鸡传染性法氏囊病病毒VP1蛋白影响病毒复制及致病性机制的研究
摘要
鸡传染性法氏囊病(IBD)是一种危害养禽业的急性、高度接触性传染病。鸡传染性法氏囊病的病原为鸡传染性法氏囊病病毒(IBDV),属于双RNA病毒科禽双RNA病毒属的成员。IBDV主要侵害3到6周龄雏鸡,破坏中枢免疫系统法氏囊中的B淋巴细胞,导致免疫抑制。IBDV基因组由A和B两条双链组成。其中A节段含有两个开放阅读框,编码VP2、VP3、VP4和VP5蛋白。B节段含有一个开放阅读框,编码VP1蛋白,具有RNA依赖的RNA聚合酶活性。
反向遗传技术的成功建立很大程度上推进了IBDV的基础研究工作。众多学者对于IBDV的A节段做了相关的研究,发现IBDV的VP2蛋白对病毒的毒力、细胞嗜性有重要作用,VP3和VP5蛋白也对病毒的复制有一定影响。但是,病毒的毒力是由多个因素共同决定的,VP1对于病毒的复制和毒力也有影响。目前,关于VP1的研究仍相对滞后,VP1对病毒复制及毒力的影响尚未见比较深入的报道。
本研究旨在研究VP1与IBDV复制及毒力的关系,并研究其影响病毒复制和毒力的分子机制。本研究首先测定并分析了七株IBDV的B节段全基因组序列,并将全基因组序列上传到GenBank中。通过遗传进化分析结合致病性实验,所测定的七株病毒均属于IBDV超强毒株。根据B节段进化分析,可将B节段划分为三个群,本研究测定毒株的B节段属于第Ⅱ群和第Ⅲ群。通过序列比对,强毒在VP1蛋白上有八个保守的氨基酸位点,分别是4V、61I、145T、287A、508K、511S、646S以及687P。在B节段的5’UTR部分,55T和63A在强毒中是保守的,在3’UTR部分,2786C是保守的。
为进一步研究VP1上的保守位点对于病毒的生物学特性是否有影响,本研究根据VP1的三维结构,构建4、61和145位点突变的N端结构域突变株,287、508、511和646位点突变的中心活性结构域突变株和C端结构域的687突变株。首先以弱毒Gt的A节段和超强毒HLJ-4的B节段为骨架构建5株针对B节段不同突变的病毒,研究其在CEF细胞上的复制动力学特性,发现将强毒HLJ-4的B节段上的4、61和145位点突变为弱毒的相应位点,可使病毒在CEF细胞上的复制能力增强。将强毒HLJ-4的B节段上的687位点P突变为弱毒的相应位点S,也可使病毒在CEF细胞上的复制能力增强。进一步拯救出4、61、145单位点突变的病毒,研究其在CEF细胞上的复制动力学特性,发现将强毒的HLJ-4的B节段上的4位点V突变为弱毒的相应位点I,也可使病毒在CEF细胞上的复制能力增强。可见V4I以及P687S突变可提高病毒在CEF细胞的复制能力。
为研究影响体外复制的位点是不是会进一步对病毒的毒力造成影响,本研究首先构建了IBDV超强毒株的反向遗传系统。以HLJ-4的A节段和HLJ-4的B节段为骨架拯救出IBDV超强毒亲本毒株。首先将A、B节段的质粒在DF1细胞上转染,转染48 h后,将转染的细胞液反复冻融3次,从尾根部接种3周龄SPF鸡的法氏囊,在接种后3天,拯救各组不同病毒的SPF鸡均出现死亡,剖检鸡法氏囊出现病变,经测序和电镜分析证实IBDV超强毒已成功拯救。将拯救的B节段上不同突变的IBDV超强毒进行SPF鸡攻毒实验,发现将强毒的4位点由V突变为I,可使病毒的致病性增强,将强毒的687位点由P突变为S,可使病毒的致病性增强。
VP1具有RNA依赖的RNA聚合酶活性,研究不同位点突变影响病毒复制及致病性的分子机制。构建了基于B节段的minigenome系统,并成功用于检测IBDV的聚合酶活性。通过聚合酶活性检测,发现将IBDV强毒的4位点V突变为弱毒的相应位点I,可使病毒的聚合酶活性升高,而将弱毒的4位点I突变为强毒的相应位点V,病毒的聚合酶活性降低。由此可见,4位点影响病毒复制及致病性的分子机制之一是由于其影响了病毒的聚合酶活性。
Infectious bursal disease is a highly contagious and immunosuppressive disease of great economic importance to the poultry industry. The causative agent of IBD is infectious bursal disease virus, which is a member of the family Birnaviridae. B lymphoid cells in the bursae of Fabricius(BF)are the target cells of IBDV, and between 3 and 6 weeks after hatching, when the BF reaches maximum development, chickens are highly susceptible to the virus. Infection results in lymphoid depletion and the final destruction of the bursae. IBDV genome consists of two segments A an B. Genome segment A of IBDV encodes VP2, VP3, VP4 and VP5, while genome segment B encodes VP1, which is the RNA dependent RNA polymerase.
