Novel isolation method of porcine epidemic diarrhea virus using suspension vero cells and immunogenicity analysis.

In February and December of 2019 separately, two large-scale outbreaks of diarrhea were observed in the same swine farm with 3000 sows in Shanghai, China. We successfully isolated two PEDV isolates (strain shxx1902 and shxx1912) from clinical samples in this farm using suspension Vero cells in February and December, respectively. The third PEDV strain (SH1302) tested positive in the other farm of Shanghai in 2013 was also isolated using suspension Vero cells. The three isolates were better adapted to growth in adherent Vero cells through serial passages in the suspension Vero cells. The three isolated strains were detected positive by an immune- fluorescence assay and observed through electron microscopy. Phylogenetic analysis of the complete genomic sequence demonstrated that shxx1902(the 5th passage) and shxx1912(the 5th passage) clustered with a new GII subgroup (GII-c), which consisted of SINDEL strains from America (e.g., OH851), and their S gene belonged to GII-a. Both the strains(the 35th passage) have occurred dramatic changes in their genomes compared with the 5th passage. The 5th and 35th SH1302 belonged to GI-b genotype. The anti-N protein antibody titer of the strain shxx1902 was elevated to the same level as the vaccine strain(CV777) in mice. The use of the suspension Vero cells to isolate and propagate PEDV provide an effective approach for studies of the epidemiological characteristics and vaccine development of this virus.