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一株具有插入和缺失突变特征鸭腺病毒B毒株的分离鉴定

郭博伦 李雅汝 郑欣 江丹丹 曾丽 陈少莺 马燕梅 陈仕龙

郭博伦,李雅汝,郑欣,等. 一株具有插入和缺失突变特征鸭腺病毒B毒株的分离鉴定 [J]. 福建农业学报,2024,39(2):1−6
引用本文: 郭博伦,李雅汝,郑欣,等. 一株具有插入和缺失突变特征鸭腺病毒B毒株的分离鉴定 [J]. 福建农业学报,2024,39(2):1−6
GUO B L, LI Y R, ZHENG X, et al. Isolation and identification of a novel duck adenovirus B2 strain with insertion and deletion mutation characteristics [J]. Fujian Journal of Agricultural Sciences,2024,39(2):1−6
Citation: GUO B L, LI Y R, ZHENG X, et al. Isolation and identification of a novel duck adenovirus B2 strain with insertion and deletion mutation characteristics [J]. Fujian Journal of Agricultural Sciences,2024,39(2):1−6

一株具有插入和缺失突变特征鸭腺病毒B毒株的分离鉴定

基金项目: 福建省科技计划公益类专项(2019R1026-11);福建省农业科学院科技创新团队建设项目(CXTD2021034);福建省农业高质量发展超越“5511”协同创新工程项目(XTCXGC2021018、XTCXGC2021012);福建农林大学科技创新专项基金(KFB23098A)
详细信息
    作者简介:

    郭博伦(1999 —),男,硕士研究生,主要从事分子病毒学研究,E-mail:754598753@qq.com

    通讯作者:

    马燕梅(1971 —),女,博士,副教授,主要从事动物病原微生物及免疫学研究,E-mail:89mym@163.com

    陈仕龙(1979 —),男,博士,研究员,主要从事动物病毒病研究,E-mail:CSL6116@163.com

  • 中图分类号: S858.32

Isolation and identification of a novel duck adenovirus B2 strain with insertion and deletion mutation characteristics

  • 摘要:   目的  对一份疑似鸭腺病毒B2(Duck adenovirus B2, DAdV B2)感染的番鸭白肝病的病例进行确诊,并对分离株进行测序,为福建省DAdV B2流行病学研究提供参考。  方法  开展病料PCR检测,并进行病毒的分离鉴定、全基因二代测序,利用 MegAlign和SnapGene软件对测序结果进行同源性及遗传进化分析,再进行动物回归试验,确定对雏番鸭的致病性。  结果  病料样本检测为DAdV B2阳性,并成功分离到一株DAdV B2毒株,命名为DAdV B2/BG48。该分离株感染鸡肝癌细胞(LMH)细胞后细胞变大变圆、最后死亡崩解,形成特征性细胞病变;感染MDEF后细胞由长梭形变为圆形并聚集,细胞间出现空隙。测序结果表明,BG48基因组在pX基因区域中有3 bp的插入,在ORF19B基因区域有33 bp的插入,在ORF64和ORF67基因区域的交界处有42 bp的缺失,ORF67基因起始密码子往后第133位碱基为G,没有突变为终止密码子,其他编码基因与 CH-GD-12-2014、实验室之前鉴定的BG27和BG18无特征性差异。动物回归试验显示,2日龄的番鸭对DAdV B2分离株BG48易感,致病率为50%,死亡率为0%,发病鸭可见与自然感染病鸭相似的临床症状和病理变化。  结论  成功从番鸭白肝病病例中分离鉴定1 株DAdV B2突变株BG48,BG48具有多位点插入和缺失特征,提示DAdV B2毒株容易突变,临床流行毒株复杂。本结果为DAdV B2的分子流行病学调查和遗传进化研究提供了参考。
  • 图  1  临床样品的PCR检测

    M:DNA标准DL 2 000;1:鸭腺病毒B2阳性对照;2:临床样品;3:DNA/RNA free水对照。

    Figure  1.  PCR detection of clinical samples

    M:DNA Marker DL 2 000;1:Duck adenovirus B2 positive control;2:Clinical samples;3:Negative.

