Isolation,Identification And Genetic Evolution Analysis Of PEDV From Hebei Province

Porcine epidemic diarrhea(PED)is an acute,highly contagious intestinal infectious disease caused by porcine epidemic diarrhea virus(PEDV).The disease is mainly harmful to suckling piglets,and the mortality rate of piglets within 5 days of age can reach 100%.However,since the re-emerge of PED in China in 2010,the vaccine immune effect was not good enough to provide protection for the pig population,which brought huge losses to the pig industry.Through continuous studies,PEDV variation was found as the main cause of vaccine failure,so it is of great significance to study the characteristics of tissue culture and molecular biology of the variant strain.Diarrhea piglet intestine and intestinal contents were collected as diseased materials in this study.The positive samples that confirmed by RT-PCR test were filtered,then inoculated on the Vero cells.The typical pathological changes were observed until the fifth generation of the blind passage.Further studies were carried out by RT-PCR detection,IFA test as well as transmission electron microscope,and eventually proved the successful separation of a PEDV strain,named HBQHD1.According to the whole gene sequence of PEDV AJ1102 strain,16 pairs of primers were designed to amplify the whole genome of HBQHD1 strain.The 16 fragments of the target genes were obtained by RNA extraction as well as RT-PCR.After cloning,sequencing and splicing,the complete gene sequence of HBQHD1 strain was obtained,then we submit it to NCBI database,the GenBank accession is MK606368.1.The genome length of HBQHD1 strain is 28038 bp.Combined with the existing sequence information of the PEDV in the NCBI database,we found that HBQHD1 strain has a distant genetic relationship with the classical strains,such as CV777.Besides,it's close to the variant strains that isolated during the recent years,such as BJ-2011.The homology between the new isolated strain in this study with other classical strains as well as other variant strains is 96.5%~97.6% and 98%~99%,respectively.Furthermore,homology and genetic evolution analysis were conducted and the phylogenetic trees of all the segment of HBQHD1 were constructed with the 25 representative strains.The results showed that HBQHD1 strain was significantly different from classical strains such as CV777 and DR13,and share a small difference as well as high affinity with the isolated mutant strains after 2010,resulting in the poor vaccine immune effect.Therefore,it is particularly important to grasp the trend of PEDV epidemic strain evolution and accelerate vaccine development.