大鲵虹彩病毒病病原的分离、鉴定、基因组测序及大鲵群体遗传多样性分析
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摘要
大鲵(Chinese giant salamander, Andrias davidianus)是我国重要的珍稀保护动物与特种养殖对象。近年来,随着大鲵养殖规模的不断扩大、养殖集约化程度的不断提高以及苗、种交流的日益频繁,大鲵的病害问题日益突出。大鲵虹彩病毒病作为新出现的病毒性疾病,对大鲵养殖业危害十分严重,已经造成巨大的经济损失。本研究对大鲵虹彩病毒病的病原进行了分离、鉴定,分析了病毒的理化特性,建立了病毒的检测方法,并且对病毒的基因组进行了测序及分析。同时,应用微卫星标记对大鲵群体的遗传多样性进行了分析。研究的主要结果如下：
1.大鲵虹彩病毒病病原的分离、鉴定
从全身水肿,腹部有出血点的幼鲵以及全身性体表出血、吻端或四肢末端溃烂的成鲵体内成功分离到一株病毒。对感染组织进行病理切片发现,患病大鲵肾脏、肝脏、脾脏和肠道受损明显,肝组织脆性增加、表面多出血点,肾脏细胞空泡变性；脾缩小,淋巴细胞减少且坏死,并在其内发现类似病毒的包涵体。病变组织的过滤匀浆液可使鲤鱼上皮瘤细胞(EPC)产生病变；对感染的组织和出现病变的EPC细胞电镜观察结果表明,细胞的线粒体变形,嵴断裂,粗面内质网扩张,细胞核破碎,染色质浓缩,细胞质内有空泡,并且可见晶格状排列、直径约140nm具典型虹彩病毒形态学特征的病毒粒子。人工回归感染实验表明,该病毒导致健康大鲵出现与自然发病大鲵相同的症状,表明其为致病病原；克隆病毒主衣壳蛋白基因(MCP)全序列并进行序列分析,结果显示该序列与虹彩病毒科蛙病毒属病毒的相似性在98%以上。结合组织病理、细胞培养、电镜观察、感染试验以及分子生物学鉴定,证明导致大鲵患病的病原为虹彩病毒科蛙病毒属病毒,命名为大鲵虹彩病毒(Chinese giant salamander iridovirus, GSIV)。
2.大鲵虹彩病毒理化与生物学特性研究及检测方法建立
采用常规的病毒理化与生物学特性研究方法,对该病毒的培养滴度、病毒对热、酸碱度、有机溶剂、胰蛋白酶的敏感性以及病毒增殖特性与最适培养温度等进行了研究。结果表明,大鲵虹彩病毒的病毒滴度TCID50/0.1mL为109.8540.48；病毒对pH3和10的酸、碱敏感；在56℃热处理30min后病毒被灭活；胰蛋白酶处理后病毒滴度显著下降；在25℃条件下病毒在EPC细胞中增殖迅速,在感染细胞6h后病毒开始增殖,72h后进入平台期。病毒的理化性质与已知虹彩病毒理化特性相符。针对病毒MCP基因,分别建立了普通PCR、巢氏PCR、荧光定量PCR、多克隆抗体和环介导的等温扩增(LAMP)共5种检测方法,通过对5种检测方法各自反应条件的优化以及对不同来源病毒进行检测验证,结果证明这5种方法都可以有效地检测大鲵虹彩病毒,可以作为大鲵虹彩病毒病原的检测方法。其中,荧光定量PCR和LAMP法检测的灵敏度高达10-9模板DNA稀释度,可以对微量病毒进行检测。
3.大鲵虹彩病毒全基因组测序及序列分析
基于De Novo测序技术,利用Illumina公司的HiSeq基因组分析仪(Illumina HiSeq2000)对大鲵虹彩病毒进行全基因测序。测序结果显示病毒基因组全长104373bp, GC含量为55.2%,有95个开放阅读框(ORFs),编码蛋白所含的氨基酸个数从49到1294个不等,分子量大小在5.2-140.9kDa之间；在基因组40219-40314nt位置有48个CA重复序列；将大鲵虹彩病毒基因组的ORFs与其它已知虹彩病毒基因组的ORFs进行比较显示,大鲵虹彩病毒病毒基因组有41个ORFs与普通产婆蟾蛙病毒(common midwife toad ranavirus, CMTV)最为相似,而且基因组点阵分析也显示大鲵虹彩病毒基因组与CMTV病毒最为相似。将大鲵虹彩病毒的26个核心基因与15株已知虹彩病毒基因组的26个核心基因进行比较,按照大鲵虹彩病毒编码蛋白的氨基酸排列顺序进行串联,构建系统进化树,进化树结果显示虹彩病毒各属成员分别聚类,感染同类宿主的病毒成员有聚在一起的趋势;大鲵虹彩病毒与蛙病毒属成员聚在一类,且与CMTV病毒关系最近。
4.大鲵微卫星引物开发及遗传多样性分析
利用磁珠富集法开发了大鲵微卫星引物70对。利用其中10对引物对野生和人工繁殖的大鲵群体遗传多样性进行分析。结果表明其中7对引物在研究群体中有多态,观察等位基因个数在6-9个之间,多态信息含量(polymorphism information content, PIC)从0.3750-0.8751不等,大部分都属于高度多态。比较而言,野生群体等位基因数量高于人工繁殖群体,人工繁殖群体存在一定的等位基因丢失现象,并且遗传多样性水平低于野生群体。
Chinese giant salamander (Andrias davidianus) is a rare protected animal and important species in aquaculture in China. Alongside the enlargement of the cultivation scale, the incease of intensification and the frequent trading of the animal, the diseases have become a great concern in recent years. The Chinese giant salamander iridovirus disease was a new emerged viral disease in farmed Chinese giant salamanders, which has caused huge economic loss and represents a great threat the industry. In this study, an irodovirus, belonging to the genus Ranavirus, family Iridovuridae was isolated and identified from the diseased Chinese giant salamander. The physic-chemical and biological properties of the virus were investigated. Moreover, the complete genome sequence of the Chinese giant salamander iridovrisus was determined and analyzed. The results are as follows:
1. Isolation and indentification a Ranavirus pathogen in diseased Chinese giant salamander, Andrias davidianus
