三株分离于新生小牛的牛病毒性腹泻病毒株的基因组特征

doi:10.3969/j.issn.1004-1524.20221234

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (8): 1814-1822.DOI: 10.3969/j.issn.1004-1524.20221234

三株分离于新生小牛的牛病毒性腹泻病毒株的基因组特征

汪梦竹¹^,²^,³(), 杨光美³, 吴玉湖¹^,²^,³, 杨宣叶¹^,²^,³, 王慧慧¹^,²^,³, 曹小安⁴, 李勇⁴, 马忠仁¹^,², 马晓霞¹^,²^,^*()

1.西北民族大学生物医学研究中心,生物工程与技术国家民委重点实验室,甘肃兰州 730030
2.西北民族大学生物医学研究中心,甘肃省动物细胞技术创新中心,甘肃兰州 730030
3.西北民族大学生命科学与工程学院,甘肃兰州 730010
4.中国农业科学院兰州兽医研究所,甘肃兰州 730046

收稿日期:2022-08-22 出版日期:2023-08-25 发布日期:2023-08-29
作者简介:汪梦竹(1997—),女,山东人,硕士,研究方向为临床兽医学。E-mail: 547493109@qq.com
通讯作者: *马晓霞,E-mail: maxiaoxia956@163.com
基金资助:
中央高校基本科研业务费专项资金(31920220134)

Genomic features of 3 bovine viral diarrhea viruses isolated from newborn claves

WANG Mengzhu¹^,²^,³(), YANG Guangmei³, WU Yuhu¹^,²^,³, YANG Xuanye¹^,²^,³, WANG Huihui¹^,²^,³, CAO Xiaoan⁴, LI Yong⁴, MA Zhongren¹^,², MA Xiaoxia¹^,²^,^*()

1. Key Laboratory of Biotechnology and Bioengineering of State Ethnic Affairs Commission, Biomedical Research Center, Northwest Minzu University, Lanzhou 730030, China
2. Gansu Tech Innovation Center of Animal Cell, Biomedical Research Center, Northwest Minzu University, Lanzhou 730030, China
3. College of Life Science and Engineering, Northwest Minzu University, Lanzhou 730010, China
4. Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China

Received:2022-08-22 Online:2023-08-25 Published:2023-08-29

摘要/Abstract

摘要：

为研究我国不同地区牛病毒性腹泻病毒(Bovine viral diarrhea virus, BVDV)感染新生小牛的遗传演化规律。以实验室保存的来自山东、内蒙古和宁夏的新生荷斯坦小牛血清为材料,利用RT-PCR方法对小牛血清进行BVDV RNA检测,并将检测为阳性的血清接种于牛肾细胞(MDBK),经分离鉴定获得3株BVDV野生株,分别命名为21NM-44、21NX-53和21SD-16。根据3株病毒对MDBK细胞的致细胞病变能力,判定21NM-44为非致细胞病变型毒株,21NX-53和21SD-16为致细胞病变型毒株。利用特异性PCR引物,克隆鉴定得到3株病毒的全基因组序列,依据全基因组序列建立系统进化树并分析其遗传演化关系。结果表明,3株来自不同地区的BVDV野生分离株与BVDV GXNN1毒株(BVDV-1c)具有高度的遗传演化关系,而BVDV-1c作为优势流行株存在于我国新生荷斯坦小牛。本研究结果可以为进一步研究BVDV在我国的演化规律奠定基础。

关键词: 牛病毒性腹泻病毒, 新生小牛, 全基因组, 牛肾细胞, 细胞病变能力

Abstract:

To study the genetic evolution of Bovine viral diarrhea virus(BVDV)-infected newborn calves in different regions of China, the newborn Holstein calf serum from Shandong, Inner Mongolia and Ningxia provinces preserved in the laboratory was used as the material, and the calf serum was detected by RT-PCR for BVDV RNA, and the positive serum was inoculated into Madin-Darby bovine kidney cells (MDBK). Three isolates were identified as BVDV and named as 21NM-44, 21NX-53 and 21SD-16, respectively. According to the cytopathic ability of the 3 isolates to MDBK cells, 21NM-44 was determined to be a non-cytopathic strain, while 21NX-53 and 21SD-16 could cause cytopathic and were determined to be cytopathic strains. Using specific PCR primers, the whole genome sequences of the 3 viruses were cloned and identified. Based on the whole genome sequences, a phylogenetic tree was established and their genetic evolution relationship was analyzed. The results showed that the 3 wild BVDV isolates from different regions had a high degree of genetic evolution relationship with the BVDV GXNN1 strain (BVDV-1c). This study made a foundation for further research on the evolution of BVDV in China.

