鸡传染性法氏囊病病毒基因突变株的构建及VP5蛋白功能研究
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摘要
鸡传染性法氏囊病(Infectious Bursal Disease, IBD)是由传染性法氏囊病病毒(Infectious Bursal Disease Virus,IBDV)引起的危害雏鸡的一种急性、高度接触性、传染性疾病。IBDV感染后能够迅速到达其靶器官——法氏囊。病毒的大量复制导致产生抗体的B淋巴细胞的崩解和缺失,使鸡体产生免疫抑制,进而引起疫苗的免疫失败和增加对其它病原微生物的易感性。IBDV有2个血清型:对鸡具有致病性的血清I型和无致病性的血清II型。血清I型IBDV根据其毒力和致病性,分为经典毒株、变异株、超强毒株和弱毒疫苗株。IBDV共编码5个蛋白(VP1-VP5),其中VP5为非结构蛋白,首先由Mundt发现仅存在于IBDV感染细胞内,不是病毒的组成成分;随后研究发现,VP5为病毒复制非必需,但缺失后可以降低病毒的复制效率和致病性。显然,VP5在IBDV的感染过程中具有重要作用,以往对VP5的功能研究取得了一定进展,但其在IBDV感染中的功能及其作用机理有待于进一步研究。
本研究首先以弱毒IBDV(rGt株)和适应CEF生长保留一定毒力的人工修饰病毒(rGx-F9VP2株)为亲本病毒,利用体外定点突变技术,突变VP5基因起始密码子,通过多重PCR在基因组两端分别引入锤头状核酶序列(HamRz)和丁肝病毒核酶序列(HdvRz)。将带有核酶序列的IBDV基因组A节段插入真核表达载体pCAGGs的β肌动蛋白启动子下游,构建了缺失表达VP5蛋白的IBDV感染性克隆pCGtA△VP5HRT和pCGx-F9VP2A△VP5HRT,将A节段感染性克隆分别与相应B节段感染性克隆pCmGtBHRT或pCmGxBHRT共转染DF I细胞。RT-PCR和IFA均显示获得重组病毒,缺失表达VP5蛋白,将其分别命名为rGt△VP5和rGx-F9VP2△VP5。体内外实验分析结果表明:缺失表达VP5蛋白降低病毒的复制效率和对细胞的毒性,以及对SPF鸡的致病性和免疫抑制作用。
为了比较强弱毒VP5蛋白功能的差异,利用融合PCR技术,分别以弱毒IBDV(rGt株)和人工修饰强毒(rGx-F9VP2株)为亲本毒,进行强弱毒VP5基因替换,构建A节段嵌合感染性克隆pCGtAGxVP5HRT和pCGx-F9VP2AGtVP5HRT,分别与pCmGtBHRT和pCmGxBHRT共转染DF I细胞。通过RT-PCR和IFA检测,获得重组嵌合病毒,将其分别命名为rGtGxVP5和rGxGtVP5。进行体内外实验分析,rGtGxVP5与rGt具有相同的复制效率和最高病毒滴度,感染后诱导细胞凋亡的能力和对细胞活力影响相同;rGxGtVP5和rGx-F9VP2感染SPF鸡,法氏囊BBIX和HBLS差异不显著,诱导产生抗体水平相同。结果表明,替换强毒VP5基因不影响弱毒株在CEF细胞的生物学特性,替换弱毒VP5不能降低强毒体内致病性,强弱毒VP5蛋白在复制和致病性方面具有相同功能,不是强弱毒株复制特性和致病性差异的决定因素。
根据VP5蛋白预测结构,利用体外定点突变技术,分段缺失表达弱毒IBDV(rGt株)VP5蛋白,并构建A节段感染性克隆pCGtA△VP5N70HRT,pCGtA△VP5N90HRT和pCGtA△VP5C51HRT,并分别与pCmGtBHRT共转染DF I细胞。RT-PCR和IFA均显示获得重组缺失病毒,将其分别命名为rGt△VP5N70,rGt△VP5N90和rGt△VP5C51。进行体外复制能力分析,rGt△VP5C51具有与亲本病毒rGt相同的复制效率,而rGt△VP5N70和rGt△VP5N90相对于rGt降低了在CEF细胞上的复制能力。结果表明,VP5蛋白影响病毒复制效率的功能区位于N端1-94个aa内,C端51个aa不影响IBDV体外复制。
为了研究VP5在IBDV感染中的分子作用机制,通过rGt和rGt△VP5感染CEF细胞诱导产生干扰素(Interferon, IFN)差异比较,发现,rGt△VP5诱导细胞产生IFN的能力高于rGt,推测VP5蛋白能够抑制宿主细胞产生IFN;并构建表达VP5蛋白的真核重组质粒pCAGGVP5,分别与在ISRE、NF-κB或Mx启动子调控下表达萤火虫荧光素酶的报告基因质粒共转染,用dsRNA刺激后,分析萤火虫荧光素酶的表达。结果,体外表达VP5蛋白抑制ISRE、NF-κB和Mx启动子的激活;表明,VP5蛋白能够抑制细胞产生IFN,推测VP5蛋白通过抑制IFN的产生影响病毒的复制和致病性。
