三株传染性法氏囊病病毒A节段全长基因组结构和编码蛋白的序列分析

用长距离RT PCR方法分别克隆了浙江地区传染性法氏囊病病毒 (IBDV)细胞致弱株HZ2、弱毒疫苗株JD1和野毒株ZJ2 0 0 0的A节段基因组全长 ,三毒株的A节段均长 32 59bp ,都包含两个相互重叠的开放阅读框架和两端的 5′ ,3′ 非编码区 (NCR)。它们在核苷酸和推导的四种病毒蛋白VP2、VP3、VP4、VP5的氨基酸水平上高度同源 ,并具有位于VP2高变区的特征性氨基酸H2 53、N2 79、T2 84、R330 ,这些氨基酸是弱毒株和几个强毒株的标志。野毒株ZJ2 0 0 0的高强毒力可能与VP2高变区和VP2 VP4剪切位点附近的几个突变有关。序列比较进一步支持VP2并非是决定IBDV毒力的唯一因素。不同毒力表型毒株的两端NCR序列高度保守提示NCR可能与IBDV毒力并不直接相关。另外 ,根据VP5在十种不同表型毒株中高度保守 ,作者提出了一种VP5与病毒毒力关系的推测     

IBDVvp2基因高变区序列测定与进化分析

本实验采用来源于江苏地区的六株不同鸡传染性法氏囊病病毒(Infectious bursal disease virus,IBDV)毒株,应用RT-PCR法对vp2基因高变区进行了扩增,构建重组质粒pMD18T-vp2,测序.与有代表性的IBDV毒株VP2基因高变区序列进行比较分析,以ClustalX软件进行序列比对,得到基因序列及氨基酸序列同源性,IBDVY3、P2G、P8G、SZ、Y5和W04(6株)IBDV与D6984(荷兰)的同源性达到99.0%以上,与其它一些超强毒株(very virulentIBDV,vvIBDV)的同源性也达到了97.8%以上,并在关键氨基酸位点符合vvIBDV特征,采用Phylip3.5软件分析作出进化树,其结果从分子水平说明六个毒株均为vvIBDV,与欧洲和日本的超强毒株有较近的亲缘关系,而与美洲株的较远,从而为IBDV分子流行病学研究和疫苗的研制提供了科学依据.     

传染性法氏囊病病毒野毒株的致病性及其vp2基因比较

对4个IBDV野毒株的致病性和它们的vp2基因高变区序列同时做了比较分析.结果表明,4个IBDV毒株在致病性程度上存在较大差异.其中有一个是真正的超强毒,即GX8/99,其他几个虽然达不到真正超强毒的毒力,但也比经典的标准毒的致死性高得多.在vp2基因高变区,这4个IBDV野毒株与超强毒参考株HK46同源性很高,在DNA水平为96.8%～99.5%,在氨基酸(aa)水平为96.6%～100%.而与疫苗毒D78有很大差异,分别只有91.7%～93.6%和91.8%～93.2%.说明IBDV毒株的vp2基因高变区确实与其致病性有一定关系.特别是SD-1/97、SD-3/98、JS-30/99株之间及其与HK46在DNA和aa水平的同源性高达98.4%和98.6%以上,SD-3/99、JS-30/99株之间及其与HK46的氨基酸同源性为100%.然而,致病性特别高的GX8/99株病毒与其他3个野毒株及HK46在DNA和氨基酸水平的同源性相对较低,在DNA和aa水平的同源性只有96.8%～97.2%和96.6%～97.9%.相对于国内的流行毒株和香港超强毒参考株HK46,GX8/99株在致病性和VP2高变区都已发生了一定的变异.     

