蓝舌病病毒反向遗传操作系统的建立
|
摘要
蓝舌病(Bluetongue)是由蓝舌病病毒(Bluetongue virus, BTV)引起的经媒介昆虫传播的一种反刍类动物急热性疾病。绵羊为本病的主要易感动物,牛、山羊和许多野生反刍动物也感染此病,但通常表现为亚临床症状或无临床症状。该病被世界动物卫生组织(OIE)列为法定通报性疾病。该病是阻碍反刍动物国际贸易和生产的重大疫病,平均每年在全世界造成超过30亿美元的经济损失。
BTV为呼肠孤病毒科(Reoviridae)环状病毒属(Orbivirus)成员。BTV粒子呈二十面体对称,无囊膜,直径约为70～80nm。BTV的核酸由10个双股RNA(double-stranded RNA, dsRNA)节段组成,按长度大小依次命名为segmentl-segment10(Seg-l-Seg-10), BTV的10个双股RNA节段总长度在19.2Kb左右,分别编码7种结构蛋白(VP1-VP7)和5种非结构蛋白(NS1、NS2、NS3、NS3a、NS4)。病毒的衣壳可以分为3层,其外层由两种结构蛋白组成,分别是VP2和VP5,其中VP2蛋白是型特异性蛋白,决定BTV的血清型；中间层由VP7蛋白构成；最内层的核心衣壳由VP3蛋白构成。病毒基因组和VP1、VP4、VP6三种酶活性结构蛋白被包裹在核心衣壳内部,另外五种非结构蛋白NS1、NS2、NS3、 NS3a、NS4在被病毒感染的细胞中合成。
目前已从非洲、欧洲、亚洲、北美、南美和大洋洲的多个热带、亚热带和温带地区国家分离到BTV,并且其分布范围不断扩大,危害也日趋严重。BTV血清型众多,到目前为止,全世界共分离到26个血清型的BTV。我国自1979年在云南首次分离到BTV以来,到现在国内共分离到BTV-1、2、3、4、12、15、16共七个血清型的BTV。
近年来,蓝舌病在欧洲许多国家相继暴发,且分布范围不断扩大,特别是2006年以来BTV-8和BTV-6在欧洲多个国家流行,对欧洲的牛羊养殖业造成了严重危害,也引起了高度重视。预防和控制蓝舌病已成为当前国际上重大动物传染病研究中的一个热点。虽然OIE将蓝舌病同禽流感等一些重要动物疫病共同列入法定通报性疾病,在国外也受到高度重视,但在国内受重视的程度还不够,投入的研究也较少。在我国虽然没有像欧洲地区那样,出现BTV强毒株的暴发,但是随着进出口贸易的日益频繁,完全存在国外的强毒株传播到国内的风险。同时,由于BTV为基因分节段的病毒,不同毒株间可以在自然界发生基因重组,所以也不能排除国内出现自然重组强毒株的可能性。因此,在加强对蓝舌病的重视程度的同时,十分有必要加强在BTV各个方面的研究工作。目前我国在蓝舌病研究领域虽然取得了一些成绩,但是在总体上仍大幅度落后于国际先进水平。在病毒的感染复制过程研究、致病机理研究,特别是新型标记疫苗研制方面一直没有突破性的进展,而缺少BTV反向遗传操作技术平台,正是制约我国深入开展蓝舌病研究的最主要原因。因此,要改变我国在蓝舌病研究领域的落后现状,就必需建立起我国自己的BTV反向遗传操作技术平台。
本研究利用全长cDNA扩增法(full-length amplification of cDNAs, FLAC)对本实验室保存的一株血清1型BTV SZ97/1株进行了全基因序列测定。测序结果表明,该毒株10个节段基因从Seg-l-Seg-10核苷酸长度分别为3944bp、2940bp、2772bp、1981bp、1772bp、1635bp、1156bp、1125bp、1049bp和822bp。编码的7个结构蛋白(VP1-VP7)的氨基酸长度分别1302aa、961aa、901aa、644aa、526aa、329aa和349aa。编码的5个非结构蛋白(NS1、NS2、NS3、NS3a、NS4)的氨基酸长度分别552aa、354aa、229aa、216aa、77aa。虽然各个节段基因的非编码区序列相互差异较大,而且长度也各不相同,但是每个节段非编码区两端的前6个核苷酸均相同,靠近5’端的前6个碱基序列GTTAAA,靠近3’端的前6个碱基序列为ACTTAC。在编码每个蛋白的基因终止密码子序列方面,编码VP1、VP2、VP5蛋白基因终止密码子为UGA,编码VP4、VP6、NS3、NS3a蛋白基因终止密码子为UAA,编码VP3、VP7、NS1、NS2、NS4蛋白基因终止密码子为UAG。本研究完成了我国血清1型BTV SZ97/1株的全基因组测序工作,这也是在我国首次获得血清1型BTV毒株的全基因序列,为进一步开展对国内外血清1型BTV的分子特征和地理起源分析的研究提供重要的参考数据。
在获得了SZ97/1株BTV的全基因组序列的基础上,本研究使用pCI-neo真核表达载体,成功构建了表达SZ97/1株BTV的VP1、VP3、VP4、VP6、VP7、NS1和NS2蛋白的7个真核表达质粒：pCI-VP1、pCI-VP3、pCI-VP4、pCI-VP6、pCI-VP7、pCI-NS1和pCI-NS2。同时又构建了克隆有SZ97/1株BTV的全部10个基因节段(Seg-1-Seg-10)的10个体外转录质粒：pBbBa-S1、pBbBa-S2、pBbBm-S3、pBbBa-S4、pBbBa-S5、pBbBa-S6、 pBbBa-S7、pBbBa-S8、pBbBa-S9、pBbBa-mS10。其中在构建Seg-10节段的体外转录质粒pBbBa-mS10时,用定点突变的方法,在Seg-10节段中引入AflⅡ限制性内切酶酶切位点序列作为分子标签。
在进行体外转录之前,分别使用限制性内切酶BsmB I或Bsa I先将环形的10个体外转录质粒进行酶切线性化处理,使用RNA体外转录试剂盒—RiboMaxTM Large Scale RNA Production Systems-T7，以酶切线性化的质粒DNA为模板,按照说明书步骤进行BTV各个基因节段RNA的体外转录,在反应结束后立即使用RNA纯化试剂盒对体外转录的RNA进行纯化,纯化后的RNA在-80℃保存。
将已经构建的表达BTV的VP1、VP3、VP4、VP6、VP7、NS1、NS2蛋白的7个真核表达质粒：pCI-VP1、pCI-VP3、pCI-VP4、pCI-VP6、pCI-VP7、pCI-NS1和pCI-NS2共同转染12孔细胞培养板中的BHK-21细胞。在18小时后,再转染10个体外转录获得的RNA产物。在体外转录RNA转染细胞48小时后,转染的BHK-21细胞已经全部病变,收集转染细胞上清和沉淀,冻融一次,离心后收获上清在-80℃冻存,将拯救出的病毒命名为rBTV1。对拯救的病毒分别进行间接免疫荧光、电镜以及基因测序检测,结果表明本研究成功拯救出了带有分子标记的BTV。对拯救毒株rBTV1进行生长曲线的测定结果表明,拯救毒rBTVl与亲本毒SZ97/1具有基本一致的生长曲线,在接毒后60～72小时内可达到最高滴度1×108TCID50/mL。
本研究首次在我国成功建立了BTV的反向遗传操作系统,为我国深入开展BTV致病机理、传播机制、基因组功能的研究以及新型疫苗的研发奠定了基础。
Bluetongue is an insect-transmitted disease of ruminants that is caused by bluetongue virus (BTV). BT occurs principally in sheep and some species of wild ruminants. BTV infection of cattle, goats and most wild ruminant species is typically asymptomatic or subclinical. Bluetongue is listed as a'notifiable disease'by the Office International des Epizooties (OIE). Bluetongue is one of the most economically important infectious animal diseases which caused directly reductions in productivity and trade losses of ruminants. The worldwide economic losses due to bluetongue have not been expressed in exact numbers, but the estimate is3billion US$a year.
BTV is the'type'species of the genus Orbivirus, within the family Reoviridae. BTV is a non-enveloped virus,70～80nm in diameter, with a triple-layered icosahedral protein capsid. The genome of BTV approximately19200base pairs consists of ten linear double-stranded RNA segments (segment1～segment10, Seg-1～Seg-10) encoding seven structural proteins (VP1-VP7), and five nonstructural proteins (NS1, NS2, NS3, NS3a and NS4). The outer layer consists of two major proteins, VP2and VP5. The VP2protein determines the serotype of BTV. The middle layer is formed by the VP7protein. The inner shell is composed of VP3protein surrounding the virus genome and viral transcriptase complexes composed of three enzymatic structural proteins (VP1, VP4and VP6). Five non-structural proteins (NS1, NS2, NS3, NS3a and NS4) were synthetized in BTV-infected cell.
BTV infection occurs throughout tropical, subtropical and some temperate regions of the world. Indeed, BTV has been isolated from ruminants and/or vector insects from all continents except Antarctica. Currently, twenty-six BTV serotypes been recognized in the world. Since1979, the first BTV strain has been isolated in Yunnan province of China, there are seven BTV serotypes, BTV-1, BTV-2, BTV-3, BTV-4, BTV-12, BTV-15, BTV-16, have been isolated from China.
In recent years, different serotypes and strains of BTV occured in many European countries, which has caused severe economical losing and damaged the cattle and sheep breeding industry in local countries, especially BTV-8and BTV-6outbreaked during2006. Prevention and control of bluetongue disease has become an international hotspot issues in research field of animal infectious diseases. Bluetongue is listed as a'notifiable disease'by OIE together with avian influenza and other important animal disease, but great importance has not been attached to the research on bluetongue in our country. Although the outbreak of virulent strain BTV has never occurred in our country, the risk of virulent strain BTV transmission to the domestic from abroad as the import and export trade is increasing. On the other hand, BTV may occur genetic recombination between different strains in nature, so the possibility of emerging recombinant virulent strain BTV in our country could not be ruled out. Particularly, it is very important to strengthen the emphasis on this area and carry out the research work in all aspects of BTV in our country. The quality of the scientific research on BTV in our country in general, is still significantly behind the international advanced level. In our country, the investigation of BTV replication and pathogenesis, as well as the research to generate new BTV vaccine strains have all been hampered by the lack of reverse genetics system. So it is necessary to establish a reverse genetics system of BTV in our country.
In the present study, the full genome of the serotype1BTV strain SZ97/1were sequenced using the full-length amplification of cDNAs (FLAC) method. The nucleotide length of SZ97/1segments1thru10are as follows:segment1,3944base pairs (bp); segment2,2940bp; segment3,2772bp; segment4,1981bp; segment5,1772bp; segment6,1635bp; segment7,1156bp; segment8,1125bp; segment9,1049bp; and segment10,822bp. The amino acid (aa) lengths of the twelve proteins of SZ97/1are as follows:VP1,1302aa; VP2,961aa; VP3,901aa; VP4,644aa; VP5,526aa; VP6,329aa; and VP7,349aa; NS1,552aa; NS2,354aa; NS3,229aa; NS3a,216aa; NS4,77aa. Although the non-coding regions (NCR) of each SZ97/1segment vary in length, the upstream and downstream terminal hexanucleotides are100%conserved as GTTAAA and ACTTAC. The stop codon of VP1, VP2and VP5is UGA, while segment VP4, VP6, NS3and NS3a use UAA, and VP3, VP7, NS1and NS2use UAG. The data presented here are the first to report the complete sequence of a BTV1strain isolated in China. These data will facilitate future investigations of the molecular characteristics and geographic origins of BTV1strains from both China and other countries.