由于VP5在IBDV感染中特殊的作用,VP5基因被认为是标记疫苗研究的重要候选基因。本研究分别对嵌合血清II型IBDV(23/82株)VP5基因的重组病毒rGt2382VP5和缺失表达VP5蛋白人工修饰强毒rGx-F9VP2△VP5进行体内外特性分析。首先,利用大肠杆菌表达的IBDV 23/82株VP5蛋白为抗原,免疫Balb/C小鼠,融合筛选获得一株分泌特异性识别血清II型IBDV23/82株VP5蛋白的杂交瘤细胞株(5D3);并利用融合PCR技术,以血清I型弱毒IBDV(rGt株)为骨架,构建替换血清II型IBDV(23/82株)VP5基因的A节段嵌合感染性克隆pCGtA2382VP5HRT,与pCmGtBHRT共转染DF I细胞。通过RT-PCR和IFA检测,获得重组嵌合病毒,将其命名为rGt2382VP5,该病毒能被抗血清II型IBDV-VP5型特异性McAb(5D3)识别。体外生物学特性分析发现,rGt2382VP5复制能力和对CEF细胞毒性低于亲本病毒rGt,表明,替换23/82株VP5基因降低了弱毒株的复制效率和细胞毒性,VP5基因是两个血清型IBDV致病性差异的因素之一。由于缺失表达VP5蛋白的强毒株rGx-F9VP2△VP5能够诱使感染SPF鸡产生中和抗体,为了验证其保护效力,分别将rGx-F9VP2△VP5与其亲本病毒rGx-F9VP2接种3周龄SPF鸡,4周后攻击vvIBDV(Gx株),统计保护效率。结果表明:两株病毒接种后,均能够保护SPF鸡抵抗vvIBDV攻击,rGx-F9VP2△VP5具有作为标记疫苗的潜力。
本研究构建一系列缺失和嵌合病毒,并进行体内外生物学特性研究,证实VP5影响病毒在体内外的复制效率和致病性,强弱毒VP5功能不存在差异,其影响病毒复制效率的功能区位于N端1-94个aa内;VP5通过抑制宿主细胞产生IFN而影响病毒的复制和致病性;VP5是血清I、II型IBDV致病性差异的决定因素之一;rGx-F9VP2△VP5能够诱使鸡体产生中和抗体,保护其抵抗vvIBDV的攻击,具有作为疫苗株的潜力。本研究进一步阐述了VP5的功能和IBDV感染的分子致病机理,为基于VP5基因的标记疫苗的研发提供理论基础和物质基础。
Infectious bursal disease virus (IBDV) is the causative agent of a highly contagious disease in young chickens known as infectious bursal disease (IBD). IBD causes significant losses in the poultry industries due to its high mortality and immunodepression in young birds by the destruction of the developing B lymphocytes in the bursa of Fabricus (BF). There are two distinct serotypes of IBDV. Viruses of the serotype I group are pathogenic to chickens, sub-divided into the classical virus, the antigenic variant virus, the very virulent virus and attenuated virus according to their virulence. Whereas the serotype II, mostly isolated from turkey, are avirulent to chickens. VP5 was first discovered only in the IBDV-infected cells but not packed into virion particles. VP5 was not essential for the viral replication, but played a key role in the viral infection of the disease. Although a few progresses of VP5 had been made because of its importance, concrete functions of VP5 and molecular pathogenic mechanisms in viral infection need be further elucidated.
