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.
[1] 冯迎春, 颜其贵, 郭万柱, 等. 2011. 山羊地方性鼻内肿瘤病毒ENTV-SC株基因组cDNA文库的构建及生物信息学分析[J]. 中国兽医科学, 41(02): 126-130. (Feng Y C, Yan Q G, Guo W Z, et a1.2011. Construction and bioinformatics analysis of cDNA library of goat Enzootic nasal tumor virus SC strain[J]. Chinese Veterinary Science, 41(02): 126-130.) [2] 耿毅, 汪开毓, 颜其贵, 等. 2010. 南江黄羊流行性鼻内腺癌的病理学观察[J]. 中国兽医学报, 30(08): 1095-1097+1102. (Geng Y, Wang K Y, Yan Q G, et a1.2010. Pathomorphologic observation of enzootic intranasal adenocarcinoma in Nanjiang yellow goats[J]. Chinese Journal of Veterinary Science, 30(08): 1095-1097+1102.) [3] 江锦秀, 林裕胜, 江斌, 等. 2017. 福建山羊地方性鼻内肿瘤的分子流行病学调查[J]. 福建农业学报, 32(08): 837-841. (Jiang J X, Lin Y S, Jiang B, et a1.2017. Molecular epidemiology of Enzootic nasal tumor virus on goats in Fujian[J]. Fujian Journal of Agricultural Sciences, 32(08): 837-841.) [4] 雷红宇, 苏建明, 宁玲忠, 等. 2006. 一起山羊地方性鼻内肿瘤的调查与诊断[J]. 动物医学进展, 27(2): 112-114. (Lei H Y, Su J M, Ning L Z, et al.2006. Investigation and diagnosis of enzootic intranasal tumour of goats[J]. Progress in Veterinary Medicine, 27(2): 112-114.) [5] 林曦, 郝先谱, 赵振华, 等. 1995. 山羊鼻内腺瘤和腺癌的病理学研究[J]. 畜牧兽医学报, (05): 456-461. (Lin X, Hao X P, Zhao Z H, et al. 1995. Pathological studies on intranasal adenoma and adenocarcinoma in goats[J]. Acta Veterinaria et Zootechnica Sinica, (05): 456-461.) [6] 刘菲, 冯迎春, 颜其贵, 等. 2011. 四川一起山羊地方性鼻内肿瘤疫情的诊断[J]. 畜牧与兽医, 43(6): 83-86. (Liu F, Feng Y C, Yan Q G, et a1.2011. Diagnosis of Enzootic intranasal tumour of goats in Sichuan[J]. Animal Husbandry & Veterinary Medicine, 43(6): 83-86.) [7] 余远迪, 韦雷飞, 黄秀君, 等. 2014. 4例山羊鼻内肿瘤的诊断[J].动物医学进展, 35(04): 129-131. (Yu Y D, Wei L F, Huang X J, et a1.2014. Diagnosis of four cases of enzootic nasal adenocarcinoma of goats[J]. Progress in Veterinary Medicine, 35(04): 129-131.) [8] 何亚鹏, 付明哲, 许信刚, 等. 2017. 山羊地方性鼻内肿瘤病毒Shaanxi株前病毒全基因组克隆及生物信息学分析[J]. 中国预防兽医学报, 39(01): 63-66. (He Y P, Fu M Z, Xu X G, et al.2017. Complete genomic cloning and bioinformatics analysis of goat Enzootic nasal tumor virus Shaanxi strain[J]. Chinese Journal of Preventive Veterinary Medicine, 39(01): 63-66.) [9] 王彬. 2016. ENTV和enENTV全基因组分析及ENA组织miRNA差异表达分析[D]. 博士学位论文, 四川农业大学, 导师: 颜其贵. pp. 90-98. (Wang B.2016. Genomic sequence analysis of oncogenic and Endogenous enzootic nasal tumor virus of goats and analysis of ENA tissue miRNA expression difference[D]. Thesis for Ph. D., Sichuan Agricultural University, Supervisor: Yan Q G, pp. 90-98.) [10] 吴保成, 张春生, 张永久, 等. 1992. 牛白血病病毒结构蛋白及抗原活性分析[J]. 中国兽医科技,(06): 5-6+49. (Wu B C, Zhang C S, Zhang Y J, et al.1992. Analysis of structural proteins and antigenic activity of Bovine leukemia virus (BLV)[J]. Chinese Journal of Veterinary Science and Technology,(06): 5-6+49.) [11] Chertova E, Chertov O, Coren L V, et al.2006. Proteomic and biochemical analysis of purified Human immunodeficiency virus type 1 produced from infected monocyte-derived macrophages[J]. Journal of Virology, 80(18): 9039-9052. [12] Cousens C, Maeda N, Murgia