Seven additional mammalian expression plasmids, pCI-VP1, pCI-VP3, pCI-VP4, pCI-NSl, pCI-VP7, pCI-NS2, and pCI-VP6, were generated, which were produced by inserting the coding region (CDR) of each of SZ97/1Seg-1, Seg-3, Seg-4, Seg-5, Seg-7, Seg-8, and Seg-9, encoding VP1, VP3, VP4, NS1, VP7, NS2, and VP6, respectively, into the mammalian expression vector pCI-neo. Ten T7plasmids for BTV transcripts used in the reverse genetics system were constructed for all10segments of the SZ97/1genome (pBbBa-S1, pBbBa-S2, pBbBm-S3, pBbBa-S4, pBbBa-S5, pBbBa-S6, pBbBa-S7, pBbBa-S8, pBbBa-S9, and pBbBa-mS10). pBbBa-mS10was altered to contain an additional AflⅡ site by site-directed mutagenesis.
T7plasmid clones were digested with BsmB Ⅰ or Bsa Ⅰ. Transcripts were generated from the digested T7plasmid clones using RiboMaxTM Large Scale RNA Production Systems-T7Kit according to the manufacturer's instructions. T7BTV transcripts were extracted using RNeasy Mini Kit according to the manufacturer's instructions and stored at-80℃.
BHK-21monolayers in12-well plates were first transfected with these seven additional mammalian expression plasmids, pCI-VP1, pCI-VP3, pCI-VP4, pCI-NS1, pCI-VP7, pCI-NS2, and pCI-VP6, followed by second transfection with a complete set of ten T7transcripts of BTV,18h after the first transfection. The cytopathy on the transfected monolayer indicated the recovery of infectious virions,48h after the second transfection. The BTV generated by reverse genetics was named rBTV1. To confirm the authenticity of the rescued virus, indirect immunofluorescence, electron microscopy and gene sequencing identification we examined, the results demonstrate that BTV mutant strain was generated by reverse genetics. Virus growth curves of the SZ97/1and mutant rBTVl were characterized. The results showed that rBTV1exhibit replication kinetis similar to the native strain SZ97/1, and the highest virus titer1×108TCID50/mL can achieve at60-72h postinfection.
In the present study, a reverse genetics system for BTV was developed which is the first to report the development of reverse genetics system for BTV in China. This system will not only facilitate future investigations of the pathogenesis, mechanism of transmission, genome function of BTV but also provide a tool to generate new BTV vaccine strains in our country.
BTV为呼肠孤病毒科(Reoviridae)环状病毒属(Orbivirus)成员。BTV粒子呈二十面体对称,无囊膜,直径约为70～80nm。BTV的核酸由10个双股RNA(double-stranded RNA, dsRNA)节段组成,按长度大小依次命名为segmentl-segment10(Seg-l-Seg-10), BTV的10个双股RNA节段总长度在19.2Kb左右,分别编码7种结构蛋白(VP1-VP7)和5种非结构蛋白(NS1、NS2、NS3、NS3a、NS4)。病毒的衣壳可以分为3层,其外层由两种结构蛋白组成,分别是VP2和VP5,其中VP2蛋白是型特异性蛋白,决定BTV的血清型；中间层由VP7蛋白构成；最内层的核心衣壳由VP3蛋白构成。病毒基因组和VP1、VP4、VP6三种酶活性结构蛋白被包裹在核心衣壳内部,另外五种非结构蛋白NS1、NS2、NS3、 NS3a、NS4在被病毒感染的细胞中合成。
目前已从非洲、欧洲、亚洲、北美、南美和大洋洲的多个热带、亚热带和温带地区国家分离到BTV,并且其分布范围不断扩大,危害也日趋严重。BTV血清型众多,到目前为止,全世界共分离到26个血清型的BTV。我国自1979年在云南首次分离到BTV以来,到现在国内共分离到BTV-1、2、3、4、12、15、16共七个血清型的BTV。
近年来,蓝舌病在欧洲许多国家相继暴发,且分布范围不断扩大,特别是2006年以来BTV-8和BTV-6在欧洲多个国家流行,对欧洲的牛羊养殖业造成了严重危害,也引起了高度重视。预防和控制蓝舌病已成为当前国际上重大动物传染病研究中的一个热点。虽然OIE将蓝舌病同禽流感等一些重要动物疫病共同列入法定通报性疾病,在国外也受到高度重视,但在国内受重视的程度还不够,投入的研究也较少。在我国虽然没有像欧洲地区那样,出现BTV强毒株的暴发,但是随着进出口贸易的日益频繁,完全存在国外的强毒株传播到国内的风险。同时,由于BTV为基因分节段的病毒,不同毒株间可以在自然界发生基因重组,所以也不能排除国内出现自然重组强毒株的可能性。因此,在加强对蓝舌病的重视程度的同时,十分有必要加强在BTV各个方面的研究工作。目前我国在蓝舌病研究领域虽然取得了一些成绩,但是在总体上仍大幅度落后于国际先进水平。在病毒的感染复制过程研究、致病机理研究,特别是新型标记疫苗研制方面一直没有突破性的进展,而缺少BTV反向遗传操作技术平台,正是制约我国深入开展蓝舌病研究的最主要原因。因此,要改变我国在蓝舌病研究领域的落后现状,就必需建立起我国自己的BTV反向遗传操作技术平台。
本研究利用全长cDNA扩增法(full-length amplification of cDNAs, FLAC)对本实验室保存的一株血清1型BTV SZ97/1株进行了全基因序列测定。测序结果表明,该毒株10个节段基因从Seg-l-Seg-10核苷酸长度分别为3944bp、2940bp、2772bp、1981bp、1772bp、1635bp、1156bp、1125bp、1049bp和822bp。编码的7个结构蛋白(VP1-VP7)的氨基酸长度分别1302aa、961aa、901aa、644aa、526aa、329aa和349aa。编码的5个非结构蛋白(NS1、NS2、NS3、NS3a、NS4)的氨基酸长度分别552aa、354aa、229aa、216aa、77aa。虽然各个节段基因的非编码区序列相互差异较大,而且长度也各不相同,但是每个节段非编码区两端的前6个核苷酸均相同,靠近5’端的前6个碱基序列GTTAAA,靠近3’端的前6个碱基序列为ACTTAC。在编码每个蛋白的基因终止密码子序列方面,编码VP1、VP2、VP5蛋白基因终止密码子为UGA,编码VP4、VP6、NS3、NS3a蛋白基因终止密码子为UAA,编码VP3、VP7、NS1、NS2、NS4蛋白基因终止密码子为UAG。本研究完成了我国血清1型BTV SZ97/1株的全基因组测序工作,这也是在我国首次获得血清1型BTV毒株的全基因序列,为进一步开展对国内外血清1型BTV的分子特征和地理起源分析的研究提供重要的参考数据。
在获得了SZ97/1株BTV的全基因组序列的基础上,本研究使用pCI-neo真核表达载体,成功构建了表达SZ97/1株BTV的VP1、VP3、VP4、VP6、VP7、NS1和NS2蛋白的7个真核表达质粒：pCI-VP1、pCI-VP3、pCI-VP4、pCI-VP6、pCI-VP7、pCI-NS1和pCI-NS2。同时又构建了克隆有SZ97/1株BTV的全部10个基因节段(Seg-1-Seg-10)的10个体外转录质粒：pBbBa-S1、pBbBa-S2、pBbBm-S3、pBbBa-S4、pBbBa-S5、pBbBa-S6、 pBbBa-S7、pBbBa-S8、pBbBa-S9、pBbBa-mS10。其中在构建Seg-10节段的体外转录质粒pBbBa-mS10时,用定点突变的方法,在Seg-10节段中引入AflⅡ限制性内切酶酶切位点序列作为分子标签。
在进行体外转录之前,分别使用限制性内切酶BsmB I或Bsa I先将环形的10个体外转录质粒进行酶切线性化处理,使用RNA体外转录试剂盒—RiboMaxTM Large Scale RNA Production Systems-T7，以酶切线性化的质粒DNA为模板,按照说明书步骤进行BTV各个基因节段RNA的体外转录,在反应结束后立即使用RNA纯化试剂盒对体外转录的RNA进行纯化,纯化后的RNA在-80℃保存。
将已经构建的表达BTV的VP1、VP3、VP4、VP6、VP7、NS1、NS2蛋白的7个真核表达质粒：pCI-VP1、pCI-VP3、pCI-VP4、pCI-VP6、pCI-VP7、pCI-NS1和pCI-NS2共同转染12孔细胞培养板中的BHK-21细胞。在18小时后,再转染10个体外转录获得的RNA产物。在体外转录RNA转染细胞48小时后,转染的BHK-21细胞已经全部病变,收集转染细胞上清和沉淀,冻融一次,离心后收获上清在-80℃冻存,将拯救出的病毒命名为rBTV1。对拯救的病毒分别进行间接免疫荧光、电镜以及基因测序检测,结果表明本研究成功拯救出了带有分子标记的BTV。对拯救毒株rBTV1进行生长曲线的测定结果表明,拯救毒rBTVl与亲本毒SZ97/1具有基本一致的生长曲线,在接毒后60～72小时内可达到最高滴度1×108TCID50/mL。
本研究首次在我国成功建立了BTV的反向遗传操作系统,为我国深入开展BTV致病机理、传播机制、基因组功能的研究以及新型疫苗的研发奠定了基础。
Bluetongue is an insect-transmitted disease of ruminants that is caused by bluetongue virus (BTV). BT occurs principally in sheep and some species of wild ruminants. BTV infection of cattle, goats and most wild ruminant species is typically asymptomatic or subclinical. Bluetongue is listed as a'notifiable disease'by the Office International des Epizooties (OIE). Bluetongue is one of the most economically important infectious animal diseases which caused directly reductions in productivity and trade losses of ruminants. The worldwide economic losses due to bluetongue have not been expressed in exact numbers, but the estimate is3billion US$a year.
BTV is the'type'species of the genus Orbivirus, within the family Reoviridae. BTV is a non-enveloped virus,70～80nm in diameter, with a triple-layered icosahedral protein capsid. The genome of BTV approximately19200base pairs consists of ten linear double-stranded RNA segments (segment1～segment10, Seg-1～Seg-10) encoding seven structural proteins (VP1-VP7), and five nonstructural proteins (NS1, NS2, NS3, NS3a and NS4). The outer layer consists of two major proteins, VP2and VP5. The VP2protein determines the serotype of BTV. The middle layer is formed by the VP7protein. The inner shell is composed of VP3protein surrounding the virus genome and viral transcriptase complexes composed of three enzymatic structural proteins (VP1, VP4and VP6). Five non-structural proteins (NS1, NS2, NS3, NS3a and NS4) were synthetized in BTV-infected cell.
BTV infection occurs throughout tropical, subtropical and some temperate regions of the world. Indeed, BTV has been isolated from ruminants and/or vector insects from all continents except Antarctica. Currently, twenty-six BTV serotypes been recognized in the world. Since1979, the first BTV strain has been isolated in Yunnan province of China, there are seven BTV serotypes, BTV-1, BTV-2, BTV-3, BTV-4, BTV-12, BTV-15, BTV-16, have been isolated from China.
In recent years, different serotypes and strains of BTV occured in many European countries, which has caused severe economical losing and damaged the cattle and sheep breeding industry in local countries, especially BTV-8and BTV-6outbreaked during2006. Prevention and control of bluetongue disease has become an international hotspot issues in research field of animal infectious diseases. Bluetongue is listed as a'notifiable disease'by OIE together with avian influenza and other important animal disease, but great importance has not been attached to the research on bluetongue in our country. Although the outbreak of virulent strain BTV has never occurred in our country, the risk of virulent strain BTV transmission to the domestic from abroad as the import and export trade is increasing. On the other hand, BTV may occur genetic recombination between different strains in nature, so the possibility of emerging recombinant virulent strain BTV in our country could not be ruled out. Particularly, it is very important to strengthen the emphasis on this area and carry out the research work in all aspects of BTV in our country. The quality of the scientific research on BTV in our country in general, is still significantly behind the international advanced level. In our country, the investigation of BTV replication and pathogenesis, as well as the research to generate new BTV vaccine strains have all been hampered by the lack of reverse genetics system. So it is necessary to establish a reverse genetics system of BTV in our country.