To study the function of VP5 in vivo and in vitro, two mutant viruses deficient in expressing VP5 protein were generated using attenuated virus (rGt strain) and cell-cultured virulent virus (rGx-F9VP2 strain) as parental viruses by RNA polymerase II system. Site-directed mutagenesis technique was used to alter start codon of VP5 gene. The 5’NCR and 3’NCR of A segments were fused by hammerhead ribozyme (HamRz) and hepatitis delta ribozyme (HdvRz) cDNA sequences respectively, which were cloned into eukaryotic expression vector, pCAGGs, to get infectious clones of A segment, pCGtA△VP5HRT and pCGx-F9VP2A△VP5HRT. The mutant viruses were rescued by co-transfection pCGtA△VP5HRT and pCmGtBHRT or pCGx-F9VP2A△VP5HRT and pCmGxBHRT into DF I cells and identified by IFA and RT-PCR. In comparison to the characteristics of parental viruses in vitro, the mutant viruses demonstrated lower viral titers and lower cytopathogenicity. To understand the role of VP5 in the pathogenesis in vivo, animal experiments were conducted. Results suggested that chickens inoculated with rGx-F9VP2△VP5 showed minimal bursal lesion with more normal bursal weight to body weight index (BBIX) and lower histopathologic bursal lesion scores (HBLS) than those inoculated with rGx-F9VP2. The titers of AIV hemagglutination inhibition (HI) antibodies of the rGx-F9VP2△VP5 inoculated group were higher than those of the rGx-F9VP2 inoculated group, indicating that deficiency of VP5 decreased the immunosupression of rGx-F9VP2△VP5 in chickens. All data indicates that VP5 plays an important role in viral replication and pathogenesis both in vitro and in vivo.
To study the functional differences between VP5 of vvIBDV and that of attenuanted virus, two mosaic viruses, rGtGxVP5 and rGxGtVP5, were constructed and rescued by exchanging the VP5 gene between virulent virus (rGx-F9VP2) and attenuated virus (rGt). In comparison to the characteristics of rGt in vitro, rGtGxVP5 demonstrated the same viral replication efficiency and cytopathogenicity. The results of animal experiments showed that both mosaic virus rGxGtVP5 and parental virus rGx-F9VP2 had the same BBIX and HBLS and induced the identical antibody level. All results indicate that there is no difference between the functions of VP5 of vvIBDV and that of attenuanted virus in replication and pathogenicity.
VP5 is highly basic, cyteine-rich and it is a class II transmembrance protein with an N-terminal cytoplasmic tail and a C-terminal extracellular domain. Three mutant infectious clones sectorial lacking expression of VP5 according to its transmembrance structure by site-directed mutagenesis technique. Mutant viruses, rGt△VP5C51, rGt△VP5N70 and rGt△VP5N90, were rescued by co-transfecting with pCmGtBHRT into DF I cells, respectively, and identified by RT-PCR and IFA. In comparison to the characteristics of parental virus rGt in vitro, the rGt△VP5C51 virus demonstrated the same replication efficiency and final viral titer, however, rGt△VP5N70 and rGt△VP5N90 showed lower replication efficiency and lower final viral titers. Results indicate that 1-94 aa of N terminal of VP5 affects the replication efficiency in IBDV infection in vitro.
