change in 13 amino acid sites(E→Q);there were 70 amino acids changed in 6 N geness.Phylogenetic tree showed that: ORF3 gene,M gene and N gene in 6 PEDV strains were divided into three groups.Including those strains,AH1304 had a closer relationship with SD-M strain and attenuanted DR13 strain,other 5 strains had a closer relationship with IA1 strain and AH2012.But,all of the 6 PEDV strains had a far affinity with CV777 strain and LZC strain.