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Journal of Southern Agriculture ; 53(8):2077-2087, 2022.
Article in Chinese | CAB Abstracts | ID: covidwho-2201259


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 TCID50and 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 6thgeneration 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 100thgeneration. After 24 h of infecting the Vero cells, virus titer of strain CH-HK-2021 reached the highest,105.6TCID50/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.