It was found that more interferon (IFN) was produced by the cell infected with rGt△VP5 than cells infected with rGt did. This result implied that VP5 might inhibit the production of IFN by host cells. The recombinant eukaryotic expression vector, pCAGGVP5, was constructed by inserting the ORF of VP5 gene into pCAGGs vector. The expression levels of firefly luciferases were analyzed by co-transfecting pCAGGVP5 with the reporter gene plasmids, pISRE-luc, pNF-κB-luc and pMx-luc, which were regulated by ISRE promoter, NF-κB promoter and Mx promoter, respectively. The results showed that the stimulations of ISRE promoter, NF-κB promoter and Mx promoter were all inhibited by the expression of VP5 in vitro. The results indicated that VP5 inhibited the production of IFN in host cells, which might be one of molecular mechanisms of viral replication and pathogenicity affected by VP5.
VP5 gene was considered as an potential marker for developing marker vaccine, due to its speciality in viral infecton. The characteristicses of rGt2382VP5 and rGx-F9VP2△VP5 was analyzed in vitro and in vivo. Fortunately, an McAb (5D3) was got which only reacted with VP5 of serotype II IBDV strains. The mosaic virus, rGt2382VP5, were generated by exchanging the VP5 of rGt with that of serotype II IBDV (23/82 strain), which was discriminated by the anti-IBDV-VP5 McAb (5D3). The analysis of mosaic virus characteristics showed that exchangement of VP5 gene decreased the mosaic virus replication efficiency and cytopathogenicity. This indicates that VP5 might contribute to the distinct pathogenesis of serotype I and serotype II strains. The VP5-deficient mutant virus rGx-F9VP2△VP5 and its parental virus rGx-F9VP2 were used to inoculate 3-week-old SPF chickens. The IBDV antibody was examined and the inoculated SPF chickens were challenged with vvIBDV (Gx strain) 4 weeks post-infection. All chichens inoculated with rGx-F9VP2△VP5 or rGx-F9VP2 were protected.
In this study, a series of deficient and mosaic viruses were rescued and analyzed in vitro and in vivo. The results indicate that VP5 plays an important role in viral infection, which affects IBDV replication and pathogenicity. The function of VP5 between virulent virus and attenuated virus show no difference. The 1-94 aa of VP5 protein N-terminal affects replication efficiency of IBDV in vitro. VP5 influences the viral replication and pathogenicity by inhibiting the IFN producd by host cells. VP5 is also one of the determinants of distinct pathogenesis between two serotypes IBDV. VP5-dificient mutant virus, rGx-F9VP2△VP5, induces protection of chickens against challenge with vvIBDV, which is a potential marker vaccine candidate. This study further elucidates the functions of VP5 and the molecular pathogenesis mechanisms for virulence and immunity of IBDV, which provides good rationales and materials to research and develop marker vaccine base on VP5 gene.
