疫苗接种对我国新城疫病毒进化的影响及新城疫LaSota疫苗株全长cDNA克隆
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摘要
新城疫是由新城疫病毒引起的一种烈性病毒性传染病，是当今全球范围内最严重的家禽传染病之一，也是我国所有鸡病中危害最大的一种，被国际兽疫局列为A类传染病。该病发病急、致死率高，对养禽业的发展构成严重的威胁。
在我国主要广泛接种新城疫疫苗来控制该病的感染，由于减毒活疫苗使用相对方便，因此它常被用于群体免疫。我国NDV免疫密度和次数也远远超过世界水平。由于大面积的疫苗使用，新城疫得到了较好的控制，但近些年来，NDV出现了一些新的疫情，在免疫过的鸡群中即使能产生高的抗体滴度，新城疫仍时有爆发。而且大量使用的疫苗株难免流失到环境中，目前那些散落在环境中的疫苗毒株对新城疫病毒本身造成的影响并没有完全了解。
为了确定在我国广泛应用的新城疫疫苗是否对该病毒进化进程产生影响，在本研究中，我们对在我国流行的新城疫病毒进行了系统发生和重组分析，获得了一系列NDV间疫苗参与的同源重组的证据，表明疫苗可能通过同源重组影响我国新城疫病毒的进程。由于疫苗与流行株之间的重组可能导致出现疫苗接种失败的后果，因此，在临床上应当尽可能避免疫苗与野生株之间的重组。故在NDV防治过程中，应该采用合适的免疫程序避免这种重组的发生。
由于负链RNA病毒重组率很低的观点被广泛接受，因此尚没有人系统研究过NDV之间是否存在重组现象。当然，这个问题在NDV疫苗使用过程中也一直被忽略，至今没有任何有关NDV疫苗参与病毒进化方面的报道。NDV间重组的机制完全未知。通过评估病毒重组在NDV生物学性状，特别是疫苗重组后对病毒毒力变化中的影响，了解重组对NDV遗传多样性方面究竟有多大作用，以确定NDV是否能够开发为一种安全的疫苗载体。因此有必要建立NDV同源重组的实验系统，为NDV疫苗株和流行株的重组提供实验室证据。反向遗传系统的建立是研究NDV同源重组的前提，因此，本研究拟以广泛用来预防新城疫的一种弱毒疫苗的NDV LaSota株为材料，构建NDV反向遗传系统。首先我们对其进行了纯化和扩增。然后针对NDV LaSota株设计了十二对引物，然后采用RT-PCR对全基因组进行分段扩增，最终获得了涵盖全基因组的十二个cDNA片段，再将它们克隆入pMD18-T载体中并进行测序。通过生物信息学软件（DNAman）分析LaSota的核苷酸序列，找出各种限制性内切酶位点的分布，然后按照一定的先后顺序，将不同的限制性内切酶位点酶切、连接、转化，最终构建出NDV LaSota株基因组全长cDNA克隆pMC18-LaSota，本研究为NDVLaSota株与流行株之间重组的研究奠定了基础。
Newcastle disease (ND), caused by ND virus (NDV), is one of the most seriousavian contagious virus disease all over the world nowadays. It is also one of the mostserious illnesses of birds in our country. NDV has posed a great threaten to the poultryindustry. Because of the severe nature of the disease and the high mortality rates. NDis included as an Office International des Epizooties list A disease.
Many commercial Newcastle disease virus (NDV) live vaccines are widely usedfor preventing infection of chickens from Newcastle disease in China. Theseattenuated live vaccines for mass vaccination is thought to be convenient and effective.Therefore, in China, the immune density and frequency of NDV far surpassinternational level, ND was controlled well as a result of the huge of vaccination area.Howover, in recent years, NDV emerged some new epidemic situation. ND stillbreaks out in those vaccinated flocks even though high titre antibody was produced.And the vaccinated strains are hard to avoid draining into the environment, however,the evolution role of a great quantity of vaccine strains is unknown in NDV. Atpresent, it is not clear whether the scattered vaccine strains have any influence on theenvironment.
In this study, phylogenetic and recombination analyses were performed and got aseries of evidences for homologous recombination between NDV strains. Severalmosaics were proposed to be partially descended from vaccine strains. These resultsindicate that vaccines may also play roles in shaping NDV evolution via homologousrecombination. Recombination between vaccines and circulating viruses may causeimmunization failure in some cases. Therefore, appropriate immunization programsshould be adopted in NDV preventive treatment so as to avoid such recombination in clinical as much as possible.
It is widely accepted that the recombination rate of negative-sense RNA virus isvery low, so the recombination phenomenon in NDV has not been systematicallystudied. This problem has not been concerned in the use of NDV vaccine. Themechanism of recombination in NDV is unknown. In order to know the influences ofrecombination in NDV biological characters, especially virulence variation, andgenetic diversity of of NDV, it is necessary to establish the experimental system ofNDV homologous recombination, to provide laboratory evidence for NDVrecombination. This study use NDV LaSota, a lentogenic vaccine strain widely usedto prevent Newcastle disease, to establish a reverse-transgenic system of NDV. First,we pretreat NDV LaSota by purification and amplification. Then we design twelvepairs of primers for NDV LaSota to amplify the whole genome, and ultimately gettwelve cDNA sections which cover the whole genome. And then, we clone thesetwelve fragments into the pMD18-T vector and sequence them. We use DNAman, abioinformatic software, to analyse the nucleotide sequence of LaSota and thedistribution of restriction enzyme sites. At last, we construct a full-length cDNA cloneof NDV strain LaSota, pMC18-NDV. This work is an important foundation for thestudy of homologous recombination between NDV LaSota strain and circulatingviruses.