In the present study, the full genome of the serotype1BTV strain SZ97/1were sequenced using the full-length amplification of cDNAs (FLAC) method. The nucleotide length of SZ97/1segments1thru10are as follows:segment1,3944base pairs (bp); segment2,2940bp; segment3,2772bp; segment4,1981bp; segment5,1772bp; segment6,1635bp; segment7,1156bp; segment8,1125bp; segment9,1049bp; and segment10,822bp. The amino acid (aa) lengths of the twelve proteins of SZ97/1are as follows:VP1,1302aa; VP2,961aa; VP3,901aa; VP4,644aa; VP5,526aa; VP6,329aa; and VP7,349aa; NS1,552aa; NS2,354aa; NS3,229aa; NS3a,216aa; NS4,77aa. Although the non-coding regions (NCR) of each SZ97/1segment vary in length, the upstream and downstream terminal hexanucleotides are100%conserved as GTTAAA and ACTTAC. The stop codon of VP1, VP2and VP5is UGA, while segment VP4, VP6, NS3and NS3a use UAA, and VP3, VP7, NS1and NS2use UAG. The data presented here are the first to report the complete sequence of a BTV1strain isolated in China. These data will facilitate future investigations of the molecular characteristics and geographic origins of BTV1strains from both China and other countries.
Seven additional mammalian expression plasmids, pCI-VP1, pCI-VP3, pCI-VP4, pCI-NSl, pCI-VP7, pCI-NS2, and pCI-VP6, were generated, which were produced by inserting the coding region (CDR) of each of SZ97/1Seg-1, Seg-3, Seg-4, Seg-5, Seg-7, Seg-8, and Seg-9, encoding VP1, VP3, VP4, NS1, VP7, NS2, and VP6, respectively, into the mammalian expression vector pCI-neo. Ten T7plasmids for BTV transcripts used in the reverse genetics system were constructed for all10segments of the SZ97/1genome (pBbBa-S1, pBbBa-S2, pBbBm-S3, pBbBa-S4, pBbBa-S5, pBbBa-S6, pBbBa-S7, pBbBa-S8, pBbBa-S9, and pBbBa-mS10). pBbBa-mS10was altered to contain an additional AflⅡ site by site-directed mutagenesis.
T7plasmid clones were digested with BsmB Ⅰ or Bsa Ⅰ. Transcripts were generated from the digested T7plasmid clones using RiboMaxTM Large Scale RNA Production Systems-T7Kit according to the manufacturer's instructions. T7BTV transcripts were extracted using RNeasy Mini Kit according to the manufacturer's instructions and stored at-80℃.
BHK-21monolayers in12-well plates were first transfected with these seven additional mammalian expression plasmids, pCI-VP1, pCI-VP3, pCI-VP4, pCI-NS1, pCI-VP7, pCI-NS2, and pCI-VP6, followed by second transfection with a complete set of ten T7transcripts of BTV,18h after the first transfection. The cytopathy on the transfected monolayer indicated the recovery of infectious virions,48h after the second transfection. The BTV generated by reverse genetics was named rBTV1. To confirm the authenticity of the rescued virus, indirect immunofluorescence, electron microscopy and gene sequencing identification we examined, the results demonstrate that BTV mutant strain was generated by reverse genetics. Virus growth curves of the SZ97/1and mutant rBTVl were characterized. The results showed that rBTV1exhibit replication kinetis similar to the native strain SZ97/1, and the highest virus titer1×108TCID50/mL can achieve at60-72h postinfection.
In the present study, a reverse genetics system for BTV was developed which is the first to report the development of reverse genetics system for BTV in China. This system will not only facilitate future investigations of the pathogenesis, mechanism of transmission, genome function of BTV but also provide a tool to generate new BTV vaccine strains in our country.
引文
[1]Hofmann MA, Renzullo S, Mader M, et al. Genetic characterization of toggenburg orbivirus, a new bluetongue virus, from goats, Switzerland[J]. Emerging Infectious Diseases,2008, 14:1855-1861.
[2]Maan S, Maan NS, Nomikou K, et al. Novel bluetongue virus serotype from Kuwait[J]. Emerging Infectious Diseases,2011,17:886-889.
[3]Tabachnick WJ. The genetics of Culicoides variipennis and the epidemiology of bluetongue disease in North America[J]. Annual Review of Entomology,1996,45:20-40.
[4]Vellema P. Bluetongue in sheep:Question marks on bluetongue virus serotype 8 in Europe[J]. Small Ruminant Research,2008,76:141-148.
[5]Mehlhorn H, Walldorf V, Klimpel S, et al. Outbreak of bluetongue disease(BTD) in Germany and the danger for Europe[J]. Parasitology Research,2008,103:79-86.
[6]MacLachlan NJ, Drew CP, Darpel KE, et al. The Pathology and Pathogenesis of Bluetongue[J]. Journal of Comparative Pathology,2009,141:1-16.
[7]Gibbs EPJ, Greiner EC. The epidemiology of bluetongue[J]. Comparative Immunology and Microbiology and Infectious Diseases,1994,17:207-220.12.
[8]Zhang N, Maclachlan NJ, Bonneau KR, et al. Identification of seven serotypes of bluetongue virus from the People's Republic of China[J]. Vet. Rec,1999,145:427-429.
[9]Clavijo A, Sepulveda L, Riva J, et al. Isolation of bluetongue virus serotype 12 from an outbreak of the disease in South America[J]. Veterinary Record,2002,151:301-302.
[10]Mellor P S, Carpenter S, Harrup L,et al. Bluetongue in Europe and the Mediterranean Basin:History of occurrence prior to 2006[J]. Preventive Veterinary Medicine,2008,87:4-20
[11]Saegerman C, Berkvens D, Mellor PS. Bluetongue epidemiology in the European Union[J]. Emerg Infect Dis,2008,14(4):539-544.
[12]Wilson AJ, Mellor PS. Bluetongue in Europe:past, present and future[J]. Philos Trans R Soc Lond B Biol Sci,2009,364(1530):2669-2681.
[13]Wilson AJ, Mellor PS. Bluetongue in Europe:vectors, epidemiology and climate change[J]. Parasitol Res,2008,103:69-77.
[14]Linden A, Gregoire F, Nahayo A, et al. Bluetongue virus in wild deer, Belgium,2005-2008[J]. Emerg Infect Dis,2010,16(5):833-836.
[15]Mellor PS, Boorman J, Baylis M. Culicoides biting midges:Their role as arbovirus vectors[J]. Annual Review of Entomology,2000,45:307-340.
[16]Bouwknegt C, van Rijn PA, Schipper JM, et al. Potential role of ticks as vectors of bluetongue virus[J]. Experimental and Applied Acarology,2010,52:183-92.
[17]Brown ES, Gorman BM, Tesh RB, et al. Isolation of bluetongue and epizootic hemorrhagic disease viruses from mosquitoes collected in Indonesia[J]. Veterinary Microbiology,1992,32: 241-251.
[18]Radostits OM, Blood DC, Gay CC. A Textbook of the Diseases of Cattle, Sheep, Pigs, Goats and Horses[M].8th ed. VB Saunders Company Ltd.1994,1028-1033.
[19]Saegerman C, Bolkaerts B, Baricalla C, et al. The impact of naturally-occuring, trans-placental bluetongue virus serotype 8 infection on reproductive performance in sheep[J]. Veterinary Journal,2011,187:72-80.
[20]Mayo CE, Crossley BM, Hietala SK, et al. Colostral transmission of bluetongue virus nucleic acid among newborn dairy calves in California[J].Transboundary and Emerging Diseases, 2010,57:277-281.
[21]White DM, Wilson WC, Blair CD, et al. Studies on overwintering of bluetongue viruses in insects[J]. J Gen Virol,2005,86(2):453-462.
[22]Tabachnick, W.J. Culicoides and the global epidemiology of bluetongue virus infection[J]. Vet. Ital.,2004,40:145-150.
[23]Toussaint JF, Toussaint F, Vandenbussche J, et al. Bluetongue in northern Europe[J]. Vet. Rec,2006,2:327.
[24]Gale P, Drew T, Phipps LP, et al. The effect of climate change on occurrence and prevalence of livestock diseases in Great Britain:a review[J]. Journal of Applied Microbiology,2009, 106:1409-1423.
[25]MacLachlan NJ. Global implications of the recent emergence of bluetongue virus in Europe[J]. Veterinary Clinics of North America:Food Animal Practise,2010,26:163-171.
[26]Haneveld J K. What BTV-8 can do, any other serotype can do, too[J]. Tijdschr Diergeneeskd, 2008,133(20):856-858.
[27]Maclachlan NJ, Guthrie AJ. Re-emergence of bluetongue, African horse sickness, and other Orbivirus diseases[J]. Vet Res.,2010,41:35.
[28]MacLachlan NJ, Osburn BI. Impact of bluetongue virus infection on the international movement and trade of ruminants[J]. J. Am. Vet. Med. Assoc.,2006,228:1346-1349.
[29]Boyle DB, Bulach DM, Amos-Ritchie R, et al. Genomic sequences of Australian bluetongue virus prototype serotypes reveal global relationships and possible routes of entry into Australia[J]. J Virol,2012,86(12):6724-31.
[30]Legisa D, Gonzalez F, De Stefano G, et al. Phylogenetic analysis of bluetongue virus serotype 4 field isolates from Argentina[J]. J Gen Virol,2013,94:652-62.
[31]Lager IA. Bluetongue virus in South America:overview of viruses, vectors, surveillance and unique features[J]. Vet Ital.2004 Jul-Sep;40(3):89-93.
[32]Gerdes GH. A South African overview of the virus, vectors, surveillance and unique features of bluetongue[J]. Vet Ital,2004,40(3):39-42.
[33]Coetzee P, Stokstad M, Venter EH, et al. Bluetongue:a historical and epidemiological perspective with the emphasis on South Africa[J]. Virol J,2012,9:198.
[34]Desai GS, Hosamane M, Kataria RS, et al. Sequence analysis of the S10 gene of six Bluetongue Virus isolates from India[J]. Transbound Emerg Dis,2009,56(8):329-36.
[35]Elbers ARW, Backx A, Mintiens K, et al. Field observations during the bluetongue serotype 8 epidemic in 2006 II. Morbidity and mortality rate, case fatality and clinical recovery in sheep and cattle in Netherlands[J]. Preventive Veterinary Medicine,2008,87:31-40.
[36]Mehlhorn H, Walldorf V, Klimpel S, et al. First occurrence of Culicoides obsoletus-transmitted Bluetongue virus epidemic in Central Europe[J]. Parasitology Research,2007, 101:219-228.
[37]Ducheyne E, De Denken R, Becu S, et al. Quitifying thewind dispersal of Culicoides species in Greece and Bulgaria[J]. Geospatial Health,2007,2:177-189.
[38]张忠信.ICTV第九次报告对病毒分类系统的一些修改[J].病毒学报,2012,5：595-599.
[39]Anthony SJ, Maan S, Maan N, et al. Genetic and phylogenetic analysis of the outer-coat proteins VP2 and VP5 of epizootic haemorrhagic disease virus (EHDV):comparison of genetic and serological data to characterise the EHDV serogroup[J]. Virus Res,2009,145(2): 200-10.
[40]Roy P. Bluetongue virus proteins and particles and their role in virus entry, assembly, and release[J].2005, Adv Virus Res,64:69-123.
[41]Belhouchet M, Mohd Jaafar F, Firth AE, et al. Detection of a fourth orbivirus non-structural protein[J]. PLoS One,2011,6:e25697. doi:10.1371/journal.pone.0025697.
[42]Ratinier M, Caporale M, Golder M, et al. Identification and characterization of a novel non-structural protein of bluetongue virus[J]. PLoS Pathog,2011,7:e1002477.