本研究首先以弱毒IBDV(rGt株)和适应CEF生长保留一定毒力的人工修饰病毒(rGx-F9VP2株)为亲本病毒,利用体外定点突变技术,突变VP5基因起始密码子,通过多重PCR在基因组两端分别引入锤头状核酶序列(HamRz)和丁肝病毒核酶序列(HdvRz)。将带有核酶序列的IBDV基因组A节段插入真核表达载体pCAGGs的β肌动蛋白启动子下游,构建了缺失表达VP5蛋白的IBDV感染性克隆pCGtA△VP5HRT和pCGx-F9VP2A△VP5HRT,将A节段感染性克隆分别与相应B节段感染性克隆pCmGtBHRT或pCmGxBHRT共转染DF I细胞。RT-PCR和IFA均显示获得重组病毒,缺失表达VP5蛋白,将其分别命名为rGt△VP5和rGx-F9VP2△VP5。体内外实验分析结果表明:缺失表达VP5蛋白降低病毒的复制效率和对细胞的毒性,以及对SPF鸡的致病性和免疫抑制作用。
为了比较强弱毒VP5蛋白功能的差异,利用融合PCR技术,分别以弱毒IBDV(rGt株)和人工修饰强毒(rGx-F9VP2株)为亲本毒,进行强弱毒VP5基因替换,构建A节段嵌合感染性克隆pCGtAGxVP5HRT和pCGx-F9VP2AGtVP5HRT,分别与pCmGtBHRT和pCmGxBHRT共转染DF I细胞。通过RT-PCR和IFA检测,获得重组嵌合病毒,将其分别命名为rGtGxVP5和rGxGtVP5。进行体内外实验分析,rGtGxVP5与rGt具有相同的复制效率和最高病毒滴度,感染后诱导细胞凋亡的能力和对细胞活力影响相同;rGxGtVP5和rGx-F9VP2感染SPF鸡,法氏囊BBIX和HBLS差异不显著,诱导产生抗体水平相同。结果表明,替换强毒VP5基因不影响弱毒株在CEF细胞的生物学特性,替换弱毒VP5不能降低强毒体内致病性,强弱毒VP5蛋白在复制和致病性方面具有相同功能,不是强弱毒株复制特性和致病性差异的决定因素。
根据VP5蛋白预测结构,利用体外定点突变技术,分段缺失表达弱毒IBDV(rGt株)VP5蛋白,并构建A节段感染性克隆pCGtA△VP5N70HRT,pCGtA△VP5N90HRT和pCGtA△VP5C51HRT,并分别与pCmGtBHRT共转染DF I细胞。RT-PCR和IFA均显示获得重组缺失病毒,将其分别命名为rGt△VP5N70,rGt△VP5N90和rGt△VP5C51。进行体外复制能力分析,rGt△VP5C51具有与亲本病毒rGt相同的复制效率,而rGt△VP5N70和rGt△VP5N90相对于rGt降低了在CEF细胞上的复制能力。结果表明,VP5蛋白影响病毒复制效率的功能区位于N端1-94个aa内,C端51个aa不影响IBDV体外复制。
为了研究VP5在IBDV感染中的分子作用机制,通过rGt和rGt△VP5感染CEF细胞诱导产生干扰素(Interferon, IFN)差异比较,发现,rGt△VP5诱导细胞产生IFN的能力高于rGt,推测VP5蛋白能够抑制宿主细胞产生IFN;并构建表达VP5蛋白的真核重组质粒pCAGGVP5,分别与在ISRE、NF-κB或Mx启动子调控下表达萤火虫荧光素酶的报告基因质粒共转染,用dsRNA刺激后,分析萤火虫荧光素酶的表达。结果,体外表达VP5蛋白抑制ISRE、NF-κB和Mx启动子的激活;表明,VP5蛋白能够抑制细胞产生IFN,推测VP5蛋白通过抑制IFN的产生影响病毒的复制和致病性。
由于VP5在IBDV感染中特殊的作用,VP5基因被认为是标记疫苗研究的重要候选基因。本研究分别对嵌合血清II型IBDV(23/82株)VP5基因的重组病毒rGt2382VP5和缺失表达VP5蛋白人工修饰强毒rGx-F9VP2△VP5进行体内外特性分析。首先,利用大肠杆菌表达的IBDV 23/82株VP5蛋白为抗原,免疫Balb/C小鼠,融合筛选获得一株分泌特异性识别血清II型IBDV23/82株VP5蛋白的杂交瘤细胞株(5D3);并利用融合PCR技术,以血清I型弱毒IBDV(rGt株)为骨架,构建替换血清II型IBDV(23/82株)VP5基因的A节段嵌合感染性克隆pCGtA2382VP5HRT,与pCmGtBHRT共转染DF I细胞。通过RT-PCR和IFA检测,获得重组嵌合病毒,将其命名为rGt2382VP5,该病毒能被抗血清II型IBDV-VP5型特异性McAb(5D3)识别。体外生物学特性分析发现,rGt2382VP5复制能力和对CEF细胞毒性低于亲本病毒rGt,表明,替换23/82株VP5基因降低了弱毒株的复制效率和细胞毒性,VP5基因是两个血清型IBDV致病性差异的因素之一。由于缺失表达VP5蛋白的强毒株rGx-F9VP2△VP5能够诱使感染SPF鸡产生中和抗体,为了验证其保护效力,分别将rGx-F9VP2△VP5与其亲本病毒rGx-F9VP2接种3周龄SPF鸡,4周后攻击vvIBDV(Gx株),统计保护效率。结果表明:两株病毒接种后,均能够保护SPF鸡抵抗vvIBDV攻击,rGx-F9VP2△VP5具有作为标记疫苗的潜力。
本研究构建一系列缺失和嵌合病毒,并进行体内外生物学特性研究,证实VP5影响病毒在体内外的复制效率和致病性,强弱毒VP5功能不存在差异,其影响病毒复制效率的功能区位于N端1-94个aa内;VP5通过抑制宿主细胞产生IFN而影响病毒的复制和致病性;VP5是血清I、II型IBDV致病性差异的决定因素之一;rGx-F9VP2△VP5能够诱使鸡体产生中和抗体,保护其抵抗vvIBDV的攻击,具有作为疫苗株的潜力。本研究进一步阐述了VP5的功能和IBDV感染的分子致病机理,为基于VP5基因的标记疫苗的研发提供理论基础和物质基础。
Infectious bursal disease virus (IBDV) is the causative agent of a highly contagious disease in young chickens known as infectious bursal disease (IBD). IBD causes significant losses in the poultry industries due to its high mortality and immunodepression in young birds by the destruction of the developing B lymphocytes in the bursa of Fabricus (BF). There are two distinct serotypes of IBDV. Viruses of the serotype I group are pathogenic to chickens, sub-divided into the classical virus, the antigenic