IBDVvp2基因高变区序列测定与进化分析

本实验采用来源于江苏地区的六株不同鸡传染性法氏囊病病毒(Infectious bursal disease virus,IBDV)毒株,应用RT-PCR法对vp2基因高变区进行了扩增,构建重组质粒pMD18T-vp2,测序。与有代表性的IBDV毒株VP2基因高变区序列进行比较分析,以ClustalX软件进行序列比对,得到基因序列及氨基酸序列同源性,IBDV Y3、P2G、P8G、SZ、Y5和W04(6株)IBDV与D6984(荷兰)的同源性达到99．0％以上,与其它一些超强毒株(very virulent IBDV,vvIBDV)的同源性也达到了97．8％以上,并在关键氨基酸位点符合vvIBDV特征,采用Phylip3．5软件分析作出进化树,其结果从分子水平说明六个毒株均为vvIBDV,与欧洲和日本的超强毒株有较近的亲缘关系,而与美洲株的较远,从而为IBDV分子流行病学研究和疫苗的研制提供了科学依据。     

三株牛源亚洲1型口蹄疫病毒VP1基因的克隆与序列分析

以3株国内分离的亚洲1型口蹄疫病毒(分别命名为F1、F2、F3)为研究目标,根据GenBank中注册的FMDV VP1基因的序列设计1对引物,采用RT-PCR方法成功地扩增出含有VP1全基因的片段,并测定了3个毒株VP1基因的序列.结果表明,3株亚洲1型FMDV毒株VP1基因长度均为633 bp,编码211个氨基酸.3株毒株彼此之间的核苷酸序列同源性在82.8% ～99.1%之间,推导氨基酸序列同源性在89.1% ～99.1%之间.从系统发生树看,F1株与我国香港2005年牛毒株序列同源性99.5%,属同一遗传谱系,F2株、F3株与2005年引起河北省万全县、北京市延庆县、甘肃静宁县疫情的毒株分属同一个基因群.     

引起产科新生儿腹泻暴发的轮状病毒株VP4 VP7编码基因与其毒力关系的探讨

克隆并测定了引起产科新生儿腹泻暴发的P2[6]、G4型轮状病毒(BN株)VP4的VP8片段和VP7编码基因的核苷酸序列,并据此推导出其氨基酸序列。与相应标准株和地方株(包括有毒株和无毒株)比较的结果表明,所测VP8序列与相同型别(P2[6])的标准株M37(无毒株)和ST3(无毒株)、地方株N16(无毒株)和VE7156(有毒株)之间的同源性为92.8％～98.6％,胰酶作用位点各毒株间相同;位于aa49、aa50、aa52、aa53、aa78处的氨基酸在有毒株与无毒株间(包括BN株)不同,但分别保守。VP7基因与同型(G4)标准株ST3(A亚型/无毒株)和VA70(B亚型/有毒株)、意大利地方株PV5249(A亚型/有毒株)和北京地方株CR117(有毒株)、同型猪有毒株Gott之间的同源性为91.4％～97.8％,其中与A亚型的同源性为95.5％～96.3％,而与B亚型的同源性为91.4％,提示VP7为G4A亚型,位于aa38、aa78、aa145、aa238位点的氨基酸在有毒株与无毒株之间不同,但分别保守。分析了虽为P2[6]型却反常地引起新生儿腹泻暴发毒株(BN)的VP8与VP7基因的变异情况,并对轮状病毒毒力与VP4、VP7基因变异的相互关系进行了讨论,为慎重确定轮状病毒疫苗候选毒株提供理论依据。     

传染性法氏囊病病毒五个抗原表位短肽的鉴定与序列分析

以5株传染性法氏囊病病毒(Infectious bursal disease virus,IBDV)单克隆抗体HNF1、HNF7、B34、2B1和2G8作为筛选分子,对噬菌体展示12肽库进行3轮"吸附-洗脱-扩增"淘洗,从每株单克隆抗体筛选到的噬菌斑中随机挑取12个单克隆蓝色噬菌斑,合计60个,用间接ELISA检测,A值大于1.00;用竞争抑制ELISA分析,单克隆抗体和IBDV抗原均能竞争抑制筛选12肽与固相包被单克隆抗体的反应,抑制率大于40%,表明在该12肽内含有IBDV抗原表位.选取35个单克隆噬菌斑,测定噬菌体gⅢ部分基因的核苷酸序列,确定了这5个含有不同IBDV抗原表位12肽的核苷酸和氨基酸序列.进一步将其与GenBank中IBDV基因组编码蛋白的氨基酸序列进行比较,发现2B1筛选肽有4个连续氨基酸残基Leu-Ala-Ser-Pro与IBDV基因组A片段编码多聚蛋白的第536-599氨基酸残基一致,推测2B1为线性表位;而HNF1、HNF7、B34和2G8筛选肽均没找到有3个以上连续氨基酸残基与IBDV蛋白序列相同之处,推测可能是构象依赖性表位.     