The establishment of reverse genetics has greatly enhanced the research towards IBDV. Many work had done to identify molecular determinants on segment A. It had been found that VP2 is the major determinant of virulence. However, it was not the sole determinant. VP3 and VP5 protein are involved in virus replication. Many factors contribute to the virulence of IBDV, and segment B is involved in pathogenicity of IBDV. Until now, studies towards segment B are rather sparse. Little had done on molecular determinants on segment B.
This study aimed to investigate the effect of conserved amino acids on VP1 on virus replication and pathogenesis, and further to investigate the molecular mechanism. First, we sequenced and analyzed the whole segment B sequence of seven IBDV isolates, and submit the sequence to Genbank. Through phylogenetic analysis and pathogenicity study, seven isolates belong to vvIBDV, and segment B of the seven isolates can be clustered into two clusters branchⅡand branchⅢ. There are eight conserved amino acids on VP1 protein, 4V, 61I, 145T, 287A, 508K, 511S, 646S and 687P. In 5’UTR, 55T and 63A are conserved, and in 3’UTR, 2786C are conserved.
In order to identify if these conserved sites are critical to virus replication and pathogenesis, a series of mutation viruses were rescued based on Gt A segment and HLJ-4 B segment. Mutations were introduced into B segment according to the three dimensional structure of VP1 protein. 4, 61 and 145 sites were mutated to be the N terminus mutation; 287, 508, 511 and 646 sites were mutated to be the central domain mutation, and 687 site was mutated to be the C terminus mutation. One step growth curve showed 687 mutation on segment B of HLJ-4 from P to S can elevate virus replication ability in CEF cells. Further, single site mutation of 4, 61 and 145 were introduced into segment B. One step growth curve showed hat 4 mutation on segment B of HLJ-4 from V to I can elevate virus replication ability in CEF cells.
To investigate if these mutations can affect virus pathogenicity, a series of vvIBDV were rescued based on both segments of HLJ-4. Animal experiment showed that 4 mutation on segment B of HLJ-4 from V to I can elevate pathogenesis, and 687 mutation on segment B of HLJ-4 from P to S can elevate virus pathogenesis.
A segment B-driven minigenome system was established to evaluate the polymerase activity. The result showed that 4 site mutation can influence the polymerase activity. 4 site mutation from V to I can elevate polymerase activity, and 4 site mutation from I to V can reduce polymerase activity.
反向遗传技术的成功建立很大程度上推进了IBDV的基础研究工作。众多学者对于IBDV的A节段做了相关的研究,发现IBDV的VP2蛋白对病毒的毒力、细胞嗜性有重要作用,VP3和VP5蛋白也对病毒的复制有一定影响。但是,病毒的毒力是由多个因素共同决定的,VP1对于病毒的复制和毒力也有影响。目前,关于VP1的研究仍相对滞后,VP1对病毒复制及毒力的影响尚未见比较深入的报道。
本研究旨在研究VP1与IBDV复制及毒力的关系,并研究其影响病毒复制和毒力的分子机制。本研究首先测定并分析了七株IBDV的B节段全基因组序列,并将全基因组序列上传到GenBank中。通过遗传进化分析结合致病性实验,所测定的七株病毒均属于IBDV超强毒株。根据B节段进化分析,可将B节段划分为三个群,本研究测定毒株的B节段属于第Ⅱ群和第Ⅲ群。通过序列比对,强毒在VP1蛋白上有八个保守的氨基酸位点,分别是4V、61I、145T、287A、508K、511S、646S以及687P。在B节段的5’UTR部分,55T和63A在强毒中是保守的,在3’UTR部分,2786C是保守的。
为进一步研究VP1上的保守位点对于病毒的生物学特性是否有影响,本研究根据VP1的三维结构,构建4、61和145位点突变的N端结构域突变株,287、508、511和646位点突变的中心活性结构域突变株和C端结构域的687突变株。首先以弱毒Gt的A节段和超强毒HLJ-4的B节段为骨架构建5株针对B节段不同突变的病毒,研究其在CEF细胞上的复制动力学特性,发现将强毒HLJ-4的B节段上的4、61和145位点突变为弱毒的相应位点,可使病毒在CEF细胞上的复制能力增强。将强毒HLJ-4的B节段上的687位点P突变为弱毒的相应位点S,也可使病毒在CEF细胞上的复制能力增强。进一步拯救出4、61、145单位点突变的病毒,研究其在CEF细胞上的复制动力学特性,发现将强毒的HLJ-4的B节段上的4位点V突变为弱毒的相应位点I,也可使病毒在CEF细胞上的复制能力增强。可见V4I以及P687S突变可提高病毒在CEF细胞的复制能力。
为研究影响体外复制的位点是不是会进一步对病毒的毒力造成影响,本研究首先构建了IBDV超强毒株的反向遗传系统。以HLJ-4的A节段和HLJ-4的B节段为骨架拯救出IBDV超强毒亲本毒株。首先将A、B节段的质粒在DF1细胞上转染,转染48 h后,将转染的细胞液反复冻融3次,从尾根部接种3周龄SPF鸡的法氏囊,在接种后3天,拯救各组不同病毒的SPF鸡均出现死亡,剖检鸡法氏囊出现病变,经测序和电镜分析证实IBDV超强毒已成功拯救。将拯救的B节段上不同突变的IBDV超强毒进行SPF鸡攻毒实验,发现将强毒的4位点由V突变为I,可使病毒的致病性增强,将强毒的687位点由P突变为S,可使病毒的致病性增强。