[43]Grimes JM, Burroughs JN, Gouet P, et al. The atomic structure of the bluetongue virus core[J]. Nature,1998,395:470-478.
[44]Nason EL, Rothagel R, Mukherjee SK, et al. Interactions between the inner and outer capsids of bluetongue virus[J]. J. Virol.,2004,78:8059-8067. [45]
[45]Schwartz-Cornil I, Mertens PP, Contreras V, et al. Bluetongue virus:virology, pathogenesis and immunity[J]. Vet Res,2008,39(5):46.
[46]White JR, Eaton BT. Conformation of the VP2 protein of bluetongue virus (BTV) determines the involvement in virus neutralization of highly conserved epitopes within the BTV serogroup[J]. J Gen Virol,1990,71(6):1325-1332.
[47]Tembhurne PA, Mondal B, Pathak K B, et al. Segment-2 sequence analysis and cross-neutralization studies on some Indian bluetongue viruses suggest isolates are VP2-variants of serotype 23[J]. Arch Virol,2010,155(1):89-95.
[48]Maan S, Maan N S, Samuel A R, et al. Analysis and phylogenetic comparisons of full-length VP2 genes of the 24 bluetongue virus serotypes[J]. J Gen Virol,2007,88(2):621-630.
[49]Maan S, Maan NS, Nomikou K, et al. Complete genome characterisation of a novel 26th bluetongue virus serotype from Kuwait[J]. PLoS One,2011,6(10):26147.
[50]Singh K.P, Maan S, Samuel AR, et al. Phylogenetic analysis of bluetongue virus genome segment 6 (encoding VP5) from different serotypes[J]. Vet Ital,2004,40(4):479-483.
[51]Cowley JA, Gorman BM. Cross-neutralization of genetic reassortants of bluetongue virus serotypes 20 and 21 [J]. Vet Microbiol,1989,19(1):37-51.
[52]Mertens PP, Pedley S, Cowley J, et al. Analysis of the roles of bluetongue virus outer capsid proteins VP2 and VP5 in determination of virus serotype[J]. Virology,1989,170(2):561-565.
[53]Hassan S. H, Wirblich C, Forzan M, et al. Expression and functional characterization of bluetongue virus VP5 protein:role in cellular permeabilization[J]. J Virol,2001,75(18): 8356-8367.
[54]Bhattacharya B, Roy P. Bluetongue virus outer capsid protein VP5 interacts with membrane lipid rafts via a SNARE domain[J]. J Virol,2008,82(21):10600-10612.
[55]Forzan M, Wirblich C, Roy P. A Capsid protein of nonenveloped bluetongue virus exhibits membrane fusion activity[J]. Proc Natl Acad Sci USA,2004,101(7):2100-2105.
[56]Grimes J, Basak A K, Roy P, et al. The crystal structure of bluetongue virus VP7[J]. Nature, 1995,373(6510):167-170.
[57]Yu Y, Fukusho A, Ritter DG, et al. Complete nucleotide sequence of the group-reactive antigen VP7 gene of bluetongue virus[J]. Nucleic Acids Res,1988,16(4):1620.
[58]Hosamani M, Shimizu S, Hirota J, et al. Expression and characterization of bluetongue virus serotype 21 VP7 antigen:C-terminal truncated protein has significantly reduced antigenicity[J]. J Vet Med Sci,2011,73(5):609-613.
[59]Nomikou K, Dovas CI, Maan S, et al. Evolution and phylogenetic analysis of full-length VP3 genes of Eastern Mediterranean bluetongue virus isolates[J]. PLoS One,2009,4(7):6437.
[60]Le Blois H, Roy P. A single point mutation in the VP7 major core protein of bluetongue virus prevents the formation of core-like particles[J]. J Virol,1993,67(1):353-359.
[61]Huismans H, Van Dijk AA, Els HJ. Uncoating of parental bluetongue virus to core and subcore particles in infected L cells[J]. Virology,1987,157:180-188.
[62]Huismans H, Van Dijk AA. Bluetongue virus structural components[J]. Curr Top Microbiol Immunol,1990,162:21-41.
[63]Stuart D I, Gouet P, Grimes J, et al. Structural studies of orbivirus particles[J]. Arch Virol Suppl,1998,14:235-250.
[64]Boyce M, Wehrfritz J, Noad R, et al. Purified recombinant bluetongue virus VP1 exhibits RNA replicase activity[J]. J Virol,2004,78(8):3994-4002.
[65]Sutton G, Grimes JM, Stuart DI, et al. Bluetongue virus VP4 is an RNA-capping assembly line[J]. Nat Struct Mol Biol,2007,14(5):449-451.
[66]Stauber N, Martinez-Costas J, Sutton G, et al. Bluetongue virus VP6 protein binds ATP and exhibits an RNA-dependent ATPase function and a helicase activity that catalyze the unwinding of double-stranded RNA substrates[J]. J Virol,1997,71(10):7220-7226.
[67]Guirakhoo F, Catalan JA, Monath TP. Adaptation of bluetongue virus in mosquito cells results in over expression of NS3 proteins and release of virus particles[J]. Archives of Virology,1995,140(5):967-974.
[68]Eaton B T, Hyatt AD, White JR. Localization of the nonstructural protein NS1 in bluetongue virus-infected cells and its presence in virus particles[J]. Virology,1988,163(2):527-537.
[69]Zhao Y, Thomas C, Bremer C, et al. Deletion and mutational analyses of bluetongue virus NS2 protein indicate that the amino but not the carboxy terminus of the protein is critical for RNA-protein interactions[J]. J Virol,1994,68:2179-2185.
[70]Horscroft NJ, Roy P. NTP binding and phosphohydrolase activity associated with purified bluetongue virus non-structural protein NS2[J]. J Gen Virol.,2000,81:1961-1965.
[71]Kar A K, Bhattacharya B, Roy P. Bluetongue virus RNA binding protein NS2 is a modulator of viral replication and assembly[J]. BMC Mol Biol,2007,8:4.
[72]Han Z, Harty RN. The NS3 protein of bluetongue virus exhibits viroporin-like properties [J]. J.Biol.Chem.,2004,279:43092-43097.
[73]Wirblich C, Bhattacharya B, Roy P. Nonstructural protein 3 of bluetongue virus assists virus release by recruiting ESCRT-1 protein Tsg101[J]. J Virol,2006,80(1):460-473.
[74]Forzan M, Marsh M, Roy P. Bluetongue virus entry into cells[J]. J Virol,2007,81:4819-4827.
[75]Mertens P P, Diprose J. The bluetongue virus core:a nano-scale transcription machine[J].
[76]Diprose J, Burroughs JN, Sutton G, et al. Translocation portals for the substrates and products of a viral transcriptase complex:the Bluetongue virus core[J]. EMBO J,2001,20:7229-7239.
[77]Gould AR, Hyatt AD. The orbivirus genus. Diversity, structure, replication and phylogenetic Relationships[J]. Comp Immunol Microbiol Infect Dis,1994,17:163-188.
[78]Roy P, Bluetongue virus proteins[J]. J Gen Virol,1992,73:3051-3064.
[79]Bhattacharya B, Noad RJ, Roy P. Interaction between bluetongue virus outer capsid protein VP2 and vimentin is necessary for virus egress[J]. Virol J,2007,4:7. [79]
[80]Mertens PP, Diprose J, Maan S, et al. Bluetongue virus replication, molecular and structural biology[J]. Vet Ital.2004,40(4):426-37.
[81]Manning J S. Bluetongue virus:detection of antiviral immunoglobulin G by means of enzyme-linked immunosorbent assay[J]. Curr Microbiol,1980,4:381-385.
[82]Afshar A, Thomas F C, Wright P F, et al. Comparison of competitive and indirect enzyme-linked immunosorbent assays for detection of bluetongue virus antibodies in serum and whole blood[J]. J Clin Microbiol,1987,25(9):1705-1710.
[83]Afshar A, Trotter H C, Dulac G C, et al. Evaluation of a commercial competitive ELISA test kit for the detection of group-specific antibodies to bluetongue virus[J]. J Vet Diagn Invest, 1993,5(3):336-340.
[84]花群义,徐自忠,周晓黎,等.竞争酶联免疫吸附试验检测蓝舌病抗体的研究[J].动物医学进展,2002,23(1)：66-69.
[85]耿宏伟,秦永丽,李俊平,等.蓝舌病病毒重组VP7蛋白单克隆抗体制备及竞争ELISA检测方法的建立[J].中国预防兽医学报,2012,5：388-392
[86]Chand K, Biswas S K, De A, et al. A polyclonal antibody-based sandwich ELISA for the detection of bluetongue virus in cell culture and blood of sheep infected experimentally[J]. J Virol Methods,2009,160(1-2):189-192.
[87]Chand K, Biswas S K, Sing B, et al. A sandwich ELISA for the detection of bluetongue virus in cell culture using antiserum against the recombinant VP7 protein[J]. Vet Ital,2009,45(3): 443-448.
[88]Anthony S, Jones H, Darpel KE, et al. A duplex RT-PCR assay for detection of genome segment 7 (VP7 gene) from 24 BTV serotypes[J]. J Virol Methods,2007,141(2):188-97,
[89]Orru G, Santis P, Solinas F, et al. Differentiation of Italian field and South African vaccine strains of bluetongue virus serotype 2 using real-time PCR[J]. J Virol Methods,2004,122 (1):37-43.
[90]Jimenez-Clavero M, Aguero M, San M, et al. High throughput detection of bluetongue virus by a new real-time fluorogenic reverse transcrtiption-polymerase chain reaction:Application on clinical samples from current Mediterranean outbreaks[J]. J Vet Diagn Invest, 2006,18(1):7-17.
[91]Orru G, Ferrando M, Meloni M, et al. Rapid detection and quantitation of bluetongue virus(BTV) using a molecular beacon fluorescent probe assay[J]. J Virol Methods, 2006,137(1):34-42.
[92]尹惠琼,张红,刘松婷,等.蓝舌病病毒荧光定量RT-PCR检测技术[J].中国动物传染病学报,2009,17(4)：7-10.
[93]Savini G, Cannas A, Casaccia C, et al. Risk factors associated with the occurrence of undesired effects in sheep and goats after field vaccination with modified-live vaccine against bluetongue virus serotypes 2,4 and 16[J]. Vet Microbiol,2010,146(1-2):44-50.
[94]Veronesi E, Hamblin C, Mellor PS. Live attenuated bluetongue vaccine viruses in Dorset Poll sheep, before and after passage in vector midges (Diptera:Ceratopogonidae)[J]. Vaccine, 2005,23:5509-5516.
[95]Savini G, MacLachlan N J, Sanchez-Vizcaino J M, et al. Vaccines against bluetongue in Europe[J]. Comp Immunol Microbiol Infect Dis,2008,31(2-3):101-120.
[96]Savini G, Hamers C, Migliaccio P, et al. Assessment of efficacy of a bivalent inactivated vaccine against bluetongue virus serotypes 2 and 4 in cattle. In:Proceedings of the 24th world buiatrics congress[C].France,2006.
[97]Savini G, Hamers C, Conte A, et al. Assessment of efficacy of a bivalent BTV-2 and BTV-4 inactivated vaccine by vaccination and challenge in cattle[J]. Vet Microbiol,2009,133(1-2): 1-8.
[98]Hamers C, Rehbein S, Hudelet P, et al. Protective duration of immunity of an inactivated bluetongue (BTV) serotype 2 vaccine against a virulent BTV serotype 2 challenge in sheep[J]. Vaccine,2009,27(21):2789-2793.
[99]Wackerlin R, Eschbaumer M, Konig P, et al. Evaluation of humoral response and protective efficacy of three inactivated vaccines against bluetongue virus serotype 8 one year after vaccination of sheep and cattle[J]. Vaccine,2010,28(27):4348-4355.
