Whole Genome Sequencing And Analysis Of Growth Characteristics Of PEDV Hefei Strain

Porcine epidemic diarrhea?PED?is a viral disease caused by porcine epidemic diarrhea virus?PEDV?,which has caused huge losses to the pig industry.In this study,pathogens and PEDV isolation and identification of suspected PED infections were carried out,whole genome sequencing of PEDV isolates,and passage in Vero cells to explore the growth characteristics and genetic stability of the isolates after passage,to understand the separation of PEDV in Anhui The molecular biological characteristics and genetic evolution of the strain provide references for PEDV related research.In this study,the disease material of piglet diarrhea was identified,PEDV was isolated by blind transmission in Vero cells,and identified by RT-PCR,indirect immunofluorescence,and ultrathin cell sections.According to the whole gene sequence of PEDV CV777 strain,9 pairs of primers covering the whole genome of PEDV were designed,and cloned and sequenced in sections to analyze the whole genome sequence.The PEDV isolates were serially passaged in Vero cells to amplify the S and ORF3 genes of 5,20,40,60 and 80 generations of virus fluid,and the growth characteristics of different generations of viruses were determined.The results showed that the diarrhea disease material was PED infection,and PEDV was successfully isolated in Vero cells when it was passed to the fifth passage,and it was named AH-2018-HF1.The whole genome sequencing results show that the AH-2018-HF1strain has a total length of 27 937 bp.Compared with the reference strain,the S gene of AH-2018-HF1 strain deleted 3 bp at positions 2319-2320?GTT?;compared with CV777and LZC strains,AH-2018-HF1 strain deleted 3 bp at positions 459-460?ATA?;compared with the domestic and foreign mutant strains,the AH-2018-HF1 strain deleted 12 bp?CAGGGTGTCAAT?at positions 174-175 and inserted 6 bp?TGGAAA?at positions461-462.The ORF3 gene of AH-2018-HF1 strain has 49 base deletions at positions245-246.The E gene of AH-2018-HF1 strain was deleted by 12 bp at positions 69-70?CCTGCTTATTAT?.The M and N genes have no deletions or insertions.Homology analysis showed that AH-2018-HF1 strain had higher nucleotide homology with SD-M,HLJBY,JS2008,CV777 and Virulebp DR13 strains,and lower nucleotide homology with mutant strains.The results of genome-wide genetic evolution tree show that AH-2018-HF1strain is in the same branch as SD-M,HLJBY,and JS2008,and has the closest genetic relationship;it is closer to CV777 and Virulebp DR13 strains;it is farther from PEDV mutant strains.S gene system evolution results are similar to whole genome system evolution results,S gene can replace whole genome as PEDV genetic evolution analysis target gene.When the AH-2018-HF1 strain was passaged in Vero cells to passage 80,the CPE time was shortened to about 20 h,the virus titer reached 10^8.2TCID₅₀/m L,and sequence comparison analysis showed that the mutation rate of S and ORF3 genes in different passages was low.The results show that the AH-2018-HF1 strain is a classic strain,but there is a certain variation.With the increase in the number of passages in Vero cells,the AH-2018-HF1strain has shortened the appearance time of CPE,the virus titer has increased,and the adaptability to cells has been continuously enhanced.The S and ORF3 genes have remained highly stable during passage.