variant virus, the very virulent virus and attenuated virus according to their virulence. Whereas the serotype II, mostly isolated from turkey, are avirulent to chickens. VP5 was first discovered only in the IBDV-infected cells but not packed into virion particles. VP5 was not essential for the viral replication, but played a key role in the viral infection of the disease. Although a few progresses of VP5 had been made because of its importance, concrete functions of VP5 and molecular pathogenic mechanisms in viral infection need be further elucidated.
To study the function of VP5 in vivo and in vitro, two mutant viruses deficient in expressing VP5 protein were generated using attenuated virus (rGt strain) and cell-cultured virulent virus (rGx-F9VP2 strain) as parental viruses by RNA polymerase II system. Site-directed mutagenesis technique was used to alter start codon of VP5 gene. The 5’NCR and 3’NCR of A segments were fused by hammerhead ribozyme (HamRz) and hepatitis delta ribozyme (HdvRz) cDNA sequences respectively, which were cloned into eukaryotic expression vector, pCAGGs, to get infectious clones of A segment, pCGtA△VP5HRT and pCGx-F9VP2A△VP5HRT. The mutant viruses were rescued by co-transfection pCGtA△VP5HRT and pCmGtBHRT or pCGx-F9VP2A△VP5HRT and pCmGxBHRT into DF I cells and identified by IFA and RT-PCR. In comparison to the characteristics of parental viruses in vitro, the mutant viruses demonstrated lower viral titers and lower cytopathogenicity. To understand the role of VP5 in the pathogenesis in vivo, animal experiments were conducted. Results suggested that chickens inoculated with rGx-F9VP2△VP5 showed minimal bursal lesion with more normal bursal weight to body weight index (BBIX) and lower histopathologic bursal lesion scores (HBLS) than those inoculated with rGx-F9VP2. The titers of AIV hemagglutination inhibition (HI) antibodies of the rGx-F9VP2△VP5 inoculated group were higher than those of the rGx-F9VP2 inoculated group, indicating that deficiency of VP5 decreased the immunosupression of rGx-F9VP2△VP5 in chickens. All data indicates that VP5 plays an important role in viral replication and pathogenesis both in vitro and in vivo.
To study the functional differences between VP5 of vvIBDV and that of attenuanted virus, two mosaic viruses, rGtGxVP5 and rGxGtVP5, were constructed and rescued by exchanging the VP5 gene between virulent virus (rGx-F9VP2) and attenuated virus (rGt). In comparison to the characteristics of rGt in vitro, rGtGxVP5 demonstrated the same viral replication efficiency and cytopathogenicity. The results of animal experiments showed that both mosaic virus rGxGtVP5 and parental virus rGx-F9VP2 had the same BBIX and HBLS and induced the identical antibody level. All results indicate that there is no difference between the functions of VP5 of vvIBDV and that of attenuanted virus in replication and pathogenicity.
