Isolation，identification，and genetic evolution analysis of variant strain CH-HK-2021 of G2a porcine epidemic diarrhea virus

【Object】 To explore genetic evolution relationship of variant porcine epidemic diarrhea virus（PEDV）and antigenic differential sites among variant strain subtypes，so as to lay a foundation for the development of novel vaccines and diagnostic kits.【Method】Three PEDV-positive porcine intestinal samples were inoculated on to confluent Vero cells to isolate PEDV.Virus identification was performed by indirect fluorescence assay（IFA），Western blotting，RT-PCR and whole genome sequencing and electron microscopic observation；virus titer was determined by TCID50 and the in vitvo proliferation dynamin curve of the virus was drawn.The genome of the isolated strain was divided into 33 segments for RT-PCR amplification，and the SeqMan of Lasergene was used to splice sequences.Then the genetic evolution analysis was performed with MEGA 7.0，and the antigenicity analysis was performed with Jameson-Wolf algorithm in Protean.【Result】Typical cytopathic effect appeared in one PEDV-positive porcine intestinal sample in Vero cells when it was blindly passaged to the 6th generation and the sample was designated as CH-HK-2021.IFA and Western blotting results showed that the strain CH-HK-2021 could react with PEDV N monoclonal antibody and expected reads were obtained through RT-PCR amplification，which demonstrated this virus was PEDV.Diameter of strain CH-HK-2021 was 80-120 nm and the surface of the virus particles were in spike-like shape，indicating it was coronavirus.The strain could be stably propagated in Vero cells，and it has been passaged to 100th generation.After 24 h of infecting the Vero cells，virus titer of strain CH-HK-2021 reached the highest，10^5.6 TCID₅₀/mL.The size whole genome of strain CH-HK-2021 not including poly（A）tail was 28034 bp，with a similarity of 96.0%-98.9% with nucleotide sequence of the PEDV reference strain and a similarity of 93.1%-99.0% with S-base nucleotide sequence of the reference strain.The strain had the highest similarity with nucleotide sequence of variant strain CH/JX/01（KX058031）and the lowest similarity with nucleotide sequence of classical strain AVCT12（LC053455）.Strain CH-HK-2021 was a subtype of G2a and it is spreading in China.Strain G2a and variant strain G2b had 42 nucleotide differential sites in S gene and 6 antigenic differential sites；and main differential sites located in subunit S2.【Conclusion】 Strain CH-HK-2021 obtained through isolation was subtype variant strain of PEDV G2a，and it is spreading in China.The strain has relatively distant relationship with the classical PEDV strain.G2a stain and G2b variant strain have multiple antigenicity differential sites on S2 subunit，and it is speculated that S2 subunit may be the main reason of the difference in antigenicity between G2a and G2b strains.