VP1具有RNA依赖的RNA聚合酶活性,研究不同位点突变影响病毒复制及致病性的分子机制。构建了基于B节段的minigenome系统,并成功用于检测IBDV的聚合酶活性。通过聚合酶活性检测,发现将IBDV强毒的4位点V突变为弱毒的相应位点I,可使病毒的聚合酶活性升高,而将弱毒的4位点I突变为强毒的相应位点V,病毒的聚合酶活性降低。由此可见,4位点影响病毒复制及致病性的分子机制之一是由于其影响了病毒的聚合酶活性。
Infectious bursal disease is a highly contagious and immunosuppressive disease of great economic importance to the poultry industry. The causative agent of IBD is infectious bursal disease virus, which is a member of the family Birnaviridae. B lymphoid cells in the bursae of Fabricius(BF)are the target cells of IBDV, and between 3 and 6 weeks after hatching, when the BF reaches maximum development, chickens are highly susceptible to the virus. Infection results in lymphoid depletion and the final destruction of the bursae. IBDV genome consists of two segments A an B. Genome segment A of IBDV encodes VP2, VP3, VP4 and VP5, while genome segment B encodes VP1, which is the RNA dependent RNA polymerase.
The establishment of reverse genetics has greatly enhanced the research towards IBDV. Many work had done to identify molecular determinants on segment A. It had been found that VP2 is the major determinant of virulence. However, it was not the sole determinant. VP3 and VP5 protein are involved in virus replication. Many factors contribute to the virulence of IBDV, and segment B is involved in pathogenicity of IBDV. Until now, studies towards segment B are rather sparse. Little had done on molecular determinants on segment B.
This study aimed to investigate the effect of conserved amino acids on VP1 on virus replication and pathogenesis, and further to investigate the molecular mechanism. First, we sequenced and analyzed the whole segment B sequence of seven IBDV isolates, and submit the sequence to Genbank. Through phylogenetic analysis and pathogenicity study, seven isolates belong to vvIBDV, and segment B of the seven isolates can be clustered into two clusters branchⅡand branchⅢ. There are eight conserved amino acids on VP1 protein, 4V, 61I, 145T, 287A, 508K, 511S, 646S and 687P. In 5’UTR, 55T and 63A are conserved, and in 3’UTR, 2786C are conserved.
In order to identify if these conserved sites are critical to virus replication and pathogenesis, a series of mutation viruses were rescued based on Gt A segment and HLJ-4 B segment. Mutations were introduced into B segment according to the three dimensional structure of VP1 protein. 4, 61 and 145 sites were mutated to be the N terminus mutation; 287, 508, 511 and 646 sites were mutated to be the central domain mutation, and 687 site was mutated to be the C terminus mutation. One step growth curve showed 687 mutation on segment B of HLJ-4 from P to S can elevate virus replication ability in CEF cells. Further, single site mutation of 4, 61 and 145 were introduced into segment B. One step growth curve showed hat 4 mutation on segment B of HLJ-4 from V to I can elevate virus replication ability in CEF cells.
To investigate if these mutations can affect virus pathogenicity, a series of vvIBDV were rescued based on both segments of HLJ-4. Animal experiment showed that 4 mutation on segment B of HLJ-4 from V to I can elevate pathogenesis, and 687 mutation on segment B of HLJ-4 from P to S can elevate virus pathogenesis.
A segment B-driven minigenome system was established to evaluate the polymerase activity. The result showed that 4 site mutation can influence the polymerase activity. 4 site mutation from V to I can elevate polymerase activity, and 4 site mutation from I to V can reduce polymerase activity.
引文
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