Isolation, Purification and Genome Sequence Analysis of Enzootic nasal tumor virus of Goats (Capra hircus) Strain ENTV-2FJ
JIANG Jin-Xiu1, LIN Yu-Sheng1, ZHANG Jin-Peng1, YOU Wei1, HUANG Li-Li2, MAO Kun-Ming3, HU Qi-Lin1*
1 Institute of Animal Husbandry & Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou 350013; 2 Fujian Normal University, Fuzhou 350108; 3 Fuqing Technical Service Center of Animal Husbandry and Veterinary Medicine, Fuzhou 350300, China
Abstract:Enzootic nasal adenocarcinoma (ENA) caused by Enzootic nasal tumour virus of goats (ENTV-2) has been reported in many provinces in China in recent years. In order to clarify the virology and genome-wide characteristics of ENTV-2FJ strain, In this study, the nasal fluid of the diseased sheep was collected, and the virus of ENTV-2FJ strain was purified by differential centrifugation combined with sucrose density gradient centrifugation, and then subjected to electron microscopic observation and SDS-PAGE; Seven cDNA fragments of ENTV-2FJ isolate were amplified by reverse transcription PCR (RT-PCR), the amplified products were cloned into pMD19-T plasmid vector and sequenced, the whole genome sequence of the ENTV-2FJ strain was obtained by splicing with DNAstar 7.1 software and the sequence was analyzed. After purification of the virus of ENTV-2FJ strain, pure virions can be observed under electron microscope. The virions were spherical, 90~110 nm in diameter, with envelope and spikes; By SDS-PAGE analysis, the structural proteins of the ENTV-2FJ strain showed 9 bands with molecular weights of 118, 79, 60, 51, 28, 20, 16, 13 and 11 ku, the genome of the ENTV-2FJ strain was 7 469 bp in length, exhibiting 95.1%, 95.4%, 95.1%, 94.9% and 95.4% homology with ENTV-2SC, ENTV-2Shaanxi, ENTV-2Shaanxi2, ENTV-2Shaanxi3, ENTV-2Shaanxi4, respectively. Phylogenetic analysis based on the nucleotide sequence of ENTV-2 whole genomes, gag and env genes revealed that the ENTV-2FJ strain was in the same evolutionary branch as ENTV-2SC, ENTV-2Shaanxi, ENTV-2Shaanxi2, ENTV-2Shaanxi3, and ENTV-2Shaanxi4. These results lay the foundation for the establishment of the ENTV-2 assay and genetic variation analysis.
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