Key words: Bovine viral diarrhea virus, newborn calf, whole genome, Madin-Darby bovine kidney cell, cytopathic ability

中图分类号:

S852.653

汪梦竹, 杨光美, 吴玉湖, 杨宣叶, 王慧慧, 曹小安, 李勇, 马忠仁, 马晓霞. 三株分离于新生小牛的牛病毒性腹泻病毒株的基因组特征[J]. 浙江农业学报, 2023, 35(8): 1814-1822.

WANG Mengzhu, YANG Guangmei, WU Yuhu, YANG Xuanye, WANG Huihui, CAO Xiaoan, LI Yong, MA Zhongren, MA Xiaoxia. Genomic features of 3 bovine viral diarrhea viruses isolated from newborn claves[J]. Acta Agriculturae Zhejiangensis, 2023, 35(8): 1814-1822.

图/表 7

参考文献 30

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引物名称 Primer name	正向引物 Forward primer sequence (5'→3')	反向引物 Reverse primer sequence (5'→3')	产物长度 Product length/bp
BVDV1	TGAATCCCCTTCAGCGAAGGC	CATGTGCCATGTACAGCAGAG	319
BVDV2	CTAAAAATCTCTGCTGTACATG	CTGGTGGCTTGTTCTTGTTGTG	759
BVDV3	GAACAAGCCACCAGAGTCACGC	GTTCTTTAAACAGCACATCAC	596
BVDV4	CGATAGTAGTGGATGGGGTCAAG	TGTTACAAGAAGTGTCATCTAC	2 184
BVDV5	CGTCGAGTCATGCAAGTGGTG	GAAGAAACTGTCCCTGGGCTCG	344
BVDV6	CTTCAGATATAAAAGATGGCTG	GCTGCCACGGTCTCATTTCTTAC	736
BVDV7	GATATCCAGGGTTGTGGCAGC	CTCCTCACTTTTAATAAACTTG	696
BVDV8	CAAGTTTATTAAAAGTGAGGAG	GTCATAGTGGTAGTCTTTTGATC	1 469
BVDV9	CGGATCAAAAGACTACCACTATG	GAAGGAAGGCTTATTTATCAC	1 049
BVDV10	CAGACAGGCAGCAGTTGACTTG	CTTCCTATTATTGAGATCCCAAC	1 572
BVDV11	GCAAGGTTAGTGGGGCAAGCAG	TATCCTTTACCCTTGGTTTCTTC	1 427
BVDV12	GTTACAGCAAGAGTAGACTAC	CTATCTCTAGTTGTTCCCATG	1 761
BVDV13	GATACTAAAAAGAATAAAGTTTC	GCTAAAGTGCTATGTGCGTTG	2 036
BVDV14	CAGAGTCAGTCTACCAATATATG	CAGTTCTGGCCATGATGTTC	1 002
BVDV15	CAACTGGCACAAGGTAACTG	TCTGTAGGCAACTTTCATC	1 351
BVDV16	GAGTCATCCAATACCCTGAAGC	TGAGGTCTTTTCCTAGTCCAAC	1 524
BVDV17	CATTACTAAGGTAATGTACAAC	GTTGATTGTAGTGTTATCTTGAG	1 423
BVDV18	CAGAGGGCCGGTCAACTATAAG	CAGTTTTCCAGGATTGAGCATC	1 586
BVDV19	CACATTAACATAATGGACAAGC	CTTTCAAATCCCCTATTCTTAAG	816
BVDV20	GAGAGGATGAAAGTTGCCTAC	GAGGTCTTTTCCTAGTCCAAC	930

