Isolation and Identification of Canine Parvovirus Xinxiang Strain and Sequence Analysis of Its VP2 gene

Abstract

【Objective】The purpose of this study was to explore the prevalence and genetic variation of canine parvovirus (CPV) in Xinxiang City, Henan Province, and provide reference for effective prevention and control of regional CPV epidemics.【Method】In this study, 23 stool samples and eye, nose, mouth and anus swabs of dogs suspected of CPV disease collected from several pet hospitals in Xinxiang were first pretreated and then PCR detected. The corresponding samples with positive PCR detection results were soaked, filtered and sterilized, and then inoculated into feline kidney cell (CRFK) for virus proliferation and culture. The isolated virus was observed under a transmission electron microscopy. Referring to the sequences of different subtypes of CPV both domestically and internationally included in GenBank, PCR was used for segmented amplification, followed by sequencing and splicing to obtain the full length genes of the virus. Molecular biology software was used for sequence analysis and amino acid homology analysis of the virus genes.【Result】One CPV was successfully isolated from the sample sent for examination and designated strain XX-2022. The virus strain was able to produce significant cytopathic changes in the CRFK cells, resulting in cell rounding, pulling and falling off. Transmission electron microscopy can observe typical canine parvovirions in a circular or hexagonal shape with a diameter of approximately 25 nm and no capsule. The full genome sequence of this virus, 4 588 bp in length, was obtained by PCR segmental amplification. Phylogenetic analysis results showed that the XX-2022 strain CPV had a close relationship with the Canine/SH/1/2019 (MN840830.1) strain CPV. The amino acid sequence of the VP2 gene of XX-2022 strain, which shares 99.7% amino acid identity with the VP2 amino acid sequence of YANJI-5 (MW715601), was analyzed using megalign software. Based on the genotyping principles of CPV, it was finally determined that the XX-2022 strain CPV belonged to CPV-2a type.【Conclusion】In this study, a CPV strain was successfully isolated from clinical material, determined as CPV-2a type, which provide a basis for the study of the epidemiology, pathogenicity, and biological characteristics of CPV in Xinxiang, as well as a reference for the regional prevalence and effective prevention and control of CPV, and to provide an endemic virus for the development of new vaccines.

Keywords

• canine parvovirus
• isolation and identification
• transmission electron microscopy
• whole gene sequence amplification
• phylogenetic analysis