鸡传染性法氏囊病病毒基因组B片段的克隆与序列分析

鸡传染性法氏囊病病毒(IBDV)属双链双节段RNA病毒科,禽双链RNA病毒属.IBDV基因组由A、B两个RNA片段组成.VP2是最主要的IBDV结构蛋白,由A片段编码,它的变异最有可能导致IBDV血清型变异.最近的研究表明:B片段对病毒的毒力也有一定的影响.而我国对B片段的研究还未见报道.为此,我们克隆了我国甘肃地区IBDV分离强毒株Ts毒株的B片段全序列,并与报道的序列进行了比较分析.     

手足口病病例肠道病毒CVA12型的鉴定及VP1基因特征分析

目的运用分子生物学方法对长沙市1例手足口病(Hand foot and mouth Disease,HFMD)患者咽拭子标本进行未分型肠道病毒的鉴定及VP1基因特征分析。方法采用兼并引物RT-PCR扩增病毒VP1区片段,BLAST比对确定其型别;特异性引物扩增VP1区基因全长,测定序列进行分析。结果所得序列BLAST比对分析为Coxsackievirus A12(CVA12)型肠道病毒;病毒VP1区基因序列全长为888bp,编码296个氨基酸,同源性分析显示与国内外CVA12毒株核苷酸同源性介于81.9%~98.4%之间;氨基酸同源性在95.3%~100.0%之间。与CVA12美国标准株Texas-12核苷酸同源性为81.9%,氨基酸同源性为95.3%。系统进化表明,长沙市CVA12与国内毒株亲缘关系较近,而与日本、美国则亲缘关系较远。结论从1例手足口病轻症病例中鉴定出CVA12型肠道病毒,该病毒VP1区基因进化特征与国内CVA12毒株亲缘关系近。     

猪细小病毒SD-68株vp2基因的克隆及序列分析

对猪细小病毒(PPV)SD-68株vp2基因进行的克隆和序列测定表明SD-68株VP2基因全长1740bp,编码579个氨基酸残基组成的多肽;PPV SD-68株与Kresse株、SY-99株、NADL-2(5075)株、NADL-2(4973)株、US-1株的VP2基因比较,核苷酸的同源性在99%以上,氨基酸的同源性在96%以上.进化树分析表明SD-68株与kresse株的亲缘关系最近;在决定毒株组织嗜性的关键氨基酸位点上(378,383及436),SD-68株与kresse株的差异最小,据此推测SD-68株的组织嗜性与Kresse株相似,即SD-68株属皮炎型PPV;而比较弱毒株NADL-2、SD-68和强毒株kresse VP2的氨基酸差异后发现,215、378和383可能是决定PPV致病性强弱的关键位点.     

Rescue of very virulent and mosaic infectious bursal disease virus from cloned cDNA: VP2 is not the sole determinant of the very virulent phenotype