    图  2  BG48感染MDEF和LMH细胞的细胞病变

    A:正常MDEF细胞(200×);B:BG48接种MDEF细胞4 d后的CPE(200×);C:正常LMH细胞(100×);D:BG48接种LMH细胞4 d后的CPE(100×)。

    Figure  2.  Cytopathic effect of BG 48 infected MDEF and LMH cells

    A: normal MDEF cells (200×); B: CPE 4 days after MDEF cells (200×); C: normal LMH cells (100×); and D: BG 48 CPE 4 days after LMH cells (100×).

    图  3  病毒全基因组序列同源性分析

    Figure  3.  Homology analysis of virus whole genome sequence

    图  4  基因插入或缺失的PCR扩增验证

    A:pX扩增产物琼脂糖凝胶电泳;M为DNA DL 2000 相对分子质量标准,1为阴性对照,2为阳性对照,3为BG48。B:ORF19B和ORF64/67扩增产物琼脂糖凝胶电泳;M为DNA DL 5000 相对分子质量标准,1~3为ORF19B引物扩增,1为阴性对照,2为阳性对照,3为BG48 ,4~6为ORF64/67引物扩增,4为阴性对照,5为阳性对照,6为BG48。

    Figure  4.  PCR amplification verification of gene insertion or deletion by PCR amplifications

    A: pX amplification; M: DNA DL 2000 marker; 1: Negative control; 2: Positive control; 3: BG 48. B: ORF19B and ORF64/67 amplification; 1–3 is the PCR amplification results of ORF19B primer. M: DNA DL 5000 marker 1: Negative Control; 2: Positive Control; 3: BG48; 4–6 is the PCR amplification results of ORF64/67 primer 4: Negative Control; 5: Positive Control; 6: BG 48.

    图  5  BG48基因组基因插入与缺失

    A:ORF19B; B:pX; C:ORF64和ORF67及其之间非编码区(蓝色箭头代表ORFs;橙色四边形代表非编码区;虚线代表缺失区域;红色代表插入区域)。

    Figure  5.  Map of gene insertions and deletions in the BG48 genome

    A: ORF19B; B: pX;C: Coding and non-coding regions between ORF 64 and ORF 67 (Blue arrows representing ORFs; orange quadrangle representing non-coding regions; dashed lines representing the delected region; red representing the inserted region).

    图  6  攻毒番鸭内脏病变

    A:正常番鸭肝脏; B~E分别为BG48攻毒番鸭肝脏、脑壳、胰腺及肾脏。

    Figure  6.  The autopsy lesion of Muscovy ducklings after BG48 infection

    A: Normal Muscovy duck liver; B–E: BG48 infected Muscovy duck liver, braincase, pancreas and kidney, respectively.

    表  1  引物序列

    Table  1.   Primer sequences

    引物名称
    Primer names
    引物序列(5′- 3')
    Primer sequences(5′-3')
    退火温度
    Annealing temperature/℃
    扩增长度
    Product length/bp
    DAdV B2 fiber 1-F TATCCCTACTGGTGGCCCTC 59.9 581
    DAdV B2 fiber 1-R TCAGTGGCTGCGTACACTTT
    ORF67-F ATGTACGCAATTCCATTCTCA 49.8 174
    ORF67-R GTTACAAATTAACTTTTGAA
    pX-F ACATCCTCATCACCAACCAT 52.3 837
    pX-R TCCAGCTGCAAGATCTATGT
    ORF64/67-F CTTAGGACTCCAAACCCAAATAGAT 56.8 2721
    ORF64/67-R TGCCATGCGCTACGCTAAG
    ORF19B-F ACAGGCTCCATGAGCTCTCAC 58.5 2292
    ORF19B-R TTGAGCCAGAGTCCGTAAAGC
    下载: 导出CSV
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  • 收稿日期:  2024-01-01
  • 修回日期:  2024-02-01
  • 网络出版日期:  2024-03-28

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