一株牦牛源牛病毒性腹泻病毒毒株的分离鉴定及其遗传特性分析

doi:10.3969/j.issn.1004-1524.2022.11.06

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (11): 2368-2378.DOI: 10.3969/j.issn.1004-1524.2022.11.06

一株牦牛源牛病毒性腹泻病毒毒株的分离鉴定及其遗传特性分析

王慧慧¹^,²(), 冯茜莉¹^,², 汪梦竹¹^,², 赵泽阳¹^,², 李易聪¹^,², 蒲飞洋¹^,², 马鹏¹^,², 李勇³, 龚真莉³, 马忠仁¹, 马晓霞¹^,^*()

1.西北民族大学生物医学研究中心,甘肃兰州 730030
2.西北民族大学生命科学与工程学院,甘肃兰州 730010
3.中国农业科学院兰州兽医研究所,甘肃兰州 730046

收稿日期:2021-10-28 出版日期:2022-11-25 发布日期:2022-11-29
通讯作者: 马晓霞
作者简介:*马晓霞,E-mail: maxiaoxia956@163.com
王慧慧(1997—),女,甘肃天水人,硕士研究生,研究方向为病毒生物分子与免疫学。E-mail: 3407975334@qq.com
基金资助:
校企横向合作项目(2021西民合(科研)字第175号)

Isolation, identification and analyses for genetic features of one bovine viral diarrhea virus strain from yak

WANG Huihui¹^,²(), FENG Xili¹^,², WANG Mengzhu¹^,², ZHAO Zeyang¹^,², LI Yicong¹^,², PU Feiyang¹^,², MA Peng¹^,², LI Yong³, GONG Zhenli³, MA Zhongren¹, MA Xiaoxia¹^,^*()

1. Biomedical Research Center, Northwest Minzu University, Lanzhou 730030, China
2. College of Life Science and Engineering, Northwest Minzu University, Lanzhou 730010, China
3. Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China

Received:2021-10-28 Online:2022-11-25 Published:2022-11-29
Contact: MA Xiaoxia

摘要/Abstract

摘要：

通过病毒分离培养技术,从牦牛血清中分离获得一株致细胞病变的牛病毒性腹泻病毒(BVDV),命名为GSTZ毒株。经电镜观察,GSTZ毒株的直径在50~60 nm。按Karber法测算,该病毒滴度为5.0×10^6.5 mL^-1[以组织半数感染量(TCID₅₀)计]。利用反转录PCR(RT-PCR)测定GSTZ毒株的完整开放阅读框(ORF),全长11 691 nt,将其与参考毒株ORF在核酸序列和同义密码子使用模式等方面进行分析,确定GSTZ毒株在遗传学特性上属于基因1型家族成员。在GSTZ毒株的完整ORF中,A+U的出现频率要高于G+C,而且,病毒同义密码子的使用模式体现出对U/A结尾同义密码子使用偏嗜性高的遗传学特征。GSTZ毒株明显降低了使用含有CpG二联核苷酸的同义密码子的频率。这有助于降低病毒对宿主细胞免疫系统的刺激强度,从而促进病毒的复制增殖。研究成果可为进一步研究BVDV的相关分子机制提供试验材料,并为BVDV不同基因型的抗原关系分析与BVDV疫苗的研制奠定基础。

关键词: 牛病毒性腹泻病毒, 牦牛, 基因1型, 同义密码子, 致细胞病变

Abstract:

In this study, one bovine viral diarrhea virus (BVDV) strain was successfully isolated and termed as GSTZ. By electron microscopy, several 50-60 nm viral particles were observed. GSTZ strain displayed the TCID₅₀ (50% tissue culture infection dose) value of 5.0×10^6.5 mL^-1 as revealed by Karber method. Via reverse transcription-PCR (RT-PCR), the whole open reading frame (ORF) of GSTZ strain was sequenced (whole length of 11 691 nt) and classified into the genotype 1 group at either nucleotide level or synonymous codon usage level. The appearance frequency of A+U was higher than that of G+C in GSTZ ORF, and the synonymous codon usage pattern of GSTZ strain displayed the obvious usage bias in synonymous codons with U/A. It was worth noting that, this strain had a tendency of avoiding the selection of the synonymous codons containing CpG dinucleotides, which strongly suggested that the GSTZ strain might weaken the immune responses from host in order to successfully generate its viral particles. The isolated BVDV strain and the above findings could provide biological materials for the related mechanism study of BVDV, and lay foundations for the analysis of antigen relationship of different genotypes of BVDV and vaccine development.

Key words: bovine viral diarrhoea virus, yak, genotype 1, synonymous codon, cytopathogenic effect

中图分类号:

S852.653

王慧慧, 冯茜莉, 汪梦竹, 赵泽阳, 李易聪, 蒲飞洋, 马鹏, 李勇, 龚真莉, 马忠仁, 马晓霞. 一株牦牛源牛病毒性腹泻病毒毒株的分离鉴定及其遗传特性分析[J]. 浙江农业学报, 2022, 34(11): 2368-2378.

WANG Huihui, FENG Xili, WANG Mengzhu, ZHAO Zeyang, LI Yicong, PU Feiyang, MA Peng, LI Yong, GONG Zhenli, MA Zhongren, MA Xiaoxia. Isolation, identification and analyses for genetic features of one bovine viral diarrhea virus strain from yak[J]. Acta Agriculturae Zhejiangensis, 2022, 34(11): 2368-2378.

