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     

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     

The 2.6-Angstrom structure of infectious bursal disease virus-derived T=1 particles reveals new stabilizing elements of the virus capsid

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a double-stranded RNA virus that causes a highly contagious disease in young chickens leading to significant economic losses in the poultry industry. The VP2 protein, the only structural component of the IBDV icosahedral capsid, spontaneously assembles into T=1 subviral particles (SVP) when individually expressed as a chimeric gene. We have determined the crystal structure of the T=1 SVP to 2.60 A resolution. Our results show that the 20 trimeric VP2 clusters forming the T=1 shell are further stabilized by calcium ions located at the threefold icosahedral axes. The structure also reveals a new unexpected domain swapping that mediates interactions between adjacent trimers: a short helical segment located close to the end of the long C-terminal arm of VP2 is projected toward the threefold axis of a neighboring VP2 trimer, leading to a complex network of interactions that increases the stability of the T=1 particles. Analysis of crystal packing shows that the exposed capsid residues, His253 and Thr284, determinants of IBDV virulence and the adaptation of the virus to grow in cell culture, are involved in particle-particle interactions.     

传染性法氏囊病毒的抗原及分子特征

用鸡胚成纤维细胞对来自野外的 5 个传染性法氏囊病毒株 (IBDV-JD1 、 JD2 、 NB 、 HZ1 、 HZ2) 进行分离,测定理化特性、致病性,同时进行血清亚型测定及 A 片段基因组的克隆分析 . 试验所用 5 个法氏囊组织悬液在鸡胚成纤维细胞盲传 2~14 代后适应细胞并产生细胞病变 . 细胞适应的 IBDV 毒株的理化和形态特征与经典传染性法氏囊病毒株一致 . 除 IBDV-HZ1 、 HZ2 属经典 IBDV 血清型外, IBDV-JD1 、 JD2 和 NB 毒株分属不同的血清亚型 . 人工感染实验结果显示,分离的 IBDV 毒株产生与野外病例相似的临床症状和病变,出现法氏囊滤泡髓质的淋巴细胞变性、坏死和消失 . 基因组序列分析显示, IBDV-NB 毒株 A 片段由 3 264 个核苷酸组成,编码由 145 个氨基酸残基组成的 VP5 和由 1 012 个氨基酸残基组成的多聚蛋白 . 与来自 GenBank 的 IBDV Ⅰ型毒株比较, NB 毒株 A 片段编码的多聚蛋白与 JD1 毒株的同源性最高,达 99.5% , VP2 与 JD1 、 CEF94 、 D78 的同源性为 99.8% , VP3 与 JD1 的同源性为 99.2% , VP4 与 JD1 的同源性为 100% , VP5 与 JD1 , HZ2 , P2 , CEF94 , CT , Cu-1 和 D78 毒株的同源性为 99.3%. NB 毒株 VP2 蛋白的第 253 、 280 、 284 位氨基酸残基与 IBDV 变异毒株和经典毒株一致,但不同于 IBDV 超强毒株 . 这些结果暗示 IBDV 的抗原表位是构象依赖性表位, IBDV 血清亚型的形成与 IBDV 弱毒疫苗病毒株密切相关 .     

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.     

Mutations of Residues 249 and 256 in VP2 Are Involved in the Replication and Virulence of Infectious Bursal Disease Virus

Infectious bursal disease virus (IBDV) is a pathogen of worldwide significance to the poultry industry. Although the P_DE and P_FG domains of the capsid protein VP2 contribute significantly to virulence and fitness, the detailed molecular basis for the pathogenicity of IBDV is still not fully understood. Because residues 253 and 284 of VP2 are not the sole determinants of virulence, we hypothesized that other residues involved in virulence and fitness might exist in the P_DE and P_FG domains of VP2. To test this, five amino acid changes selected by sequence comparison of the P_DE and P_FG domains of VP2 were introduced individually using a reverse genetics system into the virulent strain (rGx-F9VP2). Then reverse mutations of the selected residues 249 and 256 were introduced individually into the attenuated strain (rGt). Seven modified viruses were generated and evaluated in vitro (CEF cells) and in vivo (SPF chicken). For residue 249, Q249R could elevate in vitro and reduce in vivo the replication of rGx-F9VP2 while R249Q could reduce in vitro and elevate in vivo the replication of rGt; meanwhile Q249R reduced the virulence of rGx-F9VP2 while R249Q increased the virulence of rGt, which indicated that residue 249 significantly contributed to the replication and virulence of IBDV. For residue 256, I256V could elevate in vitro and reduce in vivo the replication of rGx-F9VP2 while V256I could reduce in vitro but didn’t change in vivo the replication of rGt; although V256I didn’t increase the virulence of rGt, I256V obviously reduced the virulence of virulent IBDV. The present results demonstrate for the first time, to different extent, residues 249 and 256 of VP2 are involved in the replication efficiency and virulence of IBDV; this is not only beneficial to further understanding of pathogenic mechanism but also to the design of newly tailored vaccines against IBDV.     