[100]Vitour D, Guillotin J, Sailleau C, et al. Colostral antibody induced interference of inactivated bluetongue serotype-8 vaccines in calves[J]. Vet Res,2011,42(1):18.
[101]Breard E, Belbis G, Hamers C, et al. Evaluation of humoral response and protective efficacy of two inactivated vaccines against bluetongue virus after vaccination of goats[J]. Vaccine, 2011,29(13):2495-2502.
[102]Moulin V, Noordegraaf CV, Makoschey B, et al. Clinical disease in sheep caused by bluetongue virus serotype 8, and prevention by an inactivated vaccine[J]. Vaccine,2012, 30(12):2228-35.
[103]Bhanuprakash V, Indrani B K, Hosamani M, et al. Bluetongue vaccines:the past, present and future[J]. Expert Reviews Vaccines,2009(8):191-204.
[104]Enserink M, Animal disease. Exotic disease of farm animal tests Europe's responses[J]. Science,2008,319:710-711.
[105]Roy P, French T, Erasmus BJ. Protective efficacy of virus-like particles for bluetongue disease[J]. Vaccine,1992,10:28-32.
[106]Hervas-Stubbs S, Rueda P, Lopez L, et al. Insect baculoviruses strongly potentiate adaptive immune responses by inducing type I IFN[J]. J Immunol,2007,178:2361-2369.
[107]Roy P, Bishop DH, LeBlois H, et al. Long-lasting protection of sheep against bluetongue challenge after vaccination with virus-like particles:evidence for homologous and partial heterologous Protection[J]. Vaccine,1994,12:805-811.
[108]Stewart M, Bhatia Y, Athmaran TN, et al. Validation of a novel approach for the rapid production of immunogenic virus-like particles for bluetongue virus[J]. Vaccine.2010, 28(17):3047-3054.
[109]Lobato Z I, Coupar B E, Gray C P, et al. Antibody responses and protective immunity to recombinant vaccinia virus-expressed bluetongue virus antigens[J]. Vet Immunol Immunopathol,1997,59(3-4):293-309.
[110]Wade-Evans A M, Romero C H, Mellor P. Expression of the major core structural protein(VP7) of bluetongue virus, by a recombinant capripox virus, provides partial protection of sheep against a virulent heterotypic bluetongue virus challeng[J]. Virology, 1996,220(1):227-231.
[111]Perrin A, Albina E, Breard E. Recombinant capripoxviruses expressing proteins of bluetongue virus:evaluation of immune responses and protention in small ruminants [J]. Vaccine,2007,25(37-38):6774-6783.
[112]Boone J D, Balasuriya U B, Karaca K, et al. Recombinant canarypox virus vaccine co-expressing genes encoding the VP2 and VP5 outer capsid proteins of bluetongue virus induces high level protection in sheep[J]. Vaccine,2007,25(4):672-678.
[113]Top S, Foucras G, Deplanche M, et al. Myxomavirus as a vector for the immunisation of sheep:protection study against challenge with bluetongue virus[J]. Vaccine,2012,30(9): 1609-16.
[114]Calvo-Pinilla E, Navasa N, Anguita J, et al. Multiserotype protection elicited by a combinatorial prime-boost vaccination strategy against bluetongue virus[J]. PLoS One. 2012;7(4):e34735.
[115]Dudek T, Knipe DM. Replication-defective viruses as vaccines and vaccine vectors[J]. Virology,2006,344(1):230-239.
[116]Nguyen LH, Knipe DM, Finberg RW. Replication-defective mutants of herpes simplex virus (HSV) induce cellular immunity and protect against lethal HSV infection[J]. J Virol,1992, 66:7067-7072.
[117]Morrison LA, Knipe DM. Mechanisms of immunization with a replication-defective mutant of herpes simplex virus 1[J]. Virology,1996,220:402-413.
[118]Roy P, Boyce M, Noad R. Prospects for improved bluetongue vaccines[J]. Nat Rev Microbiol,2009,7(2):120-8.
[119]Noad R, Roy P. Bluetongue vaccines[J]. Vaccine,2009,27 Suppl 4:D86-9.
[120]Matsuo E, Celma CC, Boyce M, et al. Generation of replication-defective virus-based vaccines that confer full protection in sheep against virulent bluetongue virus challenge[J]. J Virol,2011,85(19):10213-10221.
[121]Loudon PT, Blakeley DM, Boursnell ME, et al. Preclinical safety testing of DISC-hGMCSF to support phase I clinical trials in cancer patients[J]. J Gene Med,2001,3:458-467.
[122]Celma CC, Boyce M, van Rijn PA, et al. Rapid Generation of Replication-Deficient Monovalent and Multivalent Vaccines for Bluetongue Virus:Protection against Virulent Virus Challenge in Cattle and Sheep[J]. J Virol,2013,87(17):9856-64.
[123]刘光清等.动物病毒反向遗传学[M].北京：科学出版社,2009.
[124]Taniguchi T, Palmieri M, Weissmann C. QB DNA-containing hybrid plasmids giving rise to QB phage formation in the bacterial host[J]. Nature,1978,274(5668):223-8.
[125]Racaniello VR, Baltimore D. Cloned Poliovirus Complementary DNA is Infectious in Mammalian Cells [J]. Science.1981,214:916-919.
[126]Almazan F, Gonzalez JM, Penzes Z,et al. Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome[J]. Proc Natl Acad Sci U S A. 2000,97(10): 5516-21.
[127]Casais R, Thiel V, Siddell SG, et al. Reverse genetics system for the avian coronavirus infectious bronchitis virus[J]. J Virol,2001,75(24):12359-12369.
[128]Coley SE, Lavi E, Sawicki SG, et al. Recombinant mouse hepatitis virus strain A59 from cloned, full-length cDNA replicates to high titers in vitro and is fully pathogenic in vivo[J]. J Virol,2005,79(5):3097-3106.
[129]Schnell MJ, Mebatsion T, Conzelmann KK. Infectious rabies viruses from cloned cDNA[J]. EMBO J,1994,13(18):4195-203.
[130]Whelan SP, Ball LA, Barr JN, et al. Efficient recovery of infectious vesicular stomatitis virus entirely from cDNA clones[J]. Proc Natl Acad Sci USA,1995,92(18):8388-8392.
[131]Peeters BP, de Leeuw OS, Koch G, et al. Rescue of Newcastle disease virus from cloned cDNA:evidence that cleavability of the fusion protein is a major determinant for virulence[J]. J Virol,1999,73(6):5001-5009.
[132]Bridgen A, Elliott RM. Rescue of a segmented negative-strand RNA virus entirely from cloned complementary DNAs[J]. Proc Natl Acad Sci USA,1996,93(26):15400-15404.
[133]Neumann G, Watanabe T, Ito H, et al. Generation of influenza A viruses entirely from cloned cDNAs[J]. Proc Natl Acad Sci USA,1999,96(16):9345-9350.
[134]Hoffmann E, Webster RG. Unidirectional RNA polymerase I-polymerase II transcription system for the generation of influenza A virus from eight plasmids[J]. J Gen Virol,2000,81: 2843-2847.
[135]Mundt E, Vakharia VN. Synthetic transcripts of double-stranded Birnavirus genome are infectious[J]. Proc Natl Acad Sci USA,1996,93(20):11131-11136.
[136]Yao K, Vakharia VN. Generation of infectious pancreatic necrosis virus from cloned cDNA[J]. J Virol,1998,72(11):8913-8920.
[137]Kobayashi T, Antar AA, Boehme KW, et al. A plasmid-based reverse genetics system for animal double-stranded RNA viruses[J]. Cell Host Microbe,2007,1(2):147-157.
[138]Boyce M, Celma CC, Roy P. Development of reverse genetics systems for bluetongue virus: recovery of infectious virus from synthetic RNA transcripts[J], J Virol,2008,82 (17):8339-8348.
[139]Kaname Y, Celma CC, Kanai Y, et al. Recovery of African horse sickness virus from synthetic RNA[J]. J Gen Virol,2013,94:2259-2265.
[140]Kobayashi T, Ooms LS, Ikizler M, et al. An improved reverse genetics system for mammalian orthoreoviruses[J]. Virology,2010,398(2):194-200.
[141]Matsuo E, Roy P. Bluetongue virus VP6 acts early in the replication cycle and can form the basis of chimeric virus formation[J]. J Virol,2009,83(17):8842-8848.
[142]Matsuo E, Roy P. Minimum requirements for bluetongue virus primary replication in vivo[J]. J Virol,2013,87(2):882-889.
[143]Trask SD, Boehme KW, Dermody TS, et al. Comparative analysis of Reoviridae reverse genetics methods[J]. Methods,2013,59(2):199-206.
[144]Richards JE, Desselberger U, Lever AM. Experimental Pathways towards Developing a Rotavirus Reverse Genetics System:Synthetic Full Length Rotavirus ssRNAs Are Neither Infectious nor Translated in Permissive Cells[J]. PLoS One,2013,8(9):e74328.
[145]Komoto S, Sasaki J, Taniguchi K. Reverse genetics system for introduction of site-specific mutations into the double-stranded RNA genome of infectious rotavirus[J]. Proc Natl Acad Sci USA,2006,103(12):4646-4651.
[146]Trask SD, Taraporewala ZF, Boehme KW, et al. Dual selection mechanisms drive efficient single-gene reverse genetics for rotavirus[J]. Proc Natl Acad Sci USA,2010,107(43): 18652-18657.
[147]Troupin C, Dehee A, Schnuriger A, et al. Rearranged genomic RNA segments offer a new approach to the reverse genetics of rotaviruses[J]. J Virol,2010,84(13):6711-6719.
[148]Attoui H, Billoir F, Cantaloube JF, et al. Strategies for the sequence determination of viral dsRNA genomes [J]. J Virol Methods,2000,89:147-158.
[149]Maan S, Rao S, Maan NS, et al. Rapid cDNA synthesis and sequencing techniques for the genetic study of bluetongue and other dsRNA viruses[J]. J Virol Methods,2007,143:132-139.
[150]Boyce M, Celma CC and Roy P. Development of reverse genetics systems for bluetongue virus:recovery of infectious virus from synthetic RNA transcripts[J]. J Virol,2008,82: 8339-8348.
[151]Mertens PPC, Maan S, Samuel A, et al. Orbivirus Reoviridae. p.466-483. In:CM Fauquet, MA Mayo, J Maniloff, U Desselberger, and LA Ball (ed.), Virus Taxonomy Ⅷth Report of the ICTV[C]. London, Elsevier/Academic Press,2005.
[152]Gould AR, Pritchard LI. Relationships amongst bluetongue viruses revealed by comparisons of capsid and outer coat protein nucleotide sequences [J]. Virus Res,1990,17(1):31-52.
[153]Yamaguchi S, Fukusho A, Roy P. Complete sequence of VP2 gene of the bluetongue virus serotype 1 (BTV-1) [J]. Nucleic Acids Res,1988,16 (6):2725.
[154]Gould AR. Conserved and non-conserved regions of the outer coat protein, VP2, of the Australian bluetongue serotype 1 virus, revealed by sequence comparison to the VP2 of North American BTV serotype 10 [J]. Virus Res,1988,9(2-3):145-158.
[155]Boyle DB, Bulach DM, Amos-Ritchie R, et al. Genomic sequences of Australian bluetongue virus prototype serotypes reveal global relationships and possible routes of entry into Australia [J]. J Virol,2012,86(12):6724-6731.
[156]Biswas SK, Chand K, De A, et al. Isolation of bluetongue virus serotype 1 (BTV-1) from goats and its phylogenetic relationship to other BTV-1 isolates worldwide based on full-length sequence of genome segment-2[J]. Arch Virol,2010,155(12):2041-2046.
[157]Lambden PR, Cooke SJ, Caul EO, et al. Cloning of noncultivatable human rotavirus by single primer amplification[J]. J Virol,1992,66(3):1817-1822.