Cet al.2007. In vivo tumorigenesis by Jaagsiekte sheep retrovirus (JSRV) requires Y590 in Env TM, but not full-lenlgth or fX open reading flame[J]. Virology, 367(2): 413-421. [13] De Las Heras M, Ortin A, Cousens C, et a1.2003. Enzootic nasal adenocarcinoma of sheep and goats[J]. Current Topics in Microbiology and Immunology, 275(2): 01-23. [14] Hallwirth C, Maeda N, York D, et al.2005. Variable regions 1 and 2 (VR1 and VR2) in JSRV gag are not responsible for the endogenous JSRV particle release defect[J]. Virus Genes, 30(1): 59-68. [15] He Y, Zhang Q, Wang J, et al.2017. Full-length genome sequence analysis of enzootic nasal tumor virus isolated from goats in China[J]. Virology Journal, 14(1): 141. [16] Holzer T J, Allen R G, Heynen C A, et al.1990. Discrimination of HIV-2 infection from HIV-1 infection by western blot and radioimmunoprecipitation analysis[J]. AIDS Research and Human Retroviruses, 6(4): 515-524. [17] Kawasako K, Okamoto M, Kurosawa T, et a1.2005. Enzootic intranasal tumour virus infection in apparently healthy sheep in Japan[J]. The Veterinary Record, 157(4): 118-120. [18] Liu S L, Lerman M I, Miller A D.2003. Putative phosphatidylinositol 3-kinase (PI3K) binding motifs in Ovine betaretrovirus Env proteins are not essential for rodent fibroblast transformation and PI3K/Akt activation[J]. Journal of Virology, 77(14): 7924-7935. [19] Ortin A, Cousens C, Minguijon E, et a1.2003. Characterization of Enzootic nasal tumour virus of goats: Complete sequence and tissue distribution[J]. Journal of General Virology, 84(8): 2245-2252. [20] Owens R J, Dubay J W, Hunter E, et a1.1991. Human immunodeficiency virus envelope protein determines the site of virus release in polarized epithelial cells[J]. Medical Sciences, 88: 3987-3991. [21] Payne A L,Verwoerd D W, Garnett H M.1983.The morphology and morphogenesis of Jaagsiekte retrovirus (JSRV)[J]. Onderstepoort Journal of Veterinary Research, 50(4): 317-322. [22] Svara T, Gombac M, Vrecl M, et a1.2006. Enzootic nasal adenocarcinoma of sheep in Slovenia[J]. Journal of Veterinary Medicine. A, Physiology, Pathology, Clinical Medicine, 53(1): 26-29. [23] Shin Y K, Liu Q, Tikoo S K, et al.2007. Effect of the phosphatidylinositol 3-kinase/Akt pathway on Influenza A virus propagation[J]. Journal Genernal Virology, 88: 942-950. [24] Palmarini M, Hallwirth C, York D, et al.2000. Molecular cloning and functional analysis of three type D Endogenous retroviruses of sheep reveal a different cell tropism from that of the highly related exogenous Jaagsiekte sheep retrovirus[J]. Journal of Virology, 74(17). [25] Vitellozzi G, Mughetti L, Palmarini M, et al.1993. Enzootic intranasal tumour of goats in Italy[J]. Journal of Veterinary Medicine Series B-infectious Diseases and Veterinary Public Health, 40(7): 459-468. [26] Ye C, Huang Q Y, Chen T, et al.2019. First detection and genotypic analysis of goat Enzootic nasal tumor virus 2 in Chongqing, China[J]. Archives of Virology, 164(6): 1647-1650.