A single mutation in the PBC loop of VP2 is involved in the in vitro replication of infectious bursal disease virus

To test whether amino acid mutations in the P_BC and P_HI loops of VP2 are involved in the replication and virulence of infectious bursal disease virus (IBDV), a pair of viruses, namely the moderately virulent IBDV (rGx-F9VP2) and the attenuated strain (rGt), were used. Residue mutations A222P (P_BC) and S330R (P_HI), selected by sequence comparison, were introduced individually into rGx-F9VP2 by using a reverse genetics system. In addition, the reverse mutation of either P222A or R330S was introduced into rGt. The four modified viruses were then rescued and evaluated in vitro (CEF cells) and in vivo (SPF chickens). Results showed that A222P elevated the replication efficiency of rGx-F9VP2 while P222A reduced that of rGt in CEF cells. A mutation at residue 330 did not alter IBDV replication. In addition, animal experiments showed that a single mutation at either residue 222 or 330 did not significantly influence the virulence of IBDV. In conclusion, residue 222 in P_BC of VP2 is involved in the replication efficiency of IBDV in vitro but does not affect its virulence in vivo, further facilitating our understanding of the gene-function of IBDV.     

A single amino acid in VP2 is critical for the attachment of infectious bursal disease subviral particles to immobilized metal ions and DF-1 cells

VP2 protein is the primary host-protective immunogen of infectious bursal disease virus (IBDV). His249 and His253 are two surface histidine residues in IBDV subviral particles (SVP), which is formed by twenty VP2 trimers when the VP2 protein of a local isolate is expressed. Here, a systemic study was performed to investigate His249 or/and His253 on self-assembly, cell attachment and immunogenicity of SVP. Point-mutagenesis of either or both histidine residues to alanine did not affect self-assembly of the SVP, but the SVP lost its Ni-NTA binding affinity when the His253 was mutated. Indirect immunofluorescence assays and inhibitory experiments also showed that His253 is essential for SVP to attach onto the DF-1 cells and to inhibit IBDV infection of DF-1 cells. Finally, enzyme-linked immunosorbent assays and chicken protection assays demonstrated that SVP with a mutation of His253 to alanine induced comparable neutralizing antibody titers in chickens as the wild-type SVP did. It was concluded that VP2's His253, a site not significant for the overall immunogenicity induced by SVP, is crucial for the binding affinity of SVP to Ni-NTA and the attachment of an IBDV host cell line. This is the first paper to decipher the role of His253 played in receptor interaction and immunogenicity.     

Synonymous codon usage of the VP2 gene of a very virulent infectious bursal disease virus isolate serial passaged in chicken embryos

Three very virulent infectious bursal disease virus (vvIBDV) strains were isolated from a single farm and shown to be phylogenetically related to the vvIBDV isolate UK661. In this study, a comparative analysis of the synonymous codon usage in the hypervariable region of theVP2 (vVP2) gene of the vvIBDV strains was done on viruses serially passaged in chicken embryos. Sequencing demonstrated that codons change during the serial passage in the vVP2 gene of the viruses. Nine codon mutations resulted in amino acids changes. The amino acid changes were I256V, I296L 6in isolate XA1989, A222P, I242V, Q253H, I256V in isolate XA1998, and Q253H, I256V, I296L in isolate XA2004. Three of the nine amino acid changes occurred at residue 256. The codons of the amino acids A232, N233, I234, T269, T283 and H338 changed to the synonymous codons in XA1989 after the 16th passage, in XA1998 after the 24th passage and in XA2004 22nd passage viruses. These mutations change the key amino acid residues Q253H and I256V in the domains which are essential for its virulence, and the synonymous codons were observed compared to classical virulent IBDV. The results indicated that the codon changes during the serial passage comprised of synonymous codon usage in the vVP2 gene of IBDV, and this synonymous codon bias was correlated with pathotypes. The extent of synonymous codon usage bias in the IBDV-vVP2 gene maybe influence the gene expression level and secondary structure of protein as well as hydrophobicity, therefore the results provide useful perspectives for evolution and understanding of the pathogenesis of IBDV.     

Identification of a single amino acid residue in the capsid protein VP1 of coxsackievirus B4 that determines the virulent phenotype.