VP5 is highly basic, cyteine-rich and it is a class II transmembrance protein with an N-terminal cytoplasmic tail and a C-terminal extracellular domain. Three mutant infectious clones sectorial lacking expression of VP5 according to its transmembrance structure by site-directed mutagenesis technique. Mutant viruses, rGt△VP5C51, rGt△VP5N70 and rGt△VP5N90, were rescued by co-transfecting with pCmGtBHRT into DF I cells, respectively, and identified by RT-PCR and IFA. In comparison to the characteristics of parental virus rGt in vitro, the rGt△VP5C51 virus demonstrated the same replication efficiency and final viral titer, however, rGt△VP5N70 and rGt△VP5N90 showed lower replication efficiency and lower final viral titers. Results indicate that 1-94 aa of N terminal of VP5 affects the replication efficiency in IBDV infection in vitro.
It was found that more interferon (IFN) was produced by the cell infected with rGt△VP5 than cells infected with rGt did. This result implied that VP5 might inhibit the production of IFN by host cells. The recombinant eukaryotic expression vector, pCAGGVP5, was constructed by inserting the ORF of VP5 gene into pCAGGs vector. The expression levels of firefly luciferases were analyzed by co-transfecting pCAGGVP5 with the reporter gene plasmids, pISRE-luc, pNF-κB-luc and pMx-luc, which were regulated by ISRE promoter, NF-κB promoter and Mx promoter, respectively. The results showed that the stimulations of ISRE promoter, NF-κB promoter and Mx promoter were all inhibited by the expression of VP5 in vitro. The results indicated that VP5 inhibited the production of IFN in host cells, which might be one of molecular mechanisms of viral replication and pathogenicity affected by VP5.
VP5 gene was considered as an potential marker for developing marker vaccine, due to its speciality in viral infecton. The characteristicses of rGt2382VP5 and rGx-F9VP2△VP5 was analyzed in vitro and in vivo. Fortunately, an McAb (5D3) was got which only reacted with VP5 of serotype II IBDV strains. The mosaic virus, rGt2382VP5, were generated by exchanging the VP5 of rGt with that of serotype II IBDV (23/82 strain), which was discriminated by the anti-IBDV-VP5 McAb (5D3). The analysis of mosaic virus characteristics showed that exchangement of VP5 gene decreased the mosaic virus replication efficiency and cytopathogenicity. This indicates that VP5 might contribute to the distinct pathogenesis of serotype I and serotype II strains. The VP5-deficient mutant virus rGx-F9VP2△VP5 and its parental virus rGx-F9VP2 were used to inoculate 3-week-old SPF chickens. The IBDV antibody was examined and the inoculated SPF chickens were challenged with vvIBDV (Gx strain) 4 weeks post-infection. All chichens inoculated with rGx-F9VP2△VP5 or rGx-F9VP2 were protected.
In this study, a series of deficient and mosaic viruses were rescued and analyzed in vitro and in vivo. The results indicate that VP5 plays an important role in viral infection, which affects IBDV replication and pathogenicity. The function of VP5 between virulent virus and attenuated virus show no difference. The 1-94 aa of VP5 protein N-terminal affects replication efficiency of IBDV in vitro. VP5 influences the viral replication and pathogenicity by inhibiting the IFN producd by host cells. VP5 is also one of the determinants of distinct pathogenesis between two serotypes IBDV. VP5-dificient mutant virus, rGx-F9VP2△VP5, induces protection of chickens against challenge with vvIBDV, which is a potential marker vaccine candidate. This study further elucidates the functions of VP5 and the molecular pathogenesis mechanisms for virulence and immunity of IBDV, which provides good rationales and materials to research and develop marker vaccine base on VP5 gene.
引文
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