鸭坦布苏病毒分离鉴定及其生物学特性的研究
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摘要
2010年以来，中国大陆爆发了一种由未知病原引起的以雏鸭生长迟缓和死亡、蛋鸭产蛋下降为特征的传染性疫病。该病首先发现于江浙沪地区，之后迅速蔓延至中国东南部各省市，其中包括山东、北京、河北、福建、安徽、广东和广西等。即便在冬季，该疫病的传播依然相当的迅速，这给全国的养鸭业带来了巨大的威胁。流行病学调查显示对此疫病最易感的为麻鸭，感染率为100%，致死率大约为5%到30%。
为了明确该病的病原，我们从奉贤发病鸭场采集发病鸭实质脏器，经0.22um滤器过滤除菌后，经尿囊腔接种9日龄SPF鸡胚，进行病毒分离。结果鸡胚在接种后2-6天内死亡，死亡鸡胚可见尿囊膜增厚，胚体出血、水肿，经测定该死亡鸡胚尿囊液的毒价达到10~（5.5）ELD50/mL。
为了确定该病毒的分类学地位，病毒经蔗糖密度梯度离心纯化后在电镜下观察，发现该病毒粒子具有囊膜，直径约为45-50nm。提取该病毒核酸，用不同的核酸酶消化后，发现病毒核酸可被RNase消化而不被DNase降解，表明该病毒为RNA病毒。在此基础上我们构建了cDNA文库，随机挑选阳性克隆、测序，结果获得该病毒的两个基因的序列，序列分析表明该基因序列与坦布苏病毒Mm1775株基因的同源性分别为88.3%和88.0%。病毒交叉中和实验表明该病毒与乙型脑炎病毒无交叉中和活性，但该病毒可被抗坦布苏病毒Mm1775株E蛋白的血清中和。根据国际命名规则，将该病毒命名为鸭坦布苏病毒奉贤2010株（FX2010）。
研究表明，FX2010对乙醚和氯仿敏感，不具有血凝活性。FX2010可在DEF细胞上增殖，随着病毒传代增加CPE更加的明显，同时也可在Vero细胞上增殖，但不产生CPE。
FX2010经鼻腔感染和肌肉感染九周龄麻鸭后，可从感染鸭的肾脏、脑、脾脏、肺脏、胰脏、气管、血液等组织脏器中分离到较高滴度的病毒，而肝脏中的病毒含量较低；对病理学观察发现感染鸭出现中度脑炎、严重肾炎、脾脏组织细胞坏死，免疫组织化学实验显示在多个组织内皆有病毒抗原存在。
为了了解该病毒分子遗传信息，我们通过引物步移法获得覆盖FX2010病毒株cDNA全长的若干克隆，然后经3’和5’RACE获得该病毒3’和5’末端序列，序列拼接后显示该病毒全长含有10991个核苷酸，编码十个蛋白。利用生物信息学手段对FX2010株进行全基因组序列的分析。系统进化分析显示，该毒株与BYDV处于同一分支上，同属于坦布苏病毒。进一步预测该病毒的不同蛋白之间的剪切位点和N连接糖基化位点，结果可见该病毒不同蛋白之间剪切位点与同种属的其他病毒具有一定的保守性。
In2010, an outbreak of shelduck infectious disease, caused by unknown pathogen, resulted inretarded growth, egg production decline and death. In the begaining, the outbreaks happened in Jiangsu,Zhejiang, and Shanghai. Then, spread rapidly to all of the southeast provinces of China, includingShandong, Beijing, Hebei, Fujian, Anhui, Guangdong, Guangxi and so on. The disease continued totransmit until winter, all the ducks raised in China were under a huge threat by this unknown pathogen.Epizootic investigation suggested that most susceptible animals to this pathogen were shelducks, theinfection rate and morbidity of shelducks was as high as100%and mortality varied from5%to30%.
To identify this new virus, a homogenate of the spleens of sick shelducks raised in Fengxiandistrict, Shanghai, was filtered through0.22um filters, and inoculated into the allantoic sacs of9-day-old SPF embryonated chicken eggs. All of the embryos of the inoculated eggs died2–6dayspost-inoculation and were severely swollen with edema. The supernatant of the homogenate of theembryos had an infectivity titer of10~（5.5）ELD50/mL.
To identify the taxonomic placement of the virus, the purified causative agent which were treatwith sucrose density gradient centrifugation was examined by electron microscopy. Spherical andenveloped particles with a diameter of45-50nm. To define the nucleic acid type of FX2010, RNA andDNA were extracted from purified FX2010Upon treatment of the nucleic acid extracted from FX2010with RNase but not DNase, the RNA band was no longer detected, the result show that this virus wasRNA virus. At the same time, an FX2010cDNA library was made and plasmids extracted from thebacterial clones picked randomly from the cDNA library were directly sequenced. Two gene sequenceswere obtained, Sequence analysis showed that these genes of FX2010shared88.3%and88.0%nucleotide homology with those of the Tembusu virus Mm1775strain, respectively. In cross serumneutralization tests between FX2010and JEV, we found no cross-reactivity with heterologousantibodies; In cross serum neutralizing tests between FX2010and Tembusu virus, an antiserum againstthe partial E protein of Tembusu virus Mm1775strain neutralized FX2010at a serum dilution of1:90.We designated this virus Tembusu virus Fengxian2010(FX2010).
Studies have shown that the isolate were sensitive to ether, Chloroform, but it has nohaemagglutination activity. The virus could grow in duck embryo fibroblasts and caused CPE, as thepassage times increase, CPE became more regular. Aslo it can grew in Vero cells without any CPEs.
After infected six shelducks with FX2010by inoculating them i.n. or i.m., we can isolate high titerof virus from kidney, brain, spleen, lung, pancreas, trachea, blood of the infected ducks, but low titer ofvirus from liver. After observed the tissue and organs of the infected ducks, we found the infected duckswere moderate necrosis of brain tissue, severe nephrosis, pneumorrhagia, severe necrosis of the spleen.Immunohistochemistry show that several tissues had virus.
In order to understand the molecular genetics information of FX2010, several overlapping cDNAclones, covering the entire genome of FX2010strain, were obtained by primer walking RT-PCR. Thesequences of3’ and5’ termini of the viral genome were amplified by3’ and5’ RACE. Sequences complied from these clones show that FX2010strain is10991nt long, and encode ten proteins, Weanalyzed the complete genome sequence of FX2010strain with bioinformatics methods. Phylogeneticanalysis of genome sequences among FX2010strain, and other members of family flaviviridaedemonstrate that FX2010strain, BYDV are in one lineage, and both were belong Tembusu virus. Wealso predicted cleavage sites and N-linked potential glycosylation sites for the polyproteins of FX2010strain, the result show that the cleavage sites had certain conservation.
为了明确该病的病原，我们从奉贤发病鸭场采集发病鸭实质脏器，经0.22um滤器过滤除菌后，经尿囊腔接种9日龄SPF鸡胚，进行病毒分离。结果鸡胚在接种后2-6天内死亡，死亡鸡胚可见尿囊膜增厚，胚体出血、水肿，经测定该死亡鸡胚尿囊液的毒价达到10~（5.5）ELD50/mL。
为了确定该病毒的分类学地位，病毒经蔗糖密度梯度离心纯化后在电镜下观察，发现该病毒粒子具有囊膜，直径约为45-50nm。提取该病毒核酸，用不同的核酸酶消化后，发现病毒核酸可被RNase消化而不被DNase降解，表明该病毒为RNA病毒。在此基础上我们构建了cDNA文库，随机挑选阳性克隆、测序，结果获得该病毒的两个基因的序列，序列分析表明该基因序列与坦布苏病毒Mm1775株基因的同源性分别为88.3%和88.0%。病毒交叉中和实验表明该病毒与乙型脑炎病毒无交叉中和活性，但该病毒可被抗坦布苏病毒Mm1775株E蛋白的血清中和。根据国际命名规则，将该病毒命名为鸭坦布苏病毒奉贤2010株（FX2010）。
研究表明，FX2010对乙醚和氯仿敏感，不具有血凝活性。FX2010可在DEF细胞上增殖，随着病毒传代增加CPE更加的明显，同时也可在Vero细胞上增殖，但不产生CPE。
FX2010经鼻腔感染和肌肉感染九周龄麻鸭后，可从感染鸭的肾脏、脑、脾脏、肺脏、胰脏、气管、血液等组织脏器中分离到较高滴度的病毒，而肝脏中的病毒含量较低；对病理学观察发现感染鸭出现中度脑炎、严重肾炎、脾脏组织细胞坏死，免疫组织化学实验显示在多个组织内皆有病毒抗原存在。
为了了解该病毒分子遗传信息，我们通过引物步移法获得覆盖FX2010病毒株cDNA全长的若干克隆，然后经3’和5’RACE获得该病毒3’和5’末端序列，序列拼接后显示该病毒全长含有10991个核苷酸，编码十个蛋白。利用生物信息学手段对FX2010株进行全基因组序列的分析。系统进化分析显示，该毒株与BYDV处于同一分支上，同属于坦布苏病毒。进一步预测该病毒的不同蛋白之间的剪切位点和N连接糖基化位点，结果可见该病毒不同蛋白之间剪切位点与同种属的其他病毒具有一定的保守性。
In2010, an outbreak of shelduck infectious disease, caused by unknown pathogen, resulted inretarded growth, egg production decline and death. In the begaining, the outbreaks happened in Jiangsu,Zhejiang, and Shanghai. Then, spread rapidly to all of the southeast provinces of China, includingShandong, Beijing, Hebei, Fujian, Anhui, Guangdong, Guangxi and so on. The disease continued totransmit until winter, all the ducks raised in China were under a huge threat by this unknown pathogen.Epizootic investigation suggested that most susceptible animals to this pathogen were shelducks, theinfection rate and morbidity of shelducks was as high as100%and mortality varied from5%to30%.
To identify this new virus, a homogenate of the spleens of sick shelducks raised in Fengxiandistrict, Shanghai, was filtered through0.22um filters, and inoculated into the allantoic sacs of9-day-old SPF embryonated chicken eggs. All of the embryos of the inoculated eggs died2–6dayspost-inoculation and were severely swollen with edema. The supernatant of the homogenate of theembryos had an infectivity titer of10~（5.5）ELD50/mL.
To identify the taxonomic placement of the virus, the purified causative agent which were treatwith sucrose density gradient centrifugation was examined by electron microscopy. Spherical andenveloped particles with a diameter of45-50nm. To define the nucleic acid type of FX2010, RNA andDNA were extracted from purified FX2010Upon treatment of the nucleic acid extracted from FX2010with RNase but not DNase, the RNA band was no longer detected, the result show that this virus wasRNA virus. At the same time, an FX2010cDNA library was made and plasmids extracted from thebacterial clones picked randomly from the cDNA library were directly sequenced. Two gene sequenceswere obtained, Sequence analysis showed that these genes of FX2010shared88.3%and88.0%nucleotide homology with those of the Tembusu virus Mm1775strain, respectively. In cross serumneutralization tests between FX2010and JEV, we found no cross-reactivity with heterologousantibodies; In cross serum neutralizing tests between FX2010and Tembusu virus, an antiserum againstthe partial E protein of Tembusu virus Mm1775strain neutralized FX2010at a serum dilution of1:90.We designated this virus Tembusu virus Fengxian2010(FX2010).
Studies have shown that the isolate were sensitive to ether, Chloroform, but it has nohaemagglutination activity. The virus could grow in duck embryo fibroblasts and caused CPE, as thepassage times increase, CPE became more regular. Aslo it can grew in Vero cells without any CPEs.
After infected six shelducks with FX2010by inoculating them i.n. or i.m., we can isolate high titerof virus from kidney, brain, spleen, lung, pancreas, trachea, blood of the infected ducks, but low titer ofvirus from liver. After observed the tissue and organs of the infected ducks, we found the infected duckswere moderate necrosis of brain tissue, severe nephrosis, pneumorrhagia, severe necrosis of the spleen.Immunohistochemistry show that several tissues had virus.
In order to understand the molecular genetics information of FX2010, several overlapping cDNAclones, covering the entire genome of FX2010strain, were obtained by primer walking RT-PCR. Thesequences of3’ and5’ termini of the viral genome were amplified by3’ and5’ RACE. Sequences complied from these clones show that FX2010strain is10991nt long, and encode ten proteins, Weanalyzed the complete genome sequence of FX2010strain with bioinformatics methods. Phylogeneticanalysis of genome sequences among FX2010strain, and other members of family flaviviridaedemonstrate that FX2010strain, BYDV are in one lineage, and both were belong Tembusu virus. Wealso predicted cleavage sites and N-linked potential glycosylation sites for the polyproteins of FX2010strain, the result show that the cleavage sites had certain conservation.
引文
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2.兰水云. RNA病毒感染性转录体的制备国外医学(微生物学分册).1998（02）:35-40.
3.姬希文,李雪松,李国新,肖亚莉,颜丕熙,徐大伟,范钊,张七斤,李泽君.鸭坦布苏病毒E蛋白中和性单克隆抗体的制备[J].中国家禽,2012,34（4）:25-32.
4.唐维国.一例野鸭疑似鸭坦布苏病毒病的诊治[J].江西畜牧兽医杂志,2011,4:34.
5.滕巧泱,颜丕熙,张旭等.一种新的黄病毒导致蛋鸭产蛋下降及死亡[J].中国动物传染病学报,2010,18(6):1-4.
6.万春和，施少华，程龙飞，等．鸭出血性卵巢炎病毒RT-PCR检测方法的建立[J].福建农业学报,2010.
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