To identify the molecular determinants of virulence for coxsackievirus B4, a panel of recombinant, chimeric viruses were constructed from cDNA clones of a virulent virus, CB4-V, and a nonvirulent virus, CB4-P. Initial studies mapped a major determinant of virulence to the 5' end of the viral genome, which contained the 5' untranslated and the P1 regions (A. Ramsingh, A. Hixson, B. Duceman, and J. Slack, J. Virol. 64:3078-3081, 1990). To determine whether the 5' untranslated region contributed to the virulent phenotype, a chimeric virus (vCB403) containing this region of the virulent virus on an avirulent background was tested in mice. The vCB403 construct displayed a phenotype similar to that of CB4-P, suggesting that the 5' untranslated region did not significantly contribute to virulence. Analysis of the sequence data of the P1 regions of both CB4-V and CB4-P revealed five mutations that resulted in amino acid substitutions in VP1, VP2, and VP4 (A. Ramsingh, H. Araki, S. Bryant, and A. Hixson, Virus Res. 23:281-292, 1992). Analysis of individual mutations in both VP1 and VP2 revealed that a single residue (Thr-129 of VP1) determined the virulent phenotype.     

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 and structural bases for the antigenicity of VP2 of infectious bursal disease virus

Infectious bursal disease virus (IBDV), a member of the family Birnaviridae, is responsible for a highly contagious and economically important disease causing immunosuppression in chickens. IBDV variants isolated in the United States exhibit antigenic drift affecting neutralizing epitopes in the capsid protein VP2. To understand antigenic determinants of the virus, we have used a reverse-genetics approach to introduce selected amino acid changes-individually or in combination-into the VP2 gene of the classical IBDV strain D78. We thus generated a total of 42 mutants with changes in 8 amino acids selected by sequence comparison and their locations on loops P(BC) and P(HI) at the tip of the VP2 spikes, as shown by the crystal structure of the virion. The antibody reactivities of the mutants generated were assessed using a panel of five monoclonal antibodies (MAbs). Our results show that a few amino acids of the projecting domain of VP2 control the reactivity pattern. Indeed, the binding of four out of the five MAbs analyzed here is affected by mutations in these loops. Furthermore, their importance is highlighted by the fact that some of the engineered mutants display identical reactivity patterns but have different growth phenotypes. Finally, this analysis shows that a new field strain isolated from a chicken flock in Belgium (Bel-IBDV) represents an IBDV variant with a hitherto unobserved antigenic profile, involving one change (P222S) in the P(BC) loop. Overall, our data provide important new insights for devising efficient vaccines that protect against circulating IBDV strains.     

Identification and mapping of neutralizing epitopes of human parvovirus B19 by using human antibodies.

We identified and mapped the regions responsible for neutralization in the human parvovirus B19 structural protein by using region-specific human antibodies derived from seropositive blood donors. The region-specific antibodies were purified by using affinity columns coupled with synthetic peptides of the hydrophilic regions including the beta-turn structure deduced by the predicted secondary structure of VP2. Fifteen highly specific antibodies against the synthetic peptides were obtained. Ten of them were able to precipitate the radiolabeled virus. Six of them proved to be able to protect the colony-forming unit erythroid cells in human bone marrow cell cultures from injury by the virus. The sequences recognized by the six neutralizing antibodies were sites corresponding to amino acids 253 to 272, 309 to 330, 325 to 346, 359 to 382, 449 to 468, and 491 to 515 from the amino-terminal portion of VP2. These observations suggest that the neutralizing epitopes were distributed in the region from amino acid 253 in the amino-terminal portion of VP2 to the carboxyl terminus of VP2.     

Infectious Bursal Disease Virus VP5 Polypeptide: A Phosphoinositide-Binding Protein Required for Efficient Cell-to-Cell Virus Dissemination

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a major avian pathogen responsible for an immunosuppressive disease affecting juvenile chickens. The IBDV genome is formed by two dsRNA segments. The largest one harbors two partially overlapping open reading frames encoding a non-structural polypeptide, known as VP5, and a large polyprotein, respectively. VP5 is non-essential for virus replication. However, it plays a major role in IBDV pathogenesis. VP5 accumulates at the plasma membrane (PM) of IBDV-infected cells. We have analyzed the mechanism underlying the VP5 PM targeting. Updated topological prediction algorithm servers fail to identify a transmembrane domain within the VP5 sequence. However, the VP5 polycationic C-terminal region, harboring three closely spaced patches formed by two or three consecutive basic amino acid residues (lysine or arginine), might account for its PM tropism. We have found that mutations, either C-terminal VP5 deletions or replacement of basic amino acids by alanine residues, that reduce the electropositive charge of the VP5 C-terminus abolish PM targeting. Lipid overlay assays performed with an affinity-purified Flag-tagged VP5 (FVP5) protein version show that this polypeptide binds several phosphoinositides (PIP), exhibiting a clear preference for monophosphate species. Experiments performed with FVP5 mutant proteins lacking the polycationic domain demonstrate that this region is essential for PIP binding. Data gathered with IBDV mutants expressing C-terminal deleted VP5 polypeptides generated by reverse genetics demonstrate that the VP5-PIP binding domain is required both for its PM targeting in infected cells, and for efficient virus dissemination. Data presented here lead us to hypothesize that IBDV might use a non-lytic VP5-dependent cell-to-cell spreading mechanism.     

传染性法氏囊病毒VP2 蛋白的分子生物学研究进展

本文综述了IBDV的保护性抗原蛋白VP2与病毒形态、毒力变异、抗原变异间的关系及在诱导宿主细胞凋亡中的作用。同时也探讨了其在新型疫苗开发中的应用等方面的研究进展。     

Determinants of virulence of classical swine fever virus strain Brescia

Two related classical swine fever virus (CSFV) strain Brescia clones were isolated from blood samples from an infected pig. Virus C1.1.1 is a cell-adapted avirulent variant, whereas CoBrB is a virulent variant. Sequence analysis revealed 29 nucleic acid mutations in C1.1.1, resulting in 9 amino acid substitutions compared to the sequence of CoBrB (476)R. Using reverse genetics, parts of the genomes of these viruses, which contain differences that lead to amino acid changes, were exchanged. Animal experiments with chimeric viruses derived from C1.1.1 and CoBrB (476)R showed that a combination of amino acid changes in the structural and nonstructural regions reduced the virulence of CSFV in pigs. Moreover, the presence of a Leu at position 710 in structural envelope protein E2 seemed to be an important factor in the virulence of the virus. Changing the Leu at position 710 in the CoBrB (476)S variant into a His residue did not affect virulence. However, the (710)His in the C1.1.1/CoBrB virus, together with adaptive mutations (276)R, (476)R, and (477)I in E(rns), resulted in reduced virulence in pigs. These results indicated that mutations in E(rns) and E2 alone do not determine virulence in pigs. The results of in vitro experiments suggested that a high affinity for heparan sulfate of C1.1.1 E(rns) may reduce the spread of the C1.1.1/CoBrB virus in pigs and together with the altered surface structure of E2 caused by the (710)L-->H mutation may result in a less efficient infection of specific target cells in pigs. Both these features contributed to the attenuation of the C1.1.1/CoBrB virus in vivo.     

Effects of amino acid substitutions in the VP2 B-C loop on antigenicity and pathogenicity of serotype Asia1 foot-and-mouth disease virus

ABSTRACT: BACKGROUND: Foot-and-mouth disease virus (FMDV) exhibits a high degree of antigenic variability. Studies of the antigenic diversity and determination of amino acid changes involved in this diversity are important to the design of broadly protective new vaccines. Although extensive studies have been carried out to explore the molecular basis of the antigenic variation of serotype O and serotype A FMDV, there are few reports on Asia1 serotype FMDV. METHODS: Two serotype Asia1 viruses, Asia1/YS/CHA/05 and Asia1/1/YZ/CHA/06, which show differential reactivity to the neutralizing monoclonal antibody (nMAb) 1B4, were subjected to sequence comparison. Then a reverse genetics system was used to generate mutant versions of Asia1/YS/CHA/05 followed by comparative analysis of the antigenicity, growth property and pathogenicity in the suckling mice. RESULTS: Three amino acid differences were observed when the structural protein coding sequences of Asia1/1/YZ/CHA/06 were compared to that of Asia1/YS/CHA/05. Site-directed mutagenesis and Immunofluorescence analysis showed that the amino acid substitution in the B-C loop of the VP2 protein at position 72 is responsible for the antigenic difference between the two Asia1 FMDV strains. Furthermore, alignment of the amino acid sequences of VP2 proteins from serotype Asia1 FMDV strains deposited in GenBank revealed that most of the serotype Asia1 FMDV strains contain an Asn residue at position 72 of VP2. Therefore, we constructed a mutant virus carrying an Asp-to-Asn substitution at position 72 and named it rD72N. Our analysis shows that the Asp-to-Asn substitution inhibited the ability of the rD72N virus to react with the MAb 1B4 in immunofluorescence and neutralization assays. In addition, this substitution decreased the growth rate of the virus in BHK-21 cells and decreased the virulence of the virus in suckling mice compared with the Asia1/YS/CHA/05 parental strain. CONCLUSIONS: These results suggest that variations in domains other than the hyper variable VP1 G-H loop (amino acid 140 to 160) are relevant to the antigenic diversity of FMDV. In addition, amino acid substitutions in the VP2 influenced replicative ability and virulence of the virus. Thus, special consideration should be given to the VP2 protein in research on structure-function relationships and in the development of an FMDV 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.