An iridovirus was isolated form the diseased larvae and adult Chinese giant salamanders. The typical clinical signs of diseased larvae exhibited jaw and bellies swelling, subcutaneous hemorrhage, and in the diseased adults, the symptoms of included skin hemorrhages, ulceration of the hind limbs, and multiple hemorrhagic spots in the visceral organs. Light microscopy demonstrated tissue degeneration and cytoplasmic inclusions in spleen, liver and kidney, suggestive of a viral infection. The tissue necrosis included splenocytes vacuolar degeneration, necrosis of renal hemopoietic tissue cells and liver sinusoids hypertrophied. The tissue homogenates from the diseased animals were inoculated into EPC (Epithelioma papulosum cyprinid) cells and caused typical cytopathic effect. Electron microscopy observation revealed that the virion was140～180nm in diameter both in the renal tubular epithelial cells of the kidney tissue and in the virus-infected EPC cells. The virus was determined pathogenecity to the healthy animal after experimental infection and resulted in the reproduction of clinical signs as naturnaturally ocurred. The whole major capsid protein (MCP) coding gene of the isolated virus was cloned and sequenced (GenBank:JN615141) and the result of sequence analysis shown a98%～99%sililarity with other ranavirus MCP sequences in GenBank. Combined the histopathology, electron microscopy observation, virus culture in EPC cells, experimental infection and MCP sequence analyzed, the isolated virus was determined to iridovirus belonging to the genus Ranavirus, family Iridovurus and was the pathogen of diseased Chinese giant salamander. The disease was tentatively name Chinese giant salamander iridovirus disease, meanwhile the pathogen named Chinese giant salamander iridovirus (GSIV).
2. Physico-chemical and biological properties and detection methods of the Chinese giant salamander iridovirus
The physico-chemical characteristics of GSIV were investigated. The physico-chemical and biological factors included viral titer, growth, sensitivity to temperature, to acid and base, to organic solvent and trypsin. Based on the sequence of MCP gene, five methods were established to detect GSIV. They were conventional PCR, fluorogenic quantitative PCR, polyclone antibody, nested PCR and loop-mediated isothermal amplification assay (LAMP). These methods all can detect GSIV effectively. And the detection sensitivity of the fluorogenic quantitative PCR and LAMP methods can achieve10-9DNA template dilution.
3. Sequencing and characterization of GSIV genome
The complete genome of GSIV was determined by using Illumina HiSeq2000. The length of GSIV genome was104373bp. The results of sequence analysis showed that the genome encoded95predicted open reading frames (ORFs) and GC content was55.2%. The numbers of amino acid encoded proteins were between49and129. The genome had the26core genes of Iridovirus. There were48(CA) repeats in40219～40314nt. The ORF align and dot plot analysis all showed the GSIV closed with common midwife toad ranavirus (CMTV). According the amino acid order of26core genes in GSIV genome,16iridovirus genome sequences were used to construct NJ phylogenetic tree. The results revealed that the sequences clustered into five genera of Iridoviridae, respectively. The GSIV is a member of Ranavirus. In addition, the virus infecting host had the same classification status tended to cluster. The complete genome sequence was submitted to GenBank with the accession KC243313.