引物名称 Primer name	正向引物 Forward primer sequence (5'→3')	反向引物 Reverse primer sequence (5'→3')	产物长度 Product length/bp
BVDV1	TGAATCCCCTTCAGCGAAGGC	CATGTGCCATGTACAGCAGAG	319
BVDV2	CTAAAAATCTCTGCTGTACATG	CTGGTGGCTTGTTCTTGTTGTG	759
BVDV3	GAACAAGCCACCAGAGTCACGC	GTTCTTTAAACAGCACATCAC	596
BVDV4	CGATAGTAGTGGATGGGGTCAAG	TGTTACAAGAAGTGTCATCTAC	2 184
BVDV5	CGTCGAGTCATGCAAGTGGTG	GAAGAAACTGTCCCTGGGCTCG	344
BVDV6	CTTCAGATATAAAAGATGGCTG	GCTGCCACGGTCTCATTTCTTAC	736
BVDV7	GATATCCAGGGTTGTGGCAGC	CTCCTCACTTTTAATAAACTTG	696
BVDV8	CAAGTTTATTAAAAGTGAGGAG	GTCATAGTGGTAGTCTTTTGATC	1 469
BVDV9	CGGATCAAAAGACTACCACTATG	GAAGGAAGGCTTATTTATCAC	1 049
BVDV10	CAGACAGGCAGCAGTTGACTTG	CTTCCTATTATTGAGATCCCAAC	1 572
BVDV11	GCAAGGTTAGTGGGGCAAGCAG	TATCCTTTACCCTTGGTTTCTTC	1 427
BVDV12	GTTACAGCAAGAGTAGACTAC	CTATCTCTAGTTGTTCCCATG	1 761
BVDV13	GATACTAAAAAGAATAAAGTTTC	GCTAAAGTGCTATGTGCGTTG	2 036
BVDV14	CAGAGTCAGTCTACCAATATATG	CAGTTCTGGCCATGATGTTC	1 002
BVDV15	CAACTGGCACAAGGTAACTG	TCTGTAGGCAACTTTCATC	1 351
BVDV16	GAGTCATCCAATACCCTGAAGC	TGAGGTCTTTTCCTAGTCCAAC	1 524
BVDV17	CATTACTAAGGTAATGTACAAC	GTTGATTGTAGTGTTATCTTGAG	1 423
BVDV18	CAGAGGGCCGGTCAACTATAAG	CAGTTTTCCAGGATTGAGCATC	1 586
BVDV19	CACATTAACATAATGGACAAGC	CTTTCAAATCCCCTATTCTTAAG	816
BVDV20	GAGAGGATGAAAGTTGCCTAC	GAGGTCTTTTCCTAGTCCAAC	930

长度Length/nt	21NM-44	21NX-53	21SD-16
5'UTR	283	282	153
3'UTR	208	315	217
N^pro	504	504	504
Capsid	312	312	312
E^rns	684	684	684
E1	576	576	585
E2	1 122	1 122	1 122
P7	207	207	207
NS2	1 356	1 362	1 362
NS3	2 049	2 049	2 049
NS4A	192	192	192
NS4B	1 050	1 041	1 041
NS5A	1 488	1 488	1 488
NS5B	2 160	2 160	2 160

长度Length/nt	21NM-44	21NX-53	21SD-16
5'UTR	283	282	153
3'UTR	208	315	217
N^pro	504	504	504
Capsid	312	312	312
E^rns	684	684	684
E1	576	576	585
E2	1 122	1 122	1 122
P7	207	207	207
NS2	1 356	1 362	1 362
NS3	2 049	2 049	2 049
NS4A	192	192	192
NS4B	1 050	1 041	1 041
NS5A	1 488	1 488	1 488
NS5B	2 160	2 160	2 160

三株分离于新生小牛的牛病毒性腹泻病毒株的基因组特征

Genomic features of 3 bovine viral diarrhea viruses isolated from newborn claves

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