ABSTRACT
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.
ABSTRACT
The porcine deltacoronavirus (PDCoV) is a newly discovered pig enteric coronavirus that can infect cells from various species. In Haiti, PDCoV infections in children with acute undifferentiated febrile fever were recently reported. Considering the great potential of inter-species transmission of PDCoV, we performed a comprehensive analysis of codon usage patterns and host adaptation profiles of 54 representative PDCoV strains with the spike (S) gene. Phylogenetic analysis of the PDCoV S gene indicates that the PDCoV strains can be divided into five genogroups. We found a certain codon usage bias existed in the S gene, in which the synonymous codons are often ended with U or A. Heat map analysis revealed that all the PDCoV strains shared a similar codon usage trend. The PDCoV S gene with a dN/dS ratio lower than 1 reveals a negative selection on the PDCoV S gene. Neutrality analysis showed that natural selection is the dominant force in shaping the codon usage bias of the PDCoV S gene. Unexpectedly, host adaptation analysis reveals a higher adaptation level of PDCoV to Homo sapiens and Gallus gallus than to Sus scrofa. Compared to the USA lineage, the PDCoV strains in the Early China lineage and Thailand lineage were less adapted to their hosts, which indicates that the evolutionary process plays an important role in the adaptation ability of PDCoV. These findings of this study add to our understanding of PDCoV's evolution, adaptability, and inter-species transmission.