4. Development of microsatellite markers and genetic diversity analysis of Chinese giant salamander
Using the AG-enriched genomic library of the Chinese giant salamander, seventy microsatellite markers were developed for the Chinese giant salamander. Ten pairs of these microsatellite markers were evaluated the genetic diversity of cultivation and wild populations. Seven markers were polymorphic in all the samples. The number of alleles ranged from6to9. The polymorphism information content (PIC) of all populations was from0.3750to0.8751, suggesting high polymorphism at these loci. Compared with the wild populations, some alleles in the cultivation population were drifted and the PIC was lower.
1.大鲵虹彩病毒病病原的分离、鉴定
从全身水肿,腹部有出血点的幼鲵以及全身性体表出血、吻端或四肢末端溃烂的成鲵体内成功分离到一株病毒。对感染组织进行病理切片发现,患病大鲵肾脏、肝脏、脾脏和肠道受损明显,肝组织脆性增加、表面多出血点,肾脏细胞空泡变性；脾缩小,淋巴细胞减少且坏死,并在其内发现类似病毒的包涵体。病变组织的过滤匀浆液可使鲤鱼上皮瘤细胞(EPC)产生病变；对感染的组织和出现病变的EPC细胞电镜观察结果表明,细胞的线粒体变形,嵴断裂,粗面内质网扩张,细胞核破碎,染色质浓缩,细胞质内有空泡,并且可见晶格状排列、直径约140nm具典型虹彩病毒形态学特征的病毒粒子。人工回归感染实验表明,该病毒导致健康大鲵出现与自然发病大鲵相同的症状,表明其为致病病原；克隆病毒主衣壳蛋白基因(MCP)全序列并进行序列分析,结果显示该序列与虹彩病毒科蛙病毒属病毒的相似性在98%以上。结合组织病理、细胞培养、电镜观察、感染试验以及分子生物学鉴定,证明导致大鲵患病的病原为虹彩病毒科蛙病毒属病毒,命名为大鲵虹彩病毒(Chinese giant salamander iridovirus, GSIV)。
2.大鲵虹彩病毒理化与生物学特性研究及检测方法建立
采用常规的病毒理化与生物学特性研究方法,对该病毒的培养滴度、病毒对热、酸碱度、有机溶剂、胰蛋白酶的敏感性以及病毒增殖特性与最适培养温度等进行了研究。结果表明,大鲵虹彩病毒的病毒滴度TCID50/0.1mL为109.8540.48；病毒对pH3和10的酸、碱敏感；在56℃热处理30min后病毒被灭活；胰蛋白酶处理后病毒滴度显著下降；在25℃条件下病毒在EPC细胞中增殖迅速,在感染细胞6h后病毒开始增殖,72h后进入平台期。病毒的理化性质与已知虹彩病毒理化特性相符。针对病毒MCP基因,分别建立了普通PCR、巢氏PCR、荧光定量PCR、多克隆抗体和环介导的等温扩增(LAMP)共5种检测方法,通过对5种检测方法各自反应条件的优化以及对不同来源病毒进行检测验证,结果证明这5种方法都可以有效地检测大鲵虹彩病毒,可以作为大鲵虹彩病毒病原的检测方法。其中,荧光定量PCR和LAMP法检测的灵敏度高达10-9模板DNA稀释度,可以对微量病毒进行检测。
3.大鲵虹彩病毒全基因组测序及序列分析
基于De Novo测序技术,利用Illumina公司的HiSeq基因组分析仪(Illumina HiSeq2000)对大鲵虹彩病毒进行全基因测序。测序结果显示病毒基因组全长104373bp, GC含量为55.2%,有95个开放阅读框(ORFs),编码蛋白所含的氨基酸个数从49到1294个不等,分子量大小在5.2-140.9kDa之间；在基因组40219-40314nt位置有48个CA重复序列；将大鲵虹彩病毒基因组的ORFs与其它已知虹彩病毒基因组的ORFs进行比较显示,大鲵虹彩病毒病毒基因组有41个ORFs与普通产婆蟾蛙病毒(common midwife toad ranavirus, CMTV)最为相似,而且基因组点阵分析也显示大鲵虹彩病毒基因组与CMTV病毒最为相似。将大鲵虹彩病毒的26个核心基因与15株已知虹彩病毒基因组的26个核心基因进行比较,按照大鲵虹彩病毒编码蛋白的氨基酸排列顺序进行串联,构建系统进化树,进化树结果显示虹彩病毒各属成员分别聚类,感染同类宿主的病毒成员有聚在一起的趋势;大鲵虹彩病毒与蛙病毒属成员聚在一类,且与CMTV病毒关系最近。
4.大鲵微卫星引物开发及遗传多样性分析
利用磁珠富集法开发了大鲵微卫星引物70对。利用其中10对引物对野生和人工繁殖的大鲵群体遗传多样性进行分析。结果表明其中7对引物在研究群体中有多态,观察等位基因个数在6-9个之间,多态信息含量(polymorphism information content, PIC)从0.3750-0.8751不等,大部分都属于高度多态。比较而言,野生群体等位基因数量高于人工繁殖群体,人工繁殖群体存在一定的等位基因丢失现象,并且遗传多样性水平低于野生群体。
Chinese giant salamander (Andrias davidianus) is a rare protected animal and important species in aquaculture in China. Alongside the enlargement of the cultivation scale, the incease of intensification and the frequent trading of the animal, the diseases have become a great concern in recent years. The Chinese giant salamander iridovirus disease was a new emerged viral disease in farmed Chinese giant salamanders, which has caused huge economic loss and represents a great threat the industry. In this study, an irodovirus, belonging to the genus Ranavirus, family Iridovuridae was isolated and identified from the diseased Chinese giant salamander. The physic-chemical and biological properties of the virus were investigated. Moreover, the complete genome sequence of the Chinese giant salamander iridovrisus was determined and analyzed. The results are as follows:
1. Isolation and indentification a Ranavirus pathogen in diseased Chinese giant salamander, Andrias davidianus