Many recent outbreaks of infectious bursal disease in commercial chicken flocks worldwide are due to the spread of very virulent strains of infectious bursal disease virus (vvIBDV). The molecular determinants for the enhanced virulence of vvIBDV compared to classical IBDV are unknown. The lack of a reverse genetics system to rescue vvIBDV from its cloned cDNA hampers the identification and study of these determinants. In this report we describe, for the first time, the rescue of vvIBDV from its cloned cDNA. Two plasmids containing a T7 promoter and either the full-length A- or B-segment cDNA of vvIBDV (D6948) were cotransfected into QM5 cells expressing T7 polymerase. The presence of vvIBDV could be detected after passage of the transfection supernatant in either primary bursa cells (in vitro) or embryonated eggs (in vivo), but not QM5 cells. Rescued vvIBDV (rD6948) appeared to have the same virulence as the parental isolate, D6948. Segment-reassorted IBDV, in which one of the two genomic segments originated from cDNA of classical attenuated IBDV CEF94 and the other from D6948, could also be rescued by using this system. Segment-reassorted virus containing the A segment of the classical attenuated isolate (CEF94) and the B segment of the very virulent isolate (D6948) is not released until 15 h after an in vitro infection. This indicates a slightly retarded replication, as the first release of CEF94 is already found at 10 h after infection. Next to segment reassortants, we generated and analyzed mosaic IBDVs (mIBDVs). In these mIBDVs we replaced the region of CEF94 encoding one of the viral proteins (pVP2, VP3, or VP4) by the corresponding region of D6948. Analysis of these mIBDV isolates showed that tropism for non-B-lymphoid cells was exclusively determined by the viral capsid protein VP2. However, the very virulent phenotype was not solely determined by this protein, since mosaic virus containing VP2 of vvIBDV induced neither morbidity nor mortality in young chickens.     

Exchange of the C-terminal part of VP3 from very virulent infectious bursal disease virus results in an attenuated virus with a unique antigenic structure

Infectious bursal disease virus (IBDV) is the major viral pathogen in the poultry industry. Live attenuated serotype 1 vaccine strains are commonly used to protect susceptible chickens during their first 6 weeks of life. Wild-type serotype 1 IBDV strains are highly pathogenic only in chickens, whereas serotype 2 strains are apathogenic in chickens and other birds. Here we describe the replacement of the genomic double-stranded RNA (dsRNA) encoding the N- or C-terminal part of VP3 of serotype 1 very virulent IBDV (vvIBDV) (isolate D6948) with the corresponding part of serotype 2 (isolate TY89) genomic dsRNA. The modified virus containing the C-terminal part of serotype 2 VP3 significantly reduced the virulence in specific-pathogen-free chickens, without affecting the distinct bursa tropism of serotype 1 IBDV strains. Furthermore, by using serotype-specific antibodies we were able to distinguish bursas infected with wild-type vvIBDV from bursas infected with the modified vvIBDV. We are currently evaluating the potential of this recombinant strain as an attenuated live vaccine that induces a unique serological response (i.e., an IBDV marker vaccine).     

Synthesis of reassortant infectious bursal disease virus in chickens injected directly with infectious clones from different virus strains

The infectious bursal disease virus (IBDV), a member of the Birnaviridae family, containing a bisegmented double-stranded RNA genome, encodes four structural viral proteins, VP1, VP2, VP3, and VP4, as well as a non-structural protein, VP5. In the present paper, the segment A from two IBDV strains,field isolate ZJ2000 and attenuated strain HZ2, were inserted into one NaeⅠ site by site-directed silent mutagenesis and subcloned into the eukaryotic expression plasmid pCI under the control of the human cytomegalovirus (hCMV) immediate early enhancer and promoter to construct the recombinant plasmids pCI-AKZJ2000 and pCI-AKHZ2, respectively. Each of the two recombinants was combined with another recombinant pCI plasmid containing the marked segment B of strain HZ2 (pCI-mB), and injected intramuscularly into nonimmunized chickens. Two chimeric IBDV strains were recovered from the chickens. Two out of eight chickens in each of two groups showed the bursal histopathological change. The reassortant virus derived from pCI-AKZJ2000/pCI-mB can infect chicken embryos and shows relatively low virulence. We have developed a novel virus reverse genetic approach for the study of IBDV. The results also form the basis for investigating the role of VP1 in viral replication and pathogenecity.     