在我国主要广泛接种新城疫疫苗来控制该病的感染，由于减毒活疫苗使用相对方便，因此它常被用于群体免疫。我国NDV免疫密度和次数也远远超过世界水平。由于大面积的疫苗使用，新城疫得到了较好的控制，但近些年来，NDV出现了一些新的疫情，在免疫过的鸡群中即使能产生高的抗体滴度，新城疫仍时有爆发。而且大量使用的疫苗株难免流失到环境中，目前那些散落在环境中的疫苗毒株对新城疫病毒本身造成的影响并没有完全了解。
为了确定在我国广泛应用的新城疫疫苗是否对该病毒进化进程产生影响，在本研究中，我们对在我国流行的新城疫病毒进行了系统发生和重组分析，获得了一系列NDV间疫苗参与的同源重组的证据，表明疫苗可能通过同源重组影响我国新城疫病毒的进程。由于疫苗与流行株之间的重组可能导致出现疫苗接种失败的后果，因此，在临床上应当尽可能避免疫苗与野生株之间的重组。故在NDV防治过程中，应该采用合适的免疫程序避免这种重组的发生。
由于负链RNA病毒重组率很低的观点被广泛接受，因此尚没有人系统研究过NDV之间是否存在重组现象。当然，这个问题在NDV疫苗使用过程中也一直被忽略，至今没有任何有关NDV疫苗参与病毒进化方面的报道。NDV间重组的机制完全未知。通过评估病毒重组在NDV生物学性状，特别是疫苗重组后对病毒毒力变化中的影响，了解重组对NDV遗传多样性方面究竟有多大作用，以确定NDV是否能够开发为一种安全的疫苗载体。因此有必要建立NDV同源重组的实验系统，为NDV疫苗株和流行株的重组提供实验室证据。反向遗传系统的建立是研究NDV同源重组的前提，因此，本研究拟以广泛用来预防新城疫的一种弱毒疫苗的NDV LaSota株为材料，构建NDV反向遗传系统。首先我们对其进行了纯化和扩增。然后针对NDV LaSota株设计了十二对引物，然后采用RT-PCR对全基因组进行分段扩增，最终获得了涵盖全基因组的十二个cDNA片段，再将它们克隆入pMD18-T载体中并进行测序。通过生物信息学软件（DNAman）分析LaSota的核苷酸序列，找出各种限制性内切酶位点的分布，然后按照一定的先后顺序，将不同的限制性内切酶位点酶切、连接、转化，最终构建出NDV LaSota株基因组全长cDNA克隆pMC18-LaSota，本研究为NDVLaSota株与流行株之间重组的研究奠定了基础。
Newcastle disease (ND), caused by ND virus (NDV), is one of the most seriousavian contagious virus disease all over the world nowadays. It is also one of the mostserious illnesses of birds in our country. NDV has posed a great threaten to the poultryindustry. Because of the severe nature of the disease and the high mortality rates. NDis included as an Office International des Epizooties list A disease.
Many commercial Newcastle disease virus (NDV) live vaccines are widely usedfor preventing infection of chickens from Newcastle disease in China. Theseattenuated live vaccines for mass vaccination is thought to be convenient and effective.Therefore, in China, the immune density and frequency of NDV far surpassinternational level, ND was controlled well as a result of the huge of vaccination area.Howover, in recent years, NDV emerged some new epidemic situation. ND stillbreaks out in those vaccinated flocks even though high titre antibody was produced.And the vaccinated strains are hard to avoid draining into the environment, however,the evolution role of a great quantity of vaccine strains is unknown in NDV. Atpresent, it is not clear whether the scattered vaccine strains have any influence on theenvironment.
In this study, phylogenetic and recombination analyses were performed and got aseries of evidences for homologous recombination between NDV strains. Severalmosaics were proposed to be partially descended from vaccine strains. These resultsindicate that vaccines may also play roles in shaping NDV evolution via homologousrecombination. Recombination between vaccines and circulating viruses may causeimmunization failure in some cases. Therefore, appropriate immunization programsshould be adopted in NDV preventive treatment so as to avoid such recombination in clinical as much as possible.
It is widely accepted that the recombination rate of negative-sense RNA virus isvery low, so the recombination phenomenon in NDV has not been systematicallystudied. This problem has not been concerned in the use of NDV vaccine. Themechanism of recombination in NDV is unknown. In order to know the influences ofrecombination in NDV biological characters, especially virulence variation, andgenetic diversity of of NDV, it is necessary to establish the experimental system ofNDV homologous recombination, to provide laboratory evidence for NDVrecombination. This study use NDV LaSota, a lentogenic vaccine strain widely usedto prevent Newcastle disease, to establish a reverse-transgenic system of NDV. First,we pretreat NDV LaSota by purification and amplification. Then we design twelvepairs of primers for NDV LaSota to amplify the whole genome, and ultimately gettwelve cDNA sections which cover the whole genome. And then, we clone thesetwelve fragments into the pMD18-T vector and sequence them. We use DNAman, abioinformatic software, to analyse the nucleotide sequence of LaSota and thedistribution of restriction enzyme sites. At last, we construct a full-length cDNA cloneof NDV strain LaSota, pMC18-NDV. This work is an important foundation for thestudy of homologous recombination between NDV LaSota strain and circulatingviruses.
引文
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