[158]Darissa O, Willingmann P, Adam G. Optimized approaches for the sequence determination of double-stranded RNA templates[J]. J Virol Methods,2010,169(2):397-403.
[159]Potgieter AC, Page NA, Liebenberg J, et al. Improved strategies for sequence-independent amplification and sequencing of viral double-stranded RNA genomes[J]. J Gen Virol,2009, 90:1423-1432.
[160]Bonneau KR, Zhang N, Zhu J, et al. Sequence comparison of the L2 and S10 genes of bluetongue viruses from the United States and the People's Republic of China[J], Virus Res, 1999,61(2):153-160.
[161]Zhang Y, Du X, Li W, et al. Genetic diversity of the S10 RNA segment of field and vaccine strains of bluetongue virus from the P. R. China[J]. Arch Virol,2010,155(2):281-286.
[162]Lee F, Ting LJ, Lee MS, et al. Genetic analysis of two Taiwanese bluetongue viruses[J]. Vet Microbiol,2011,148(2-4):140-149.
[163]Yang T, Liu N, Xu Q, et al. Complete genomic sequence of bluetongue virus serotype 16 from China[J]. J Virol,2011,85(24):13472.
[164]Lim BL, Cao Y, Yu T, et al. Adaptation of very virulent infectious bursal disease virus to chicken embryonic fibroblasts by site-directed mutagenesis of residues 279 and 284 of viral coat protein VP2[J]. J Virol,1999,73(4):2854-2862.
[165]Boot HJ, ter Huurne AA, Peeters BP, et al. Efficient rescue of infectious bursal disease virus from cloned cDNA:evidence for involvement of the 3'-terminal sequence in genome replication[J]. Virology,1999,265(2):330-341.
[166]Qi XL, Gao YL, Gao HL, et al. An improved method for infectious bursal disease virus rescue using RNA polymeraseⅡsystem. J Virol Meth,2007,142:81-88.
[167]Brochu-Lafontaine V, Lemay G. Addition of exogenous polypeptides on the mammalian reovirus outer capsid using reverse genetics[J]. J Virol Methods,2012,179(2):342-350.
[2]Maan S, Maan NS, Nomikou K, et al. Novel bluetongue virus serotype from Kuwait[J]. Emerging Infectious Diseases,2011,17:886-889.
[3]Tabachnick WJ. The genetics of Culicoides variipennis and the epidemiology of bluetongue disease in North America[J]. Annual Review of Entomology,1996,45:20-40.
[4]Vellema P. Bluetongue in sheep:Question marks on bluetongue virus serotype 8 in Europe[J]. Small Ruminant Research,2008,76:141-148.
[5]Mehlhorn H, Walldorf V, Klimpel S, et al. Outbreak of bluetongue disease(BTD) in Germany and the danger for Europe[J]. Parasitology Research,2008,103:79-86.
[6]MacLachlan NJ, Drew CP, Darpel KE, et al. The Pathology and Pathogenesis of Bluetongue[J]. Journal of Comparative Pathology,2009,141:1-16.
[7]Gibbs EPJ, Greiner EC. The epidemiology of bluetongue[J]. Comparative Immunology and Microbiology and Infectious Diseases,1994,17:207-220.12.
[8]Zhang N, Maclachlan NJ, Bonneau KR, et al. Identification of seven serotypes of bluetongue virus from the People's Republic of China[J]. Vet. Rec,1999,145:427-429.
[9]Clavijo A, Sepulveda L, Riva J, et al. Isolation of bluetongue virus serotype 12 from an outbreak of the disease in South America[J]. Veterinary Record,2002,151:301-302.
[10]Mellor P S, Carpenter S, Harrup L,et al. Bluetongue in Europe and the Mediterranean Basin:History of occurrence prior to 2006[J]. Preventive Veterinary Medicine,2008,87:4-20
[11]Saegerman C, Berkvens D, Mellor PS. Bluetongue epidemiology in the European Union[J]. Emerg Infect Dis,2008,14(4):539-544.
[12]Wilson AJ, Mellor PS. Bluetongue in Europe:past, present and future[J]. Philos Trans R Soc Lond B Biol Sci,2009,364(1530):2669-2681.
[13]Wilson AJ, Mellor PS. Bluetongue in Europe:vectors, epidemiology and climate change[J]. Parasitol Res,2008,103:69-77.
[14]Linden A, Gregoire F, Nahayo A, et al. Bluetongue virus in wild deer, Belgium,2005-2008[J]. Emerg Infect Dis,2010,16(5):833-836.
[15]Mellor PS, Boorman J, Baylis M. Culicoides biting midges:Their role as arbovirus vectors[J]. Annual Review of Entomology,2000,45:307-340.
[16]Bouwknegt C, van Rijn PA, Schipper JM, et al. Potential role of ticks as vectors of bluetongue virus[J]. Experimental and Applied Acarology,2010,52:183-92.
[17]Brown ES, Gorman BM, Tesh RB, et al. Isolation of bluetongue and epizootic hemorrhagic disease viruses from mosquitoes collected in Indonesia[J]. Veterinary Microbiology,1992,32: 241-251.
[18]Radostits OM, Blood DC, Gay CC. A Textbook of the Diseases of Cattle, Sheep, Pigs, Goats and Horses[M].8th ed. VB Saunders Company Ltd.1994,1028-1033.
[19]Saegerman C, Bolkaerts B, Baricalla C, et al. The impact of naturally-occuring, trans-placental bluetongue virus serotype 8 infection on reproductive performance in sheep[J]. Veterinary Journal,2011,187:72-80.
[20]Mayo CE, Crossley BM, Hietala SK, et al. Colostral transmission of bluetongue virus nucleic acid among newborn dairy calves in California[J].Transboundary and Emerging Diseases, 2010,57:277-281.
[21]White DM, Wilson WC, Blair CD, et al. Studies on overwintering of bluetongue viruses in insects[J]. J Gen Virol,2005,86(2):453-462.
[22]Tabachnick, W.J. Culicoides and the global epidemiology of bluetongue virus infection[J]. Vet. Ital.,2004,40:145-150.
[23]Toussaint JF, Toussaint F, Vandenbussche J, et al. Bluetongue in northern Europe[J]. Vet. Rec,2006,2:327.
[24]Gale P, Drew T, Phipps LP, et al. The effect of climate change on occurrence and prevalence of livestock diseases in Great Britain:a review[J]. Journal of Applied Microbiology,2009, 106:1409-1423.
[25]MacLachlan NJ. Global implications of the recent emergence of bluetongue virus in Europe[J]. Veterinary Clinics of North America:Food Animal Practise,2010,26:163-171.
[26]Haneveld J K. What BTV-8 can do, any other serotype can do, too[J]. Tijdschr Diergeneeskd, 2008,133(20):856-858.
[27]Maclachlan NJ, Guthrie AJ. Re-emergence of bluetongue, African horse sickness, and other Orbivirus diseases[J]. Vet Res.,2010,41:35.
[28]MacLachlan NJ, Osburn BI. Impact of bluetongue virus infection on the international movement and trade of ruminants[J]. J. Am. Vet. Med. Assoc.,2006,228:1346-1349.
[29]Boyle DB, Bulach DM, Amos-Ritchie R, et al. Genomic sequences of Australian bluetongue virus prototype serotypes reveal global relationships and possible routes of entry into Australia[J]. J Virol,2012,86(12):6724-31.
[30]Legisa D, Gonzalez F, De Stefano G, et al. Phylogenetic analysis of bluetongue virus serotype 4 field isolates from Argentina[J]. J Gen Virol,2013,94:652-62.
[31]Lager IA. Bluetongue virus in South America:overview of viruses, vectors, surveillance and unique features[J]. Vet Ital.2004 Jul-Sep;40(3):89-93.
[32]Gerdes GH. A South African overview of the virus, vectors, surveillance and unique features of bluetongue[J]. Vet Ital,2004,40(3):39-42.
[33]Coetzee P, Stokstad M, Venter EH, et al. Bluetongue:a historical and epidemiological perspective with the emphasis on South Africa[J]. Virol J,2012,9:198.
[34]Desai GS, Hosamane M, Kataria RS, et al. Sequence analysis of the S10 gene of six Bluetongue Virus isolates from India[J]. Transbound Emerg Dis,2009,56(8):329-36.
[35]Elbers ARW, Backx A, Mintiens K, et al. Field observations during the bluetongue serotype 8 epidemic in 2006 II. Morbidity and mortality rate, case fatality and clinical recovery in sheep and cattle in Netherlands[J]. Preventive Veterinary Medicine,2008,87:31-40.
[36]Mehlhorn H, Walldorf V, Klimpel S, et al. First occurrence of Culicoides obsoletus-transmitted Bluetongue virus epidemic in Central Europe[J]. Parasitology Research,2007, 101:219-228.
[37]Ducheyne E, De Denken R, Becu S, et al. Quitifying thewind dispersal of Culicoides species in Greece and Bulgaria[J]. Geospatial Health,2007,2:177-189.
[38]张忠信.ICTV第九次报告对病毒分类系统的一些修改[J].病毒学报,2012,5：595-599.
[39]Anthony SJ, Maan S, Maan N, et al. Genetic and phylogenetic analysis of the outer-coat proteins VP2 and VP5 of epizootic haemorrhagic disease virus (EHDV):comparison of genetic and serological data to characterise the EHDV serogroup[J]. Virus Res,2009,145(2): 200-10.
[40]Roy P. Bluetongue virus proteins and particles and their role in virus entry, assembly, and release[J].2005, Adv Virus Res,64:69-123.
[41]Belhouchet M, Mohd Jaafar F, Firth AE, et al. Detection of a fourth orbivirus non-structural protein[J]. PLoS One,2011,6:e25697. doi:10.1371/journal.pone.0025697.
[42]Ratinier M, Caporale M, Golder M, et al. Identification and characterization of a novel non-structural protein of bluetongue virus[J]. PLoS Pathog,2011,7:e1002477.
[43]Grimes JM, Burroughs JN, Gouet P, et al. The atomic structure of the bluetongue virus core[J]. Nature,1998,395:470-478.
[44]Nason EL, Rothagel R, Mukherjee SK, et al. Interactions between the inner and outer capsids of bluetongue virus[J]. J. Virol.,2004,78:8059-8067. [45]
[45]Schwartz-Cornil I, Mertens PP, Contreras V, et al. Bluetongue virus:virology, pathogenesis and immunity[J]. Vet Res,2008,39(5):46.
[46]White JR, Eaton BT. Conformation of the VP2 protein of bluetongue virus (BTV) determines the involvement in virus neutralization of highly conserved epitopes within the BTV serogroup[J]. J Gen Virol,1990,71(6):1325-1332.
[47]Tembhurne PA, Mondal B, Pathak K B, et al. Segment-2 sequence analysis and cross-neutralization studies on some Indian bluetongue viruses suggest isolates are VP2-variants of serotype 23[J]. Arch Virol,2010,155(1):89-95.
[48]Maan S, Maan N S, Samuel A R, et al. Analysis and phylogenetic comparisons of full-length VP2 genes of the 24 bluetongue virus serotypes[J]. J Gen Virol,2007,88(2):621-630.
[49]Maan S, Maan NS, Nomikou K, et al. Complete genome characterisation of a novel 26th bluetongue virus serotype from Kuwait[J]. PLoS One,2011,6(10):26147.
[50]Singh K.P, Maan S, Samuel AR, et al. Phylogenetic analysis of bluetongue virus genome segment 6 (encoding VP5) from different serotypes[J]. Vet Ital,2004,40(4):479-483.
[51]Cowley JA, Gorman BM. Cross-neutralization of genetic reassortants of bluetongue virus serotypes 20 and 21 [J]. Vet Microbiol,1989,19(1):37-51.
[52]Mertens PP, Pedley S, Cowley J, et al. Analysis of the roles of bluetongue virus outer capsid proteins VP2 and VP5 in determination of virus serotype[J]. Virology,1989,170(2):561-565.