图/表 8

表1 引物序列

Table 1 The sequences of primers

引物 Primers	正向引物序列 Forward primer sequences(5'→3')	反向引物序列 Reverse primer sequences(5'→3')	产物长度 Product length/bp
BVDV1	CCACCCACTACAAAGAGCAG	CACCGTAGGTATGTTTCAGATTAG	541
BVDV2	GATAGCAGGACTAAACCACC	CCATGTAGGCTCCTATTGAC	293
BVDV3	AACAGACCCACGCTACTGA	CCTGCCCAACTTCTTCAAAT	728
BVDV4	CGAAAGCCTTACCACCGTCTG	CGCCCTTTAGGAATCTGTAACCAC	578
BVDV5	AACTTTAACATTGTTGGTGGCT	CCCTGGGTGTAGTTCCTCTT	1 283
BVDV6	CATCATACGGATATTTCTGCC	TTCCTCTATTGGGTGCTTTT	221
BVDV7	TTGTAAAGTCCCAAGCAGAA	AGTCCATACCCAGTAGTTCG	954
BVDV8	GCAGCAGTTGATTTGGTAGT	GTTCGGGTTATTGTCAGCAT	1 638
BVDV9	GCGGAAGGGTAGTTGGTAGA	GCAAGTCCTCGGAGATGAGC	1 233
BVDV10	AAAGCAAGTGATGGGTCTGT	ACTGGAGGTGGGTGGTATCT	158
BVDV11	GGAAAGGGAAGAGTGATGGA	AGCCTGGTCAGGTTGGAGAT	1 069
BVDV12	TGGATTAGATGATGGCAGTT	TTTATTTGATGTTTGGTGGG	586
BVDV13	TATCGGAGAAATCCAGAAGT	TGGATTAGATGAGGCAGTT	449

图1 RT-PCR扩增结果 M,DL2000 DNA marker;N,阴性对照;1~16,血清样品。

Fig.1 Amplification result of RT-PCR M, DL2000 DNA marker; N, Negative control; 1-16, Serum samples.

图2 GSTZ在MDBK细胞上的病变情况(5×) A,阴性对照的正常MDBK细胞(5 d);B,感染分离株后MDBK细胞发生明显病变(5 d)。

Fig.2 Cytopathogenic effects caused by the isolated strain on MDBK cells(5×) A, Cell culture after incubation for 5 days in the negative control; B, MDBK cells exhibited cytopathogenic effects after infection with the isolated strain for 5 days.

图3 接种分离株5 d后纯化MDBK细胞培养物在电镜下观察到的病毒粒子在电子显微镜下可以观察到数个直径在50~60 nm的病毒粒子(白箭头所示)。

Fig.3 Virus particles observed under electron microscope in purified MDBK cell cultures inoculated with isolated strain after 5 days Several 50-60 nm viral particles (white arrows show) were observed under electron microscopy.

图4 GSTZ分离株基因组的RT-PCR扩增结果 M,DL2000 DNA marker;1~13,RT-PCR扩增产物(分别对应于表1中的引物BVDV1—BVDV13)。

Fig.4 RT-PCR amplification result of GSTZ strain genome M, DL2000 DNA marker; 1-13, RT-PCR amplification product (in accordance with the primers BVDV1-BVDV13 in Table 1).

图5 GSTZ分离株的基因组系统发育分析 gt1和gt2别表示基因1型和基因2型。

Fig.5 Phylogenetic analysis of GSTZ isolates gt1 and gt2 represents genotype 1 and genotype 2, respectively.

表2 BVDV GSTZ毒株同义密码子使用模式的遗传特征

Table 2 Synonymous codon usage patterns of BVDV GSTZ strain

同义密码子(氨基酸) Synonymous codon (Amino acid)	RSCU	同义密码子(氨基酸) Synonymous codon (Amino acid)	RSCU
UUU(F)	0.98	GCC(A)	1.24
UUC(F)	1.02	GCA(A)	1.52
UUA(L)	0.90	GCG(A)	0.31
UUG(L)	1.34	UAU(Y)	0.92
CUU(L)	0.50	UAC(Y)	1.08
CUC(L)	0.53	CAU(H)	0.72
CUA(L)	1.34	CAC(H)	1.27
CUG(L)	1.39	CAA(Q)	0.97
AUU(I)	0.62	CAG(Q)	1.03
AUC(I)	0.69	AAU(N)	0.86
AUA(I)	1.69	AAC(N)	1.14
GUU(V)	0.59	AAA(K)	0.99
GUC(V)	1.01	AAG(K)	1.01
GUA(V)	0.95	GAU(D)	1.14
GUG(V)	1.45	GAC(D)	0.86
UCU(S)	0.79	GAA(E)	1.06
UCC(S)	0.71	GAG(E)	0.94
UCA(S)	1.74	UGU(C)	1.13
UCG(S)	0.09	UGC(C)	0.87
AGU(S)	1.29	CGU(R)	0.06
AGC(S)	1.38	CGC(R)	0.19
CCU(P)	1.01	CGA(R)	0.19
CCC(P)	0.92	CGG(R)	0.30
CCA(P)	1.66	AGA(R)	2.70
CCG(P)	0.41	AGG(R)	2.56
ACU(T)	0.91	GGU(G)	0.82
ACC(T)	1.04	GGC(G)	0.72
ACA(T)	1.58	GGA(G)	0.95
ACG(T)	0.46	GGG(G)	1.51
GCU(A)	0.93

图6 基于RSCU数据主成分分析的GSTZ毒株ORF同义密码子使用模式遗传进化解析

Fig.6 Visualization of genetic diversity for GSTZ ORF at synonymous codon usage pattern based on RSCU data via principal component analysis

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一株牦牛源牛病毒性腹泻病毒毒株的分离鉴定及其遗传特性分析

Isolation, identification and analyses for genetic features of one bovine viral diarrhea virus strain from yak

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