Exchange of the VP5 of infectious bursal disease virus in a serotype I strain with that of a serotype II strain reduced the viral replication and cytotoxicity

Infectious bursal disease virus (IBDV), belonging to Avibirnavirus genus in the Birnaviridae family, consists of two segments of double-strand RNA. There are two distinct serotypes of IBDV, the pathogenic serotype I and the non-pathogenic serotype II. Comparison of the deduced amino acid sequences of a panel of VP5 genes retrieved from GenBank revealed a high identity among strains within the serotype I or serotype II group but a low identity between strains across two serotypes. In this study, we rescued two mosaic viruses, rGtGxVP5 and rGt2382VP5 by exchanging the VP5 gene of a cell culture-adapted serotype I Gt strain with its counterpart of the very virulent IBDV Gx strain, or a non-pathogenic 23/82 strain of the serotype II. In comparison to the parental strain rGt virus, the rGtGxVP5 showed the similar viral replication, cytotoxicity and the ability of inducing apoptosis; however, the other mosaic virus rGt2382VP5 had a lower titer and a reduced cytotoxicity. Although exchange of VP5 within serotype I group did not alter the viral replication and cytotoxicity of Gt strain, exchange of VP5 in the serotype I with that of a serotype II reduced the viral replication and cytotoxicity on chicken embryo fibroblast (CEF) cells. Therefore, the VP5 of serotype II may be one of the factors responsible for the distinct pathogenic features of two serotypes.     

传染性法氏囊病病毒中国强毒株A节段cDNA基因的克隆和序列分析

以来自哈尔滨传染性法氏囊病病毒（ＩＢＤＶ） 强毒株（Ｈａｒｂｉｎ 毒株,Ｈ） 的基因组ＲＮＡ为模板,用反转录聚合酶链反应（ＲＴ－ ＰＣＲ） 的方法得到了其Ａ 节段的全长ｃＤＮＡ 片段,分５＇端（１ ６５９ｂｐ） 和３＇端（１ ４４４ｂｐ） 上下两段分别克隆到ｐＧＥＭＢ○Ｒ － Ｔ 载体上,测定了其核苷酸顺序,在长为３ １０１ ｂｐ 中含有两个阅读框ＯＲＦＡ１ 和ＯＲＦＡ２ ,分别编码１ ０１２ 个氨基酸的前体蛋白（ＶＰ２ － ４ －３） 和１４５ 个氨基酸的ＶＰ５,ＯＲＦＡ１ 和ＯＲＦＡ２ 有部分的重叠。将核苷酸序列及推测出的氨基酸序列与已报道的ＩＢＤＶ 血清Ⅰ型和Ⅱ型毒株的相应序列进行了比较,结果表明：Ｈ 毒株与其它血清Ⅰ型毒株之间,在核苷酸水平上存在２５ｂｐ － ２６７ｂｐ 的差异;在氨基酸水平上存在１７ ～４０ 个氨基酸的差异。在ＶＰ２ － ４ － ３ 内比较显示,Ｈ 毒株与Ｐ２ 、Ｃｕ－ １ 之间氨基酸的差异最小为１ ．７％ ,Ｈ 毒株与ＵＫ６６１ 之间氨基酸的差异最大为３ ．９ ％ 。变异主要发生在ＶＰ２ 的可变区（２０６ － ３５０ 位氨基酸） ,在Ｈ 毒株所特有的１２ 个氨基酸当中,该区就占５ 个,代表１ ．７６ ％ 的变异。ＶＰ４、ＶＰ３ 和ＶＰ５区各有     

Apoptotic response of chicken embryonic fibroblast cells to infectious bursal disease virus infections reflects viral pathogenicity

Infectious bursal disease virus (IBDV) induces immunodeficiency in young chickens and apoptosis in chicken embryos. To understand the relation between the viral pathogenesis and the induction of cell death, chicken embryonic fibroblast (CEF) cells were infected with IBDV intermediate (im) and very virulent (vv) strains at different MOIs. The cell viability and DNA fragmentation were evaluated in infected cells. The cellular apoptotic pathway involve was investigated by determining the activities of caspase cascade. The imIBDV strain was replicated well in CEF cells and shown higher viral titers than vvIBDV. Apoptosis changes were observed only in vvIBDV-infected CEF cells at higher MOI 48 h post infection. Efflux of cytochrome c suggests that the intrinsic pathway of the apoptotic process induced by vvIBDV infection independently of virus replication. Prediction of caspase substrates cleavage sites revealed that different IBDV strains have conserved cleavage motif pattern for VP2 and VP5 viral proteins. These findings suggest the pathogenicity of IBDV strains might be involved in the induction of apoptosis in host cells.     

Molecular determinants of virulence, cell tropism, and pathogenic phenotype of infectious bursal disease virus.

Infectious bursal disease viruses (IBDVs), belonging to the family Birnaviridae, exhibit a wide range of immunosuppressive potential, pathogenicity, and virulence for chickens. The genomic segment A encodes all the structural (VP2, VP4, and VP3) and nonstructural proteins, whereas segment B encodes the viral RNA-dependent RNA polymerase (VP1). To identify the molecular determinants for the virulence, pathogenic phenotype, and cell tropism of IBDV, we prepared full-length cDNA clones of a virulent strain, Irwin Moulthrop (IM), and constructed several chimeric cDNA clones of segments A and B between the attenuated vaccine strain (D78) and the virulent IM or GLS variant strain. Using the cRNA-based reverse-genetics system developed for IBDV, we generated five chimeric viruses after transfection by electroporation procedures in Vero or chicken embryo fibroblast (CEF) cells, one of which was recovered after propagation in embryonated eggs. To evaluate the characteristics of the recovered viruses in vivo, we inoculated 3-week-old chickens with D78, IM, GLS, or chimeric viruses and analyzed their bursae for pathological lesions 3 days postinfection. Viruses in which VP4, VP4-VP3, and VP1 coding sequences of the virulent strain IM were substituted for the corresponding region in the vaccine strain failed to induce hemorrhagic lesions in the bursa. In contrast, viruses in which the VP2 coding region of the vaccine strain was replaced with the variant GLS or virulent IM strain caused rapid bursal atrophy or hemorrhagic lesions in the bursa, as seen with the variant or classical virulent strain, respectively. These results show that the virulence and pathogenic-phenotype markers of IBDV reside in VP2. Moreover, one of the chimeric viruses containing VP2 sequences of the virulent strain could not be recovered in Vero or CEF cells but was recovered in embryonated eggs, suggesting that VP2 contains the determinants for cell tropism. Similarly, one of the chimeric viruses containing the VP1 segment of the virulent strain could not be recovered in Vero cells but was recovered in CEF cells, suggesting that VP1 contains the determinants for cell-specific replication in Vero cells. By comparing the deduced amino acid sequences of the D78 and IM strains and their reactivities with monoclonal antibody 21, which binds specifically to virulent IBDV, the putative amino acids involved in virulence and cell tropism were identified. Our results indicate that residues Gln at position 253 (Gln253), Asp279, and Ala284 of VP2 are involved in the virulence, cell tropism, and pathogenic phenotype of virulent IBDV.     

VP1, the Putative RNA-Dependent RNA Polymerase of Infectious Bursal Disease Virus, Forms Complexes with the Capsid Protein VP3, Leading to Efficient Encapsidation into Virus-Like Particles

A cDNA corresponding to the coding region of VP1, the putative RNA-dependent RNA polymerase, of infectious bursal disease virus (IBDV) was cloned and inserted into the genome of a vaccinia virus inducible expression vector. The molecular mass and antigenic reactivity of VP1 expressed in mammalian cells are identical to those of its counterpart expressed in IBDV-infected cells. The results presented here demonstrate that VP1 is efficiently incorporated into IBDV virus-like particles (VLPs) produced in mammalian cells coexpressing the IBDV polyprotein and VP1. Incorporation of VP1 into VLPs requires neither the presence of IBDV RNAs nor that of the nonstructural polypeptide VP5. Immunofluorescence, confocal laser scanning microscopy, and immunoprecipitation analyses conclusively showed that VP1 forms complexes with the structural polypeptide VP3. Formation of VP1-VP3 complexes is likely to be a key step for the morphogenesis of IBDV particles.