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准噶尔盆地东南部亚洲璃眼蜱携带蜱媒病毒的宏基因组学研究

Metagenomic study of tick-borne viruses carried by Hyalomma asiaticum in southeastern Junggar basin

    摘要
  • 摘要:
    目的 调查新疆维吾尔自治区（新疆）准噶尔盆地东南部地区蜱媒病毒的多样性。
    方法 2023年4月底在新疆准噶尔盆地东南部地区阜康县（44°23′ N，88°8′ E）和奇台县（44°11′ N， 89°37′ E）两地的半沙漠采样点采集蜱样本，并进行形态与分子生物学鉴定；分别按采样地点以及蜱种不同将采集到的蜱分为若干组，每组选取15只同种蜱，提取总的病毒RNA，构建cDNA文库后在Illumina Hiseq 2500 平台上进行测序，运用生物信息学工具对测序结果进行病毒组分分析，探究病毒的多样性；使用G-test检验方法比较两地蜱媒病毒组成的差异；使用聚合酶链式反应（PCR） 方法对分析的病毒序列进行验证。
    结果 本研究共采集蜱189只，经鉴定均为亚洲璃眼蜱。 按采样地点不同将蜱样本分为FK和QT两组进行宏基因组学研究，病毒物种注释结果显示，两组样本共有57条contigs与病毒基因组序列相匹配，匹配到的病毒基因组分别属于楚病毒科、白蛉纤细病毒科、全病毒科、弹状病毒科、戊型肝炎病毒科和未分类的病毒科；经组间差异比较，除Kashgar Totiv tick virus 1外， 博乐蜱病毒 1（ P=1×10−15）、博乐蜱病毒 3（P=3.71×10−3）、泰顺蜱病毒（P=5.95×10−5）、博乐蜱病毒 4（P=7.45×10−4）、Bole hyalomma asiaticum virus 1（P=1.72×10−8），Lonestar tick totivirus（P=1.11×10−6）在两样本间丰度差异有统计学意义；为了验证上述结果，根据已鉴定的7种病毒的基因组序列设计9对引物进行PCR扩增验证，最终证明了博乐蜱病毒 1、博乐蜱病毒 3、 Taishun Tick Virus以及Bole hyalomma asiaticum virus 这4种病毒的存在。
    结论 准噶尔盆地东南部地区亚洲璃眼蜱所携带的蜱媒病毒组成复杂，阜康和奇台两县蜱媒病毒丰度存在明显差异，通过PCR技术证实了其中 4种病毒的存在。

     

    Abstract:
    Objective To investigate the diversity of tick-borne viruses in southeastern part of Junggar basin, Xinjiang Uyghur Autonomous Region.
    Methods In late April 2023, we collected tick samples from semi-desert sampling sites in Fukang (latitude 44°23′ N, longitude 88°8′ E) and Qitai (latitude 44°11′ N, longitude 89°37′ E) counties in southeastern Junggar basin. The collected ticks were identified morphologically and molecularly. Subsequently, the samples were categorized based on the collection sites and tick species. In each group, 15 ticks of same species were randomly selected for the extraction of total viral RNA. cDNA libraries were constructed from these extractions and the sequencing analysis was conducted on Illumina HiSeq 2500 platform. Bioinformatics tools were used to analyze the viral components of the sequencing data, exploring the diversity of the viruses. G-test was used to compare the differences in abundance composition of tick-borne viruses between two sampling sites. Additionally, polymerase chain reaction(PCR) was used to verify the viruses identified through metagenomics.
    Results A total of 189 tick samples were collected, all were identified as Hyalomma asiaticum. The virus species annotation results revealed that both sample groups had 57 contigs matching viral genome sequences of Chuvirudae, Phenuiviridae, Totiviridae, Rhabdoviridae, Hepeviridae and other unclassified virus families. Through inter-group difference comparison, except for Kashgar Totiv tick virus 1, there were significant abundance differences observed between the two sample groups for Bole Tick virus 1 (P= 1×10−15), Bole Tick virus 3 (P= 3.71×10−3), Taishun Tick virus 1 (P= 5.95×10−5), Bole Tick virus 4 (P= 7.45×10−4), Bole Hyalomma asiaticum virus 1 (P= 1.72×10−8), and Lonestar tick totivirus (P= 1.11×10−6). To verify the above results, 9 pairs of primers were designed based on the genomic sequences of the 7 identified viruses for PCR amplification verification. Finally, the presences of Bole Tick Virus 1, Bole Tick virus 3, Taishun Tick virus, and Bole hyalomma asiaticum virus were confirmed.
    Conclusion The tick-borne viruses carried by Hyalomma asiaticum in southeastern Junggar basin were complex, and the abundance of tick-borne viruses in Fukang and Qitai varied significantly, and the presences of four virus species were confirmed by PCR.

     

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