An iridovirus was isolated form the diseased larvae and adult Chinese giant salamanders. The typical clinical signs of diseased larvae exhibited jaw and bellies swelling, subcutaneous hemorrhage, and in the diseased adults, the symptoms of included skin hemorrhages, ulceration of the hind limbs, and multiple hemorrhagic spots in the visceral organs. Light microscopy demonstrated tissue degeneration and cytoplasmic inclusions in spleen, liver and kidney, suggestive of a viral infection. The tissue necrosis included splenocytes vacuolar degeneration, necrosis of renal hemopoietic tissue cells and liver sinusoids hypertrophied. The tissue homogenates from the diseased animals were inoculated into EPC (Epithelioma papulosum cyprinid) cells and caused typical cytopathic effect. Electron microscopy observation revealed that the virion was140～180nm in diameter both in the renal tubular epithelial cells of the kidney tissue and in the virus-infected EPC cells. The virus was determined pathogenecity to the healthy animal after experimental infection and resulted in the reproduction of clinical signs as naturnaturally ocurred. The whole major capsid protein (MCP) coding gene of the isolated virus was cloned and sequenced (GenBank:JN615141) and the result of sequence analysis shown a98%～99%sililarity with other ranavirus MCP sequences in GenBank. Combined the histopathology, electron microscopy observation, virus culture in EPC cells, experimental infection and MCP sequence analyzed, the isolated virus was determined to iridovirus belonging to the genus Ranavirus, family Iridovurus and was the pathogen of diseased Chinese giant salamander. The disease was tentatively name Chinese giant salamander iridovirus disease, meanwhile the pathogen named Chinese giant salamander iridovirus (GSIV).
2. Physico-chemical and biological properties and detection methods of the Chinese giant salamander iridovirus
The physico-chemical characteristics of GSIV were investigated. The physico-chemical and biological factors included viral titer, growth, sensitivity to temperature, to acid and base, to organic solvent and trypsin. Based on the sequence of MCP gene, five methods were established to detect GSIV. They were conventional PCR, fluorogenic quantitative PCR, polyclone antibody, nested PCR and loop-mediated isothermal amplification assay (LAMP). These methods all can detect GSIV effectively. And the detection sensitivity of the fluorogenic quantitative PCR and LAMP methods can achieve10-9DNA template dilution.
3. Sequencing and characterization of GSIV genome
The complete genome of GSIV was determined by using Illumina HiSeq2000. The length of GSIV genome was104373bp. The results of sequence analysis showed that the genome encoded95predicted open reading frames (ORFs) and GC content was55.2%. The numbers of amino acid encoded proteins were between49and129. The genome had the26core genes of Iridovirus. There were48(CA) repeats in40219～40314nt. The ORF align and dot plot analysis all showed the GSIV closed with common midwife toad ranavirus (CMTV). According the amino acid order of26core genes in GSIV genome,16iridovirus genome sequences were used to construct NJ phylogenetic tree. The results revealed that the sequences clustered into five genera of Iridoviridae, respectively. The GSIV is a member of Ranavirus. In addition, the virus infecting host had the same classification status tended to cluster. The complete genome sequence was submitted to GenBank with the accession KC243313.
4. Development of microsatellite markers and genetic diversity analysis of Chinese giant salamander
Using the AG-enriched genomic library of the Chinese giant salamander, seventy microsatellite markers were developed for the Chinese giant salamander. Ten pairs of these microsatellite markers were evaluated the genetic diversity of cultivation and wild populations. Seven markers were polymorphic in all the samples. The number of alleles ranged from6to9. The polymorphism information content (PIC) of all populations was from0.3750to0.8751, suggesting high polymorphism at these loci. Compared with the wild populations, some alleles in the cultivation population were drifted and the PIC was lower.
引文
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