[53]Hassan S. H, Wirblich C, Forzan M, et al. Expression and functional characterization of bluetongue virus VP5 protein:role in cellular permeabilization[J]. J Virol,2001,75(18): 8356-8367.
[54]Bhattacharya B, Roy P. Bluetongue virus outer capsid protein VP5 interacts with membrane lipid rafts via a SNARE domain[J]. J Virol,2008,82(21):10600-10612.
[55]Forzan M, Wirblich C, Roy P. A Capsid protein of nonenveloped bluetongue virus exhibits membrane fusion activity[J]. Proc Natl Acad Sci USA,2004,101(7):2100-2105.
[56]Grimes J, Basak A K, Roy P, et al. The crystal structure of bluetongue virus VP7[J]. Nature, 1995,373(6510):167-170.
[57]Yu Y, Fukusho A, Ritter DG, et al. Complete nucleotide sequence of the group-reactive antigen VP7 gene of bluetongue virus[J]. Nucleic Acids Res,1988,16(4):1620.
[58]Hosamani M, Shimizu S, Hirota J, et al. Expression and characterization of bluetongue virus serotype 21 VP7 antigen:C-terminal truncated protein has significantly reduced antigenicity[J]. J Vet Med Sci,2011,73(5):609-613.
[59]Nomikou K, Dovas CI, Maan S, et al. Evolution and phylogenetic analysis of full-length VP3 genes of Eastern Mediterranean bluetongue virus isolates[J]. PLoS One,2009,4(7):6437.
[60]Le Blois H, Roy P. A single point mutation in the VP7 major core protein of bluetongue virus prevents the formation of core-like particles[J]. J Virol,1993,67(1):353-359.
[61]Huismans H, Van Dijk AA, Els HJ. Uncoating of parental bluetongue virus to core and subcore particles in infected L cells[J]. Virology,1987,157:180-188.
[62]Huismans H, Van Dijk AA. Bluetongue virus structural components[J]. Curr Top Microbiol Immunol,1990,162:21-41.
[63]Stuart D I, Gouet P, Grimes J, et al. Structural studies of orbivirus particles[J]. Arch Virol Suppl,1998,14:235-250.
[64]Boyce M, Wehrfritz J, Noad R, et al. Purified recombinant bluetongue virus VP1 exhibits RNA replicase activity[J]. J Virol,2004,78(8):3994-4002.
[65]Sutton G, Grimes JM, Stuart DI, et al. Bluetongue virus VP4 is an RNA-capping assembly line[J]. Nat Struct Mol Biol,2007,14(5):449-451.
[66]Stauber N, Martinez-Costas J, Sutton G, et al. Bluetongue virus VP6 protein binds ATP and exhibits an RNA-dependent ATPase function and a helicase activity that catalyze the unwinding of double-stranded RNA substrates[J]. J Virol,1997,71(10):7220-7226.
[67]Guirakhoo F, Catalan JA, Monath TP. Adaptation of bluetongue virus in mosquito cells results in over expression of NS3 proteins and release of virus particles[J]. Archives of Virology,1995,140(5):967-974.
[68]Eaton B T, Hyatt AD, White JR. Localization of the nonstructural protein NS1 in bluetongue virus-infected cells and its presence in virus particles[J]. Virology,1988,163(2):527-537.
[69]Zhao Y, Thomas C, Bremer C, et al. Deletion and mutational analyses of bluetongue virus NS2 protein indicate that the amino but not the carboxy terminus of the protein is critical for RNA-protein interactions[J]. J Virol,1994,68:2179-2185.
[70]Horscroft NJ, Roy P. NTP binding and phosphohydrolase activity associated with purified bluetongue virus non-structural protein NS2[J]. J Gen Virol.,2000,81:1961-1965.
[71]Kar A K, Bhattacharya B, Roy P. Bluetongue virus RNA binding protein NS2 is a modulator of viral replication and assembly[J]. BMC Mol Biol,2007,8:4.
[72]Han Z, Harty RN. The NS3 protein of bluetongue virus exhibits viroporin-like properties [J]. J.Biol.Chem.,2004,279:43092-43097.
[73]Wirblich C, Bhattacharya B, Roy P. Nonstructural protein 3 of bluetongue virus assists virus release by recruiting ESCRT-1 protein Tsg101[J]. J Virol,2006,80(1):460-473.
[74]Forzan M, Marsh M, Roy P. Bluetongue virus entry into cells[J]. J Virol,2007,81:4819-4827.
[75]Mertens P P, Diprose J. The bluetongue virus core:a nano-scale transcription machine[J].
[76]Diprose J, Burroughs JN, Sutton G, et al. Translocation portals for the substrates and products of a viral transcriptase complex:the Bluetongue virus core[J]. EMBO J,2001,20:7229-7239.
[77]Gould AR, Hyatt AD. The orbivirus genus. Diversity, structure, replication and phylogenetic Relationships[J]. Comp Immunol Microbiol Infect Dis,1994,17:163-188.
[78]Roy P, Bluetongue virus proteins[J]. J Gen Virol,1992,73:3051-3064.
[79]Bhattacharya B, Noad RJ, Roy P. Interaction between bluetongue virus outer capsid protein VP2 and vimentin is necessary for virus egress[J]. Virol J,2007,4:7. [79]
[80]Mertens PP, Diprose J, Maan S, et al. Bluetongue virus replication, molecular and structural biology[J]. Vet Ital.2004,40(4):426-37.
[81]Manning J S. Bluetongue virus:detection of antiviral immunoglobulin G by means of enzyme-linked immunosorbent assay[J]. Curr Microbiol,1980,4:381-385.
[82]Afshar A, Thomas F C, Wright P F, et al. Comparison of competitive and indirect enzyme-linked immunosorbent assays for detection of bluetongue virus antibodies in serum and whole blood[J]. J Clin Microbiol,1987,25(9):1705-1710.
[83]Afshar A, Trotter H C, Dulac G C, et al. Evaluation of a commercial competitive ELISA test kit for the detection of group-specific antibodies to bluetongue virus[J]. J Vet Diagn Invest, 1993,5(3):336-340.
[84]花群义,徐自忠,周晓黎,等.竞争酶联免疫吸附试验检测蓝舌病抗体的研究[J].动物医学进展,2002,23(1)：66-69.
[85]耿宏伟,秦永丽,李俊平,等.蓝舌病病毒重组VP7蛋白单克隆抗体制备及竞争ELISA检测方法的建立[J].中国预防兽医学报,2012,5：388-392
[86]Chand K, Biswas S K, De A, et al. A polyclonal antibody-based sandwich ELISA for the detection of bluetongue virus in cell culture and blood of sheep infected experimentally[J]. J Virol Methods,2009,160(1-2):189-192.
[87]Chand K, Biswas S K, Sing B, et al. A sandwich ELISA for the detection of bluetongue virus in cell culture using antiserum against the recombinant VP7 protein[J]. Vet Ital,2009,45(3): 443-448.
[88]Anthony S, Jones H, Darpel KE, et al. A duplex RT-PCR assay for detection of genome segment 7 (VP7 gene) from 24 BTV serotypes[J]. J Virol Methods,2007,141(2):188-97,
[89]Orru G, Santis P, Solinas F, et al. Differentiation of Italian field and South African vaccine strains of bluetongue virus serotype 2 using real-time PCR[J]. J Virol Methods,2004,122 (1):37-43.
[90]Jimenez-Clavero M, Aguero M, San M, et al. High throughput detection of bluetongue virus by a new real-time fluorogenic reverse transcrtiption-polymerase chain reaction:Application on clinical samples from current Mediterranean outbreaks[J]. J Vet Diagn Invest, 2006,18(1):7-17.
[91]Orru G, Ferrando M, Meloni M, et al. Rapid detection and quantitation of bluetongue virus(BTV) using a molecular beacon fluorescent probe assay[J]. J Virol Methods, 2006,137(1):34-42.
[92]尹惠琼,张红,刘松婷,等.蓝舌病病毒荧光定量RT-PCR检测技术[J].中国动物传染病学报,2009,17(4)：7-10.
[93]Savini G, Cannas A, Casaccia C, et al. Risk factors associated with the occurrence of undesired effects in sheep and goats after field vaccination with modified-live vaccine against bluetongue virus serotypes 2,4 and 16[J]. Vet Microbiol,2010,146(1-2):44-50.
[94]Veronesi E, Hamblin C, Mellor PS. Live attenuated bluetongue vaccine viruses in Dorset Poll sheep, before and after passage in vector midges (Diptera:Ceratopogonidae)[J]. Vaccine, 2005,23:5509-5516.
[95]Savini G, MacLachlan N J, Sanchez-Vizcaino J M, et al. Vaccines against bluetongue in Europe[J]. Comp Immunol Microbiol Infect Dis,2008,31(2-3):101-120.
[96]Savini G, Hamers C, Migliaccio P, et al. Assessment of efficacy of a bivalent inactivated vaccine against bluetongue virus serotypes 2 and 4 in cattle. In:Proceedings of the 24th world buiatrics congress[C].France,2006.
[97]Savini G, Hamers C, Conte A, et al. Assessment of efficacy of a bivalent BTV-2 and BTV-4 inactivated vaccine by vaccination and challenge in cattle[J]. Vet Microbiol,2009,133(1-2): 1-8.
[98]Hamers C, Rehbein S, Hudelet P, et al. Protective duration of immunity of an inactivated bluetongue (BTV) serotype 2 vaccine against a virulent BTV serotype 2 challenge in sheep[J]. Vaccine,2009,27(21):2789-2793.
[99]Wackerlin R, Eschbaumer M, Konig P, et al. Evaluation of humoral response and protective efficacy of three inactivated vaccines against bluetongue virus serotype 8 one year after vaccination of sheep and cattle[J]. Vaccine,2010,28(27):4348-4355.
[100]Vitour D, Guillotin J, Sailleau C, et al. Colostral antibody induced interference of inactivated bluetongue serotype-8 vaccines in calves[J]. Vet Res,2011,42(1):18.
[101]Breard E, Belbis G, Hamers C, et al. Evaluation of humoral response and protective efficacy of two inactivated vaccines against bluetongue virus after vaccination of goats[J]. Vaccine, 2011,29(13):2495-2502.
[102]Moulin V, Noordegraaf CV, Makoschey B, et al. Clinical disease in sheep caused by bluetongue virus serotype 8, and prevention by an inactivated vaccine[J]. Vaccine,2012, 30(12):2228-35.
[103]Bhanuprakash V, Indrani B K, Hosamani M, et al. Bluetongue vaccines:the past, present and future[J]. Expert Reviews Vaccines,2009(8):191-204.
[104]Enserink M, Animal disease. Exotic disease of farm animal tests Europe's responses[J]. Science,2008,319:710-711.
[105]Roy P, French T, Erasmus BJ. Protective efficacy of virus-like particles for bluetongue disease[J]. Vaccine,1992,10:28-32.
[106]Hervas-Stubbs S, Rueda P, Lopez L, et al. Insect baculoviruses strongly potentiate adaptive immune responses by inducing type I IFN[J]. J Immunol,2007,178:2361-2369.
[107]Roy P, Bishop DH, LeBlois H, et al. Long-lasting protection of sheep against bluetongue challenge after vaccination with virus-like particles:evidence for homologous and partial heterologous Protection[J]. Vaccine,1994,12:805-811.
[108]Stewart M, Bhatia Y, Athmaran TN, et al. Validation of a novel approach for the rapid production of immunogenic virus-like particles for bluetongue virus[J]. Vaccine.2010, 28(17):3047-3054.
[109]Lobato Z I, Coupar B E, Gray C P, et al. Antibody responses and protective immunity to recombinant vaccinia virus-expressed bluetongue virus antigens[J]. Vet Immunol Immunopathol,1997,59(3-4):293-309.
