Isolation,Identification and Genetic Evolution Analysis of a Strain of Bovine Parvovirus Type 1 in Guangxi
[Objective]The aim of this study was to understand the biological characteristics and genetic variation of Bovine parvovirus 1(BPV1)in Guangxi,and provide theoretical basis and support for the prevention and control of BPV1.[Method]A total of 409 fecal samples of calf diarrhea were collected from cattle farms in Guangxi region,RCR was detected by BPV1 specific primer,and virus was isolated from positive samples by bovine turbinate bone cells(BT).The supernatant of BT cells with cytopathic effect(CPE)was collected,and the virus was identified by PCR amplification,indirect immunofluorescence assay(IFA)and electron microscope observation.The median tissue culture infectious dose(TCID5o)was measured at different times after BPV1 isolates infected BT cells,and the multi-step growth curve was drawn.The whole genome sequence of the virus was obtained by high throughput sequencing and its genetic evolution was analyzed.[Result]One positive BPV1 was detected in 409 fecal samples,with a positive rate of 0.24％.After the blind transfer of BT cells to the third generation,the phenomenon of cell circle shrinkage,bending,formation of cell mass and dissolution was observed.PCR amplification results showed that specific bands could be produced by amplification of CPE supernatant.The round,membraneless virions with a diameter of about 24 nm were observed by electron microscopy.IFA results showed that specific green fluorescence could be observed in BT cells inoculated with the isolate,and a strain of BPV1 named GXBS2209 was successfully isolated.The titer of the 6th generation isolates was 105.75 TCID50/mL,and the multi-step growth curve showed that the virus titer peaked at 120 h after the isolate infected BT cells,and then gradually decreased.The sequencing results showed that the full length of the isolated genome was 5 515 bp(GenBank accession No.:PP158225).The sequence similarity between the isolates and the American Bovine parvovirus strain was the highest(98.7％).The genetic and evolutionary analysis showed that the isolate and Bovine parvovirus strain were in the same branch and were closely related.The nucleotide sequence similarities of NS1 and VP2 genes were 98.8％-99.4％and 94.4％-98.1％,and the amino acid sequence similarities were 98.8％-99.5％and 90.3％-99.4％,respectively.There are 2 and 3 amino acid sites substitutions in NS1 and VP2 sequences,respectively.[Conclusion]A strain of BPV1 was successfully isolated and its whole gene sequence was analyzed,which laid a foundation for the subsequent pathogenesis,vaccine research and prevention and control of BPV1.
Bovine parvovirus type 1(BPV1)virus isolationsequence analysis
万方数据
维普
