东北地区蜱传内罗毕羊病毒鉴定及全基因组克隆与序列分析

发布时间：2018-04-18 05:37

本文选题：内罗毕羊病毒 + 蜱组织　；参考：《吉林农业大学》2015年硕士论文

【摘要】：众所周知,蜱是一种寄生在动物体表的吸血寄生虫,是多种人畜共患传染病的重要传播媒介。蜱虫分类上属于动物界节肢动物门、蛛形纲、螨亚纲、寄螨目、蜱总科。全世界已经发现18个属897种,而中国已发现10属,119种。蜱可引起多种重要的自然疫源性疾病和人兽共患病,如Q热、发热伴血小板减少综合征、森林脑炎、出血热等。内罗毕羊病毒(Nairobi sheep disease virus)在分类上属于布尼亚病毒科。其主要传播媒介是蜱虫,对公众危害较为广泛,能够引发急性出血性胃肠炎,绵羊和山羊的死亡率高达90%。该病于1910年在肯尼亚的内罗毕附近首次发现,并在1917年确定其病原为内罗毕羊病毒。1994年首次报道发现人感染内罗毕羊病毒发病。内罗毕羊病主要发生在非洲东部的肯尼亚、乌干达、坦桑尼亚、埃塞尔比亚和索马里。目前,内罗毕羊病尚未在中国乃至东亚报道过,因此,对中国东北部内罗毕羊病毒(NSDV)进行鉴定及全基因组序列分析具有重要意义。基于目前东北地区蜱类分布不清、病毒本底尚不明确等因素,本研究在吉林省的集安、敬信、春化、南山、松林,黑龙江省的哈尔滨、呼玛和辽宁省的丹东等10余个县市进行了蜱类的采集。通过形态学鉴定,主要发现四个蜱虫物种,丰富的是长角血蜱属longicornis(84.8%),其次是矩头蝉属silvarum(7.2%),矩头蝉属nuttalli(5.5%)和Ixodes persulcatus(2.5%)。通过病毒宏基因组学分析,共获得26411条reads,进一步拼接得到平均长度为108碱基的18997条重叠序列。其中,动物性病毒科数量最多,占病毒序列总数的65%(7505/11547),包括:细小病毒科(Parvoviridae),黄病毒科(Flaviviridae),布尼亚病毒科(Bunyaviridae)等;植物性病毒占19%(2189/11547);噬菌体序列占16%(1853/11547)。本研究进一步对蜱组织进行裂解,采用RT-PCR手段再次对内罗毕羊病毒(NSDV)进行验证,结果得出吉林集安、敬信及辽宁丹东地区蜱虫均携带内罗毕羊病毒。通过以病毒宏基因组学分析数据及国内外NSDVs病毒序列为参考,本研究成功克隆了内罗毕羊病毒(NSDV)的全基因组。该序列与国外NCBI已收录的全基因组序列进行核苷酸同源性比较,基因片段序列比较显示:在核苷酸水平上相比于其他NSDVs同源性为75.1~89.6%之间,推导出氨基酸同源性在81.3~96.7%之间。相比于其他物种的成员NSDVs(Dugbe,Kupe,Hazara and Crimean Congo hemorrhagic fever viruses),同源性在37.5-68.6%之间。本研究在东亚地区首次证实了蜱传内罗毕羊病毒的存在。另外,基于对以上阳性地区蜱虫形态学鉴定,首次发现了长角血蜱可携带内罗毕羊病毒,同时为开展东北地区内罗毕羊病毒(NSDV)致病性及疫苗的研究奠定了良好的基础。
[Abstract]:It is well known that tick is a blood-sucking parasite parasitic on animal surface and an important transmission medium of zoonotic infectious diseases.Ticks are classified as Arthropoda, Arachne, Acari, Acaridae, Acaridae.There are 897 species of 18 genera in the world, while 119 species of 10 genera have been found in China.Ticks can cause many important natural diseases and zoonosis, such as Q fever, fever with thrombocytopenia syndrome, forest encephalitis, hemorrhagic fever and so on.Nairobi sheep disease virus belongs to the Bunia virus family.Its main transmission vector is ticks, which are widely harmful to the public and can cause acute hemorrhagic gastroenteritis. The mortality rate of sheep and goats is as high as 90%.The disease was first found near Nairobi, Kenya, in 1910, and was identified as Nairobi sheep virus in 1917. It was first reported in 1994 that people were infected with Nairobi sheep virus.Nairobi sheep disease mainly occurs in eastern Africa Kenya, Uganda, Tanzania, Ethiopia and Somalia.At present, Nairobi sheep disease has not been reported in China or even in East Asia, so it is of great significance to identify and analyze the whole genome sequence of Nairobi sheep virus (NSDV) in northeastern China.Due to the unclear distribution of ticks and the unclear background of the virus in Northeast China, this study was conducted in Ji'an, Jingxin, Chunhua, Nanshan, Pine Forest and Harbin, Heilongjiang Province.More than 10 counties and cities, such as Huma and Dandong of Liaoning Province, collected ticks.By morphological identification, four species of ticks were found, which were abundant in the genus Haemaphysalis longicornis84.8, followed by Silvarum 7.2dicum, nuttallius 5.5) and Ixodes persulcatus 2.5.A total of 26411 readswere obtained by virus macrogenomics analysis, and 18997 overlapping sequences with an average length of 108bp were obtained by further splicing.Among them, the animal viridae has the largest number, accounting for 65% of the total number of virus sequences, including Parvoviridaeae, Flaviviridaeae, Bunyaviridaeae and so on; plant viruses account for 191899 / 11547; bacteriophage sequences account for 161853 / 11547; phage sequences account for 161853 / 11547.In this study, the tissues of ticks were further cracked, and the Nairobi sheep virus (NSDV) was verified again by RT-PCR. The results showed that Jilin Ji'an, Jingxin and Dandong of Liaoning all carried Nairobi sheep virus.The whole genome of Nairobi Sheep virus (NSDV) was successfully cloned by reference to the data of viral macrogenomics analysis and the sequences of NSDVs virus at home and abroad.The nucleotide homology of this sequence was compared with that of the whole genome sequence of foreign NCBI. Compared with other NSDVs, the nucleotide homology of this sequence was 75.1% and the deduced amino acid homology was 81.33% and 96.7% respectively.The homology of Hazara and Crimean Congo hemorrhagic fever virusesus was 37.5-68.6% compared with other species.In this study, the existence of tick-borne Nairobi sheep virus was first confirmed in East Asia.In addition, based on the morphological identification of the above positive ticks, Haemaphysalis longicornis was found to carry the Nairobi sheep virus for the first time, which laid a good foundation for the study of the pathogenicity and vaccine of NSDV in Northeast China.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.654

【参考文献】