鸡传染性法氏囊病病毒Gt株VP5基因缺失株感染性克隆的构建

传染性法氏囊病病毒 (IBDV)是双链,双节段RNA病毒,其基因组由A、B两个节段组成,编码结构蛋白VP1-VP4和非结构蛋白VP5。【目的】利用反向遗传操作构建拯救VP5基因缺失重组IBDV。【方法】利用体外定点突变技术,缺失IBDV Gt株VP5基因,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基因组插入载体pCAGG的b肌动蛋白启动子下游,构建了IBDV感染性克隆pCAGGmGtA △VP5HRT,将该感染性克隆与pCAGGmGtBHRT共转染DFⅠ细胞。【结果】RT-PCR和间接免疫荧光均显示获得重组病毒,将其命名为rmGtA △VP5。IBDV VP5基因缺失感染性克隆的成功构建为从分子水平上深入研究vp5基因功能奠定了基础。     

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.     

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％o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％～9712％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。     

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.     

乙型脑炎病毒减毒中间株SA14—12—1—7基因组全序列的测定

本研究通过对乙型脑炎活疫苗减毒过程中间株SA14 12 1 7株进行全序列测定和分析 ,进一步了解乙脑活疫苗减毒及其稳定性的分子机制。根据已发表的SA14 14 2株及SA14 株的序列 ,设计 6对重叠引物 ,涵括整个乙脑病毒的基因组 ,通过RT PCR扩增出SA14 12 1 7株的各cDNA片段 ,分别克隆到pGEM T载体 ,转化至TG1受体菌中 ,挑取阳性克隆进行鉴定后测序。结果表明SA14 12 1 7株基因组全序列长 10 976个核苷酸 ,从 96到 10 394为一个长开放读码框 ,编码 3432个氨基酸。与野毒株SA14 和疫苗株SA14 14 2的核苷酸序列和氨基酸序列相比 ,同源性均在 99%以上 ,突变位点分散于各个区域 ,E区有 5个位点与疫苗株一致而与野毒株不同 ,3个位点与野毒株一致而与疫苗株不同 ,推测与其容易产生回复突变、恢复毒力有关。此外 ,NS3、NS5和 3′NTR的几个位点可能与病毒毒力稳定性相关。综上所述 ,乙脑病毒减毒中间株的基因组全序列基本类似于已发表的序列 ,若干突变位点影响病毒的弱毒性及毒力的稳定性。全序列的测定对于研究疫苗株的减毒机理具有重要意义     

我国禽脑脊髓炎病毒分离株全基因组的测定

测定了我国禽脑脊髓炎病毒(avian encephalomyelitis virus,AEV)分离株L2Z株的全基因组核苷酸序列.该病毒株的3′和5′非编码区核苷酸序列用3′和5′RACE(cDNA末端快速扩增)法获得.基因组全长为7 059个核苷酸残基,包括494个核苷酸残基的5′非编码区、6 402个核苷酸残基的开放阅读框和136个核苷酸残基的3′非编码区及poly(A)尾巴.与已发表的AEV疫苗株1 143的基因组序列比较发现,它们之间核苷酸和氨基酸的同源性分别为98%和97.6%.结构蛋白(VP1～VP4)中,主要宿主保护性免疫原蛋白VP1氨基酸之间差异较小.与小RNA病毒科其它病毒属相比,在非结构蛋白3D中,预测的8个RNA依赖性RNA聚合酶主要结构域中的4个高度保守.从而进一步确认了AEV的分子特性.     

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。     

不同致病型马立克氏病病毒1．8kb基因家族序列比较

为了分析鸡马立克氏病病毒(Marek′sdiseasevirus,MDV)的致病性与其1.8kb基因家族的关系,本研究比较了四个不同致病型12个毒株的该基因家族的同源性关系,即弱毒疫苗株、强毒株、超强毒株、特超强毒株和中国野毒株。结果表明:不同毒株间1.8kb基因家族的上游调控序列的同源性在1.8kb基因家族转录方向上大于92.5%,序列间有缺失突变,其中大多数突变发生在弱毒株上。CVI988在TATAbox和上游SP1位点间丢失小片段“5′CTCGG3′”。1.8kb基因家族中存在132bp串连重复序列,CVI988包含37个132bp串连重复序列拷贝,强毒株、超强毒株、特超强毒株通常只包含2个串连重复序列。但是已知的中国疫苗毒株814株也只有2个重复序列,表明重复序列的拷贝数不是引起病毒毒力变化的主要原因。各毒株的1.8kb基因家族同源性在97%以上。其中未剪切的1.69kbcDNA中有两个阅读框,分别编码63和64个氨基酸组成的多肽。不同毒株的这二个多肽都很保守,虽然也有一些氨基酸变异,但这些变异与病毒的致病型或地域分布没有明显的关系。     

三株牛源亚洲1型口蹄疫病毒聊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633bp,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,雅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。     

2007―2008年肠道病毒71型北京地区分离株的VP4基因序列分析

为了解2007 ― 2008 年北京地区流行的肠道病毒71 型( EV71) 是否存在基因序列变异及其与病毒毒力的关系, 我们选择2007 年分离的3 株EV71( 其中1 株分离自重症手足口病患儿的咽拭子标本, 其余2 株分离自普通手足口病患儿咽拭子标本) 和2008 年分离的5 株EV71( 其中3 株分离自重症手足口病患儿的咽拭子或鼻拭子标本, 2 株分离自普通手足口病患儿的疱疹液标本) , 提取基因组RNA, 经反转录-聚合酶链反应( RT-PCR) 扩增得到VP4 基因片段, 并进行核苷酸序列测定, 使用生物信息软件与GenBank 中的EV71 VP4 基因进行序列及病毒型别分析。结果表明, 所测得的8 株EV71 VP4 基因全长均为207 bp, 编码69 个氨基酸, 理论相对分子质量( Mr) 为7 ×10³。8 株EV71 病毒VP4 基因的核苷酸同源性在94% ～100% , 与GenBank 中其他EV71 病毒株VP4 的核苷酸同源性为82% ～100% , 与阜阳、深圳和台湾等地区流行的EV71 VP4 的核苷酸同源性比其他地区高。除了与印度报道的VP4 编码的氨基酸在第7 和54 位不同外( 印度株: 7 位蛋氨酸, 54位苏氨酸; 其余株7 位苏氨酸,54 位丙氨酸) , 这8 株EV71 VP4 编码的氨基酸序列之间以及与其他EV71 VP4编码的氨基酸同源性均为100%。8 株EV71 病毒VP4 与文献报道的3 株重症感染病毒株VP4 ( BrCr、MS 和NCKU9822) 核苷酸有较大差别, 而8 株病毒株中从重症感染( BJ97、BJ110B、BJ110Y 和BJ4243) 与轻症感染( BJ25、BJ47、BJ65 和BJ67) 分离到的毒株之间VP4 基因序列未见明显改变, 只有几个核苷酸存在差别。VP4 核苷酸序列的进化树分析表明, 这8 株EV71 均属于C4 亚型, 显示2007 ― 2008 年北京地区流行的EV71的VP4 基因相当保守, 分离自伴有神经系统感染的重症手足口病和普通手足口病患儿的EV71 的VP4 基因之间在核苷酸水平未出现同样的变异。结果提示, 近2 年来北京地区所流行的EV71 属C4 亚型。     

Genetic reassortment of infectious bursal disease virus in nature

Infectious bursal disease virus (IBDV), a double-stranded RNA virus, is a member of the Birnaviridae family. Four pathotypes of IBDV, attenuated, virulent, antigenic variant, and very virulent (vvIBDV), have been identified. We isolated and characterized the genomic reassortant IBDV strain ZJ2000 from severe field outbreaks in commercial flocks. Full-length genomic sequence analysis showed that ZJ2000 is a natural genetic reassortant virus with segments A and B derived from attenuated and very virulent strains of IBDV, respectively. ZJ2000 exhibited delayed replication kinetics as compared to attenuated strains. However, ZJ2000 was pathogenic to specific pathogen free (SPF) chickens and chicken embryos. Similar to a standard virulent IBDV strain, ZJ2000 caused 26.7% mortality, 100% morbidity, and severe bursal lesions at both gross and histopathological levels. Taken together, our data provide direct evidence for genetic reassortment of IBDV in nature, which may play an important role in the evolution, virulence, and host range of IBDV. Our data also suggest that VP2 is not the sole determinant of IBDV virulence, and that the RNA-dependent RNA polymerase protein, VP1, may play an important role in IBDV virulence. The discovery of reassortant viruses in nature suggests an additional risk of using live IBDV vaccines, which could act as genetic donors for genome reassortment.     

Molecular determinants of infectious pancreatic necrosis virus virulence and cell culture adaptation

Infectious pancreatic necrosis viruses (IPNVs) exhibit a wide range of virulence in salmonid species. In previous studies, we have shown that the amino acid residues at positions 217 and 221 in VP2 are implicated in virulence. To pinpoint the molecular determinants of virulence in IPNV, we generated recombinant IPNV strains using the cRNA-based reverse-genetics system. In two virulent strains, residues at positions 217 and 247 were replaced by the corresponding amino acids of a low-virulence strain. The growth characteristics of the recovered chimeric strains in cell culture were similar to the low-virulence strains, and these viruses induced significantly lower mortality in Atlantic salmon fry than the parent strains did in in vivo challenge studies. Furthermore, the virulent strain was serially passaged in CHSE-214 cells 10 times and was completely characterized by nucleotide sequencing. Deduced amino acid sequence analyses revealed a single amino acid substitution of Ala to Thr at position 221 in VP2 of this virus, which became highly attenuated and induced 15% cumulative mortality in Atlantic salmon fry, compared to 68% mortality induced by the virulent parent strain. The attenuated strain grows to higher titers in CHSE cells and can be distinguished antigenically from the wild-type virus by use of a monoclonal antibody. However, the virulent strain passaged 10 times in RTG-2 cells was stable, and it retained its antigenicity and virulence. Our results indicate that residues Thr at position 217 (Thr217) and Ala221 of VP2 are the major determinants of virulence in IPNV of the Sp serotype. Highly virulent isolates possess residues Thr217 and Ala221; moderate- to low-virulence strains have Pro217 and Ala221; and strains containing Thr221 are almost avirulent, irrespective of the residue at position 217.     

Sequence of the fourth gene of human rotaviruses recovered from asymptomatic or symptomatic infections.

The complete nucleotide sequence of the fourth gene of symptomatic (Wa, DS-1, P, and VA70) and asymptomatic (M37, 1076, McN13, and ST3) rotaviruses of serotype 1, 2, 3, or 4 was determined by the dideoxy chain termination method. In each strain, the fourth gene, which encodes the outer capsid protein VP3, is 2,359 base pairs in length and has 5'- and 3'-noncoding regions of 9 and 25 nucleotides, respectively. The gene has a single long open reading frame of 2,325 base pairs that is capable of coding for a protein of 775 amino acids. A total of 14 N-terminal and 12 C-terminal amino acids are completely conserved or almost completely conserved, respectively, among nine human rotavirus VP3 genes that have been sequenced. In addition, there is conservation of arginine at the two trypsin cleavage sites as well as conservation of clusters of amino acids in different regions of the two VP3 cleavage products, VP8 and VP5. Three distinct forms of VP3 were identified among the nine human rotavirus strains analyzed. Three symptomatic rotaviruses (serotypes 1, 3, and 4) possess highly related VP3 genes (92.2 to 97% nucleotide identity). Two symptomatic serotype 2 rotaviruses possess VP3 genes which are even more closely related to each other (98.6% nucleotide identity) and only moderately related to the aforementioned VP3 genes of serotypes 1, 3, and 4 (87.4 to 88.2% nucleotide identity). The four asymptomatic rotaviruses, which constitute the third group, possess highly related VP3 genes (95.5 to 97.5% nucleotide identity) which are distinct from those of the virulent rotaviruses (73 to 74.8% nucleotide identity). At 91 positions in the protein sequence of VP3, an amino acid is conserved among the asymptomatic rotaviruses, while a different amino acid is conserved among the symptomatic rotaviruses. Notably, five