山羊Aichivirus C的分离鉴定及演化分析

doi:10.11843/j.issn.0366-6964.2024.08.032

摘要/Abstract

摘要：

C型爱知病毒(Aichivirus C)是新近证实的致山羊腹泻病毒，本研究旨在分离山羊Aichivirus C并分析其基因组特征。在四川某暴发腹泻的山羊场采集腹泻粪便9份，1只病死羔羊的组织样本13份，进行常见的腹泻病原检测。采用Vero细胞系对山羊Aichivirus C阳性样本进行病毒分离，以PCR、间接免疫荧光试验和电镜技术对分离毒株进行鉴定，并扩增获得其基因组序列。结果显示：通过RT-PCR方法从9份山羊腹泻粪便样本中检出Aichivirus C阳性8份，羔羊的组织样本中检出Aichivirus C阳性8份，其它常见的致山羊腹泻病原在上述粪便和组织样本中均未检出。使用Vero细胞系成功从阳性样品中分离到2株山羊Aichivirus C，其病毒滴度分别为10^6.5 TCID₅₀·mL^-1和10^5.9 TCID₅₀·mL^-1。获得两条近乎全长基因组序列，与GenBank中山羊源Aichivirus C毒株的核苷酸相似性为78.8%~97.4%，与国内SWUN/F11/2019毒株的核苷酸相似性为97.0%~97.4%。此外，本研究还从5份临床阳性样本中扩增出VP0、VP3和VP1基因完整序列。基于基因组的演化分析显示：获得的两个病毒株与SWUN/F11/2019毒株在系统发育树上聚为一支。并且，这些毒株的VP0和VP1基因编码蛋白也有独特的氨基酸突变。综上所述，本研究分离得到2株山羊Aichivirus C，获得了其近乎全长基因组序列，与国内SWUN/F11/2019毒株亲缘关系最近，其VP0和VP1基因具有独特的分子特征。本结果丰富了国内山羊源Aichivirus C的分子遗传信息，为进一步研究山羊Aichivirus C的致病性和分子生物学特征提供了参考。

关键词: 山羊, 腹泻, C型爱知病毒, 分离鉴定, 分子特征

Abstract:

Aichivirus C is a newly confirmed virus associated with caprine diarrhoea. The purpose of this study was to isolate goat Aichivirus C and analyze the genomic characteristics. Nine fecal samples and 13 tissue samples from a dead kid were collected from a goat farm with an outbreak of diarrhoea in Sichuan province, and used for testing the common pathogens associated with diarrhoea. Virus was isolated from Aichivirus C positive samples using Vero cells and identified by PCR, indirect immunofluorescence and electron microscopy. The complete genomes of the isolates were amplified and analyzed. Results were as follows: 8 fecal samples with diarrhoea and 8 tissue samples from a lamb were detected as positive for Aichivirus C by RT-PCR, and no other common diarrhoea-causing pathogens were detected in the above samples. Two Aichivirus C strains were successfully isolated from positive samples using Vero cells, and the virus titers were 10^6.5TCID₅₀·mL^-1 and 10^5.9TCID₅₀·mL^-1, respectively. The nearly full-length genomic sequences of both strains were obtained, which shared 78.8%-97.4% nucleotide identity with the known goat Aichivirus C genomes from GenBank and 97.0%-97.4% nucleotide identity with the SWUN/F11/2019 isolate from China. Furthermore, the VP0, VP3 and VP1 genes were successfully amplified from 5 clinical positive samples. Phylogenetic analysis based on the genome revealed that the two isolates were clustered with the SWUN/F11/2019 strain. Unique amino acid mutations were also revealed in the VP0 and VP1 genes. In summary, two strains of goat Aichivirus C were successfully isolated and the nearly full-length genomic sequences were obtained. Phylogentic analysis demonstrated that these two strains are closely related to the SWUN/F11/2019 strain from China, with a unique molecular characteristics in the VP0 and VP1 genes. The results enrich the molecular genetic information of Aichivirus C from goats in China and laid the foundation for further studies on the pathogenicity and molecular biological characteristics of goat Aichivirus C.

Key words: goat, diarrhea, Aichivirus C, isolation, molecular characterisation

中图分类号:

S852.659.6

程玉婷, 阿比克哈莫, 杨晨, 张丁中, 任云鑫, 岳华, 汤承. 山羊Aichivirus C的分离鉴定及演化分析[J]. 畜牧兽医学报, 2024, 55(8): 3612-3622.

Yuting CHENG, ABI-Kehamo, Chen YANG, Dingzhong ZHANG, Yunxin REN, Hua YUE, Cheng TANG. Isolation and Molecular Characterization of Aichivirus C from Goats[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3612-3622.

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引物名称 Primer Name	上游引物(F) Forward	上游引物(R) Reverse	片段大小/bp Amplification size
1	5′-TCTTTTCCGGTGGCCA-3′	5′-TCGGCATCAGTGGTGAAAGA-3′	1 065
2	5′-CCCACCATTCCTCCCACCTT-3′	5′-AGGGTAGGCTCGCACAAAGG-3′	1 009
3	5′-CCCACCATTCCTCCCACCTT-3′	5′-CGGTAGAGCCCGTCGTAATC-3′	738
4	5′-AGTGGACTGCCCTTCAACAA-3′	5′-CACAGGTGGGCGATAGGTTT-3′	626
5	5′-AATGCGTTTGGTTCTGTTGTG-3′	5′-ACATCGGTCTCGCCATCGTA-3′	1 203
6	5′-CCAGGTTGTCCCGCTTTCAT-3′	5′-TCGGGTGAACGGCACGCAAT-3′	440
7	5′-TCTCCGCTGTCGTCGCTTAC-3′	5′-AGGGCTTGTGGGTCCGTCTT-3′	886
8	5′-CAGTCAATGCGGTGAAGAACA-3′	5′-AAACAGCACGCTCAAAGGAA-3′	1 092
9	5′-GTGCTGTTTCCCAGAACGCTCCC-3′	5′-CGAGTTTGGATTTGCGGTTG-3′	912
10	5′-GGGTGAATGTCAACCGCAAAT-3′	5′-CCCGTAAATGTGACGGGA-3′	803
11	5′-AAACTAACTGGAGATGAAAGGGTC-3′	5′-TCTCCTGCCTCAGCCTCCCT	784
12	5′-CCAGGGAGGCTGAGGCAGGAGAAT-3′	5′-AAGAACTTAATGCTCTCTAATTTTTTT-3′	153

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