[110]Wade-Evans A M, Romero C H, Mellor P. Expression of the major core structural protein(VP7) of bluetongue virus, by a recombinant capripox virus, provides partial protection of sheep against a virulent heterotypic bluetongue virus challeng[J]. Virology, 1996,220(1):227-231.
[111]Perrin A, Albina E, Breard E. Recombinant capripoxviruses expressing proteins of bluetongue virus:evaluation of immune responses and protention in small ruminants [J]. Vaccine,2007,25(37-38):6774-6783.
[112]Boone J D, Balasuriya U B, Karaca K, et al. Recombinant canarypox virus vaccine co-expressing genes encoding the VP2 and VP5 outer capsid proteins of bluetongue virus induces high level protection in sheep[J]. Vaccine,2007,25(4):672-678.
[113]Top S, Foucras G, Deplanche M, et al. Myxomavirus as a vector for the immunisation of sheep:protection study against challenge with bluetongue virus[J]. Vaccine,2012,30(9): 1609-16.
[114]Calvo-Pinilla E, Navasa N, Anguita J, et al. Multiserotype protection elicited by a combinatorial prime-boost vaccination strategy against bluetongue virus[J]. PLoS One. 2012;7(4):e34735.
[115]Dudek T, Knipe DM. Replication-defective viruses as vaccines and vaccine vectors[J]. Virology,2006,344(1):230-239.
[116]Nguyen LH, Knipe DM, Finberg RW. Replication-defective mutants of herpes simplex virus (HSV) induce cellular immunity and protect against lethal HSV infection[J]. J Virol,1992, 66:7067-7072.
[117]Morrison LA, Knipe DM. Mechanisms of immunization with a replication-defective mutant of herpes simplex virus 1[J]. Virology,1996,220:402-413.
[118]Roy P, Boyce M, Noad R. Prospects for improved bluetongue vaccines[J]. Nat Rev Microbiol,2009,7(2):120-8.
[119]Noad R, Roy P. Bluetongue vaccines[J]. Vaccine,2009,27 Suppl 4:D86-9.
[120]Matsuo E, Celma CC, Boyce M, et al. Generation of replication-defective virus-based vaccines that confer full protection in sheep against virulent bluetongue virus challenge[J]. J Virol,2011,85(19):10213-10221.
[121]Loudon PT, Blakeley DM, Boursnell ME, et al. Preclinical safety testing of DISC-hGMCSF to support phase I clinical trials in cancer patients[J]. J Gene Med,2001,3:458-467.
[122]Celma CC, Boyce M, van Rijn PA, et al. Rapid Generation of Replication-Deficient Monovalent and Multivalent Vaccines for Bluetongue Virus:Protection against Virulent Virus Challenge in Cattle and Sheep[J]. J Virol,2013,87(17):9856-64.
[123]刘光清等.动物病毒反向遗传学[M].北京：科学出版社,2009.
[124]Taniguchi T, Palmieri M, Weissmann C. QB DNA-containing hybrid plasmids giving rise to QB phage formation in the bacterial host[J]. Nature,1978,274(5668):223-8.
[125]Racaniello VR, Baltimore D. Cloned Poliovirus Complementary DNA is Infectious in Mammalian Cells [J]. Science.1981,214:916-919.
[126]Almazan F, Gonzalez JM, Penzes Z,et al. Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome[J]. Proc Natl Acad Sci U S A. 2000,97(10): 5516-21.
[127]Casais R, Thiel V, Siddell SG, et al. Reverse genetics system for the avian coronavirus infectious bronchitis virus[J]. J Virol,2001,75(24):12359-12369.
[128]Coley SE, Lavi E, Sawicki SG, et al. Recombinant mouse hepatitis virus strain A59 from cloned, full-length cDNA replicates to high titers in vitro and is fully pathogenic in vivo[J]. J Virol,2005,79(5):3097-3106.
[129]Schnell MJ, Mebatsion T, Conzelmann KK. Infectious rabies viruses from cloned cDNA[J]. EMBO J,1994,13(18):4195-203.
[130]Whelan SP, Ball LA, Barr JN, et al. Efficient recovery of infectious vesicular stomatitis virus entirely from cDNA clones[J]. Proc Natl Acad Sci USA,1995,92(18):8388-8392.
[131]Peeters BP, de Leeuw OS, Koch G, et al. Rescue of Newcastle disease virus from cloned cDNA:evidence that cleavability of the fusion protein is a major determinant for virulence[J]. J Virol,1999,73(6):5001-5009.
[132]Bridgen A, Elliott RM. Rescue of a segmented negative-strand RNA virus entirely from cloned complementary DNAs[J]. Proc Natl Acad Sci USA,1996,93(26):15400-15404.
[133]Neumann G, Watanabe T, Ito H, et al. Generation of influenza A viruses entirely from cloned cDNAs[J]. Proc Natl Acad Sci USA,1999,96(16):9345-9350.
[134]Hoffmann E, Webster RG. Unidirectional RNA polymerase I-polymerase II transcription system for the generation of influenza A virus from eight plasmids[J]. J Gen Virol,2000,81: 2843-2847.
[135]Mundt E, Vakharia VN. Synthetic transcripts of double-stranded Birnavirus genome are infectious[J]. Proc Natl Acad Sci USA,1996,93(20):11131-11136.
[136]Yao K, Vakharia VN. Generation of infectious pancreatic necrosis virus from cloned cDNA[J]. J Virol,1998,72(11):8913-8920.
[137]Kobayashi T, Antar AA, Boehme KW, et al. A plasmid-based reverse genetics system for animal double-stranded RNA viruses[J]. Cell Host Microbe,2007,1(2):147-157.
[138]Boyce M, Celma CC, Roy P. Development of reverse genetics systems for bluetongue virus: recovery of infectious virus from synthetic RNA transcripts[J], J Virol,2008,82 (17):8339-8348.
[139]Kaname Y, Celma CC, Kanai Y, et al. Recovery of African horse sickness virus from synthetic RNA[J]. J Gen Virol,2013,94:2259-2265.
[140]Kobayashi T, Ooms LS, Ikizler M, et al. An improved reverse genetics system for mammalian orthoreoviruses[J]. Virology,2010,398(2):194-200.
[141]Matsuo E, Roy P. Bluetongue virus VP6 acts early in the replication cycle and can form the basis of chimeric virus formation[J]. J Virol,2009,83(17):8842-8848.
[142]Matsuo E, Roy P. Minimum requirements for bluetongue virus primary replication in vivo[J]. J Virol,2013,87(2):882-889.
[143]Trask SD, Boehme KW, Dermody TS, et al. Comparative analysis of Reoviridae reverse genetics methods[J]. Methods,2013,59(2):199-206.
[144]Richards JE, Desselberger U, Lever AM. Experimental Pathways towards Developing a Rotavirus Reverse Genetics System:Synthetic Full Length Rotavirus ssRNAs Are Neither Infectious nor Translated in Permissive Cells[J]. PLoS One,2013,8(9):e74328.
[145]Komoto S, Sasaki J, Taniguchi K. Reverse genetics system for introduction of site-specific mutations into the double-stranded RNA genome of infectious rotavirus[J]. Proc Natl Acad Sci USA,2006,103(12):4646-4651.
[146]Trask SD, Taraporewala ZF, Boehme KW, et al. Dual selection mechanisms drive efficient single-gene reverse genetics for rotavirus[J]. Proc Natl Acad Sci USA,2010,107(43): 18652-18657.
[147]Troupin C, Dehee A, Schnuriger A, et al. Rearranged genomic RNA segments offer a new approach to the reverse genetics of rotaviruses[J]. J Virol,2010,84(13):6711-6719.
[148]Attoui H, Billoir F, Cantaloube JF, et al. Strategies for the sequence determination of viral dsRNA genomes [J]. J Virol Methods,2000,89:147-158.
[149]Maan S, Rao S, Maan NS, et al. Rapid cDNA synthesis and sequencing techniques for the genetic study of bluetongue and other dsRNA viruses[J]. J Virol Methods,2007,143:132-139.
[150]Boyce M, Celma CC and Roy P. Development of reverse genetics systems for bluetongue virus:recovery of infectious virus from synthetic RNA transcripts[J]. J Virol,2008,82: 8339-8348.
[151]Mertens PPC, Maan S, Samuel A, et al. Orbivirus Reoviridae. p.466-483. In:CM Fauquet, MA Mayo, J Maniloff, U Desselberger, and LA Ball (ed.), Virus Taxonomy Ⅷth Report of the ICTV[C]. London, Elsevier/Academic Press,2005.
[152]Gould AR, Pritchard LI. Relationships amongst bluetongue viruses revealed by comparisons of capsid and outer coat protein nucleotide sequences [J]. Virus Res,1990,17(1):31-52.
[153]Yamaguchi S, Fukusho A, Roy P. Complete sequence of VP2 gene of the bluetongue virus serotype 1 (BTV-1) [J]. Nucleic Acids Res,1988,16 (6):2725.
[154]Gould AR. Conserved and non-conserved regions of the outer coat protein, VP2, of the Australian bluetongue serotype 1 virus, revealed by sequence comparison to the VP2 of North American BTV serotype 10 [J]. Virus Res,1988,9(2-3):145-158.
[155]Boyle DB, Bulach DM, Amos-Ritchie R, et al. Genomic sequences of Australian bluetongue virus prototype serotypes reveal global relationships and possible routes of entry into Australia [J]. J Virol,2012,86(12):6724-6731.
[156]Biswas SK, Chand K, De A, et al. Isolation of bluetongue virus serotype 1 (BTV-1) from goats and its phylogenetic relationship to other BTV-1 isolates worldwide based on full-length sequence of genome segment-2[J]. Arch Virol,2010,155(12):2041-2046.
[157]Lambden PR, Cooke SJ, Caul EO, et al. Cloning of noncultivatable human rotavirus by single primer amplification[J]. J Virol,1992,66(3):1817-1822.
[158]Darissa O, Willingmann P, Adam G. Optimized approaches for the sequence determination of double-stranded RNA templates[J]. J Virol Methods,2010,169(2):397-403.
[159]Potgieter AC, Page NA, Liebenberg J, et al. Improved strategies for sequence-independent amplification and sequencing of viral double-stranded RNA genomes[J]. J Gen Virol,2009, 90:1423-1432.
[160]Bonneau KR, Zhang N, Zhu J, et al. Sequence comparison of the L2 and S10 genes of bluetongue viruses from the United States and the People's Republic of China[J], Virus Res, 1999,61(2):153-160.
[161]Zhang Y, Du X, Li W, et al. Genetic diversity of the S10 RNA segment of field and vaccine strains of bluetongue virus from the P. R. China[J]. Arch Virol,2010,155(2):281-286.
[162]Lee F, Ting LJ, Lee MS, et al. Genetic analysis of two Taiwanese bluetongue viruses[J]. Vet Microbiol,2011,148(2-4):140-149.
[163]Yang T, Liu N, Xu Q, et al. Complete genomic sequence of bluetongue virus serotype 16 from China[J]. J Virol,2011,85(24):13472.
[164]Lim BL, Cao Y, Yu T, et al. Adaptation of very virulent infectious bursal disease virus to chicken embryonic fibroblasts by site-directed mutagenesis of residues 279 and 284 of viral coat protein VP2[J]. J Virol,1999,73(4):2854-2862.
[165]Boot HJ, ter Huurne AA, Peeters BP, et al. Efficient rescue of infectious bursal disease virus from cloned cDNA:evidence for involvement of the 3'-terminal sequence in genome replication[J]. Virology,1999,265(2):330-341.
[166]Qi XL, Gao YL, Gao HL, et al. An improved method for infectious bursal disease virus rescue using RNA polymeraseⅡsystem. J Virol Meth,2007,142:81-88.
[167]Brochu-Lafontaine V, Lemay G. Addition of exogenous polypeptides on the mammalian reovirus outer capsid using reverse genetics[J]. J Virol Methods,2012,179(2):342-350.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |