随着基因测序技术的创新和应用,新的高通量测序技术不断涌现,以Pacific Biosciences(PacBio)公司的单分子实时测序(single molecule real time sequencing)为代表的第三代测序(third generation sequencing,TGS)技术开始逐渐应用于基因...随着基因测序技术的创新和应用,新的高通量测序技术不断涌现,以Pacific Biosciences(PacBio)公司的单分子实时测序(single molecule real time sequencing)为代表的第三代测序(third generation sequencing,TGS)技术开始逐渐应用于基因组研究,包括大型基因组拼装、基因结构变异和表观遗传研究等方面。本文主要对TGS技术的原理、特点和应用,特别是在病毒研究中的应用进行介绍,并与第二代测序(next generation sequencing,NGS)技术进行比较,为基因组测序技术的选择及其临床应用提供一定参考。展开更多
从广东省河源市连平县采集3份疑似双生病毒侵染引起的叶片表现黄花叶症状构树样品,提取总DNA,利用Bego-movirus通用引物AV494/CoPR进行PCR检测表明,疑似病样中均检测到菜豆金色黄花叶病毒属病毒。进一步选取PCR检测为阳性的样品进行RCA...从广东省河源市连平县采集3份疑似双生病毒侵染引起的叶片表现黄花叶症状构树样品,提取总DNA,利用Bego-movirus通用引物AV494/CoPR进行PCR检测表明,疑似病样中均检测到菜豆金色黄花叶病毒属病毒。进一步选取PCR检测为阳性的样品进行RCA扩增、酶切、克隆及测序,获得侵染广东构树的病毒分离物基因组全长序列。广东构树分离物(GS-2021)为一个双组分病毒,包含DNA-A和DNA-B两组分。DNA-A组分(GS-2021-A)全长为2777 nt,编码7个ORFs;DNA-B组分(GS-2021-B)为2742 nt,编码2个ORFs。GS-2021与已报道的中国大青金色花叶病毒(clerodendrum golden mosaic China virus,ClGMCNV)各分离物的DNA-A、DNA-B均有较高的一致性,全长序列的一致性分别为93.0%~93.9%和86.3%~89.6%,其中与福建Fz7分离物DNA-A(GenBank登录号:FJ011668)和DNA-B(GenBank登录号:FJ011669)的相一致性最高,为93.9%和89.6%。GS-2021与ClGMCNV福建、浙江、江苏和美国的5个分离物亲缘关系近,同属一个分支,其中与福建Fz7分离物聚集在一个小分支,亲缘关系最近。基因重组分析显示,GS-2021无明显的基因重组事件存在。根据ICTV对菜豆金色黄花叶病毒属病毒最新分类标准,GS-2021属ClGMCNV的一个新株系。本研究首次在构树上检测到菜豆金色花叶病毒属病毒,获得其病毒基因组全序列,明确其为ClGMCNV的新株系。因此,构树是菜豆金色黄花叶病毒属病毒的新自然寄主。展开更多
[目的]2016年以来,新疆阿克苏等地区出现了一种新的枣树病害,严重威胁当地及周边红枣产业。本研究旨在鉴定引起此次病害的病原,探究病原体的传播方式,为生物防治策略的开发提供研究基础。[方法]对发病植株进行小RNA测序以鉴定病原体;对...[目的]2016年以来,新疆阿克苏等地区出现了一种新的枣树病害,严重威胁当地及周边红枣产业。本研究旨在鉴定引起此次病害的病原,探究病原体的传播方式,为生物防治策略的开发提供研究基础。[方法]对发病植株进行小RNA测序以鉴定病原体;对新鉴定的病毒,通过RNAseq和反转录PCR获取病毒全序列;体外表达重组的病毒结构蛋白并制备特异性抗体,通过Western斑点杂交法在发病植株中确证病毒蛋白;收集发病区域的媒介昆虫,通过反转录PCR在昆虫体内检测病毒的基因组,鉴定可能的传毒介体。[结果]本研究鉴定一种新的欧洲山梣环斑病毒属病毒为新疆新发枣树病害可能的病原体,命名为中国枣树花叶伴随病毒(Chinese date mosaic-associated virus,CDMaV)。CDMaV是一种多分段单链RNA病毒,基因组由5条负义RNA组成;RNA1-RNA5大小分别为7160、2224、1230、1493、971 nt,每条基因组RNA的互补链包含一个开放阅读框,共编码5个蛋白,依次为依赖RNA的RNA聚合酶、包膜糖蛋白、核衣壳蛋白和两个未知功能蛋白。在枣树寄生虫枣瘿螨体内扩增到病毒序列,表明该病毒可能以枣瘿螨为介体在枣树间进行传播。[结论]本研究为新疆新发枣树病害鉴定了相关病原体CDMaV,完成CDMaV全基因组测序,并鉴定枣瘿螨为可能的传毒介体。鉴定病原体和传播介体是建立病害防治方法的必要基础。展开更多
Porcine reproductive and respiratory syndrome( PRRS) is one of viral diseases with severe reproductive obstacle of pregnant sows and respiratory tract symptoms and higher mortality of piglets as characteristics,which ...Porcine reproductive and respiratory syndrome( PRRS) is one of viral diseases with severe reproductive obstacle of pregnant sows and respiratory tract symptoms and higher mortality of piglets as characteristics,which is caused by porcine reproductive and respiratory syndrome virus( PRRSV). PRRS has brought great threats to swine industry in the world. The advances of studies on the viral proteins of PRRSV were reviewed from the genome,non-structural proteins and structural proteins of PRRSV.展开更多
Objective Knowledge of an enterovirus genome sequence is very important in epidemiological investigation to identify transmission patterns and ascertain the extent of an outbreak. The MinION sequencer is increasingly ...Objective Knowledge of an enterovirus genome sequence is very important in epidemiological investigation to identify transmission patterns and ascertain the extent of an outbreak. The MinION sequencer is increasingly used to sequence various viral pathogens in many clinical situations because of its long reads, portability, real-time accessibility of sequenced data, and very low initial costs. However, information is lacking on MinION sequencing of enterovirus genomes. Methods In this proof-of-concept study using Enterovirus 71 （EV71） and Coxsackievirus A16 （CA16） strains as examples, we established an amplicon-based whole genome sequencing method using MinION. We explored the accuracy, minimum sequencing time, discrimination and high-throughput sequencing ability of MinION, and compared its performance with Sanger sequencing. Results Within the first minute （min） of sequencing, the accuracy of MinION was 98.5% for the single EV71 strain and 94.12%-97.33% for 10 genetically-related CA16 strains. In as little as 14 min, 99% identity was reached for the single EV71 strain, and in 17 min （on average）, 99% identity was achieved for 10 CA16 strains in a single run. Conclusion MinION is suitable for whole genome sequencing of enteroviruses with sufficient accuracy and fine discrimination and has the potential as a fast, reliable and convenient method for routine use.展开更多
文摘随着基因测序技术的创新和应用,新的高通量测序技术不断涌现,以Pacific Biosciences(PacBio)公司的单分子实时测序(single molecule real time sequencing)为代表的第三代测序(third generation sequencing,TGS)技术开始逐渐应用于基因组研究,包括大型基因组拼装、基因结构变异和表观遗传研究等方面。本文主要对TGS技术的原理、特点和应用,特别是在病毒研究中的应用进行介绍,并与第二代测序(next generation sequencing,NGS)技术进行比较,为基因组测序技术的选择及其临床应用提供一定参考。
文摘从广东省河源市连平县采集3份疑似双生病毒侵染引起的叶片表现黄花叶症状构树样品,提取总DNA,利用Bego-movirus通用引物AV494/CoPR进行PCR检测表明,疑似病样中均检测到菜豆金色黄花叶病毒属病毒。进一步选取PCR检测为阳性的样品进行RCA扩增、酶切、克隆及测序,获得侵染广东构树的病毒分离物基因组全长序列。广东构树分离物(GS-2021)为一个双组分病毒,包含DNA-A和DNA-B两组分。DNA-A组分(GS-2021-A)全长为2777 nt,编码7个ORFs;DNA-B组分(GS-2021-B)为2742 nt,编码2个ORFs。GS-2021与已报道的中国大青金色花叶病毒(clerodendrum golden mosaic China virus,ClGMCNV)各分离物的DNA-A、DNA-B均有较高的一致性,全长序列的一致性分别为93.0%~93.9%和86.3%~89.6%,其中与福建Fz7分离物DNA-A(GenBank登录号:FJ011668)和DNA-B(GenBank登录号:FJ011669)的相一致性最高,为93.9%和89.6%。GS-2021与ClGMCNV福建、浙江、江苏和美国的5个分离物亲缘关系近,同属一个分支,其中与福建Fz7分离物聚集在一个小分支,亲缘关系最近。基因重组分析显示,GS-2021无明显的基因重组事件存在。根据ICTV对菜豆金色黄花叶病毒属病毒最新分类标准,GS-2021属ClGMCNV的一个新株系。本研究首次在构树上检测到菜豆金色花叶病毒属病毒,获得其病毒基因组全序列,明确其为ClGMCNV的新株系。因此,构树是菜豆金色黄花叶病毒属病毒的新自然寄主。
文摘[目的]2016年以来,新疆阿克苏等地区出现了一种新的枣树病害,严重威胁当地及周边红枣产业。本研究旨在鉴定引起此次病害的病原,探究病原体的传播方式,为生物防治策略的开发提供研究基础。[方法]对发病植株进行小RNA测序以鉴定病原体;对新鉴定的病毒,通过RNAseq和反转录PCR获取病毒全序列;体外表达重组的病毒结构蛋白并制备特异性抗体,通过Western斑点杂交法在发病植株中确证病毒蛋白;收集发病区域的媒介昆虫,通过反转录PCR在昆虫体内检测病毒的基因组,鉴定可能的传毒介体。[结果]本研究鉴定一种新的欧洲山梣环斑病毒属病毒为新疆新发枣树病害可能的病原体,命名为中国枣树花叶伴随病毒(Chinese date mosaic-associated virus,CDMaV)。CDMaV是一种多分段单链RNA病毒,基因组由5条负义RNA组成;RNA1-RNA5大小分别为7160、2224、1230、1493、971 nt,每条基因组RNA的互补链包含一个开放阅读框,共编码5个蛋白,依次为依赖RNA的RNA聚合酶、包膜糖蛋白、核衣壳蛋白和两个未知功能蛋白。在枣树寄生虫枣瘿螨体内扩增到病毒序列,表明该病毒可能以枣瘿螨为介体在枣树间进行传播。[结论]本研究为新疆新发枣树病害鉴定了相关病原体CDMaV,完成CDMaV全基因组测序,并鉴定枣瘿螨为可能的传毒介体。鉴定病原体和传播介体是建立病害防治方法的必要基础。
基金Supported by Program of National Natural Science Foundation of China(No.31272564)The Joint Fund of NSFC-Guangdong(U0931003)+1 种基金Special Program of Modern Agricultural Industry Technology System(CARS-36)Hainan Program of Scientific Operating Expenses(Qiong Cai Yu[2013]131)
文摘Porcine reproductive and respiratory syndrome( PRRS) is one of viral diseases with severe reproductive obstacle of pregnant sows and respiratory tract symptoms and higher mortality of piglets as characteristics,which is caused by porcine reproductive and respiratory syndrome virus( PRRSV). PRRS has brought great threats to swine industry in the world. The advances of studies on the viral proteins of PRRSV were reviewed from the genome,non-structural proteins and structural proteins of PRRSV.
基金supported by the National key research and development plan(2016TFC1202700,2016YFC1200900)Beijing Municipal Science&Technology Commission project(grant numbers D151100002115003)Guangzhou Municipal Science&Technology Commission project(grant numbers 2015B2150820)
文摘Objective Knowledge of an enterovirus genome sequence is very important in epidemiological investigation to identify transmission patterns and ascertain the extent of an outbreak. The MinION sequencer is increasingly used to sequence various viral pathogens in many clinical situations because of its long reads, portability, real-time accessibility of sequenced data, and very low initial costs. However, information is lacking on MinION sequencing of enterovirus genomes. Methods In this proof-of-concept study using Enterovirus 71 （EV71） and Coxsackievirus A16 （CA16） strains as examples, we established an amplicon-based whole genome sequencing method using MinION. We explored the accuracy, minimum sequencing time, discrimination and high-throughput sequencing ability of MinION, and compared its performance with Sanger sequencing. Results Within the first minute （min） of sequencing, the accuracy of MinION was 98.5% for the single EV71 strain and 94.12%-97.33% for 10 genetically-related CA16 strains. In as little as 14 min, 99% identity was reached for the single EV71 strain, and in 17 min （on average）, 99% identity was achieved for 10 CA16 strains in a single run. Conclusion MinION is suitable for whole genome sequencing of enteroviruses with sufficient accuracy and fine discrimination and has the potential as a fast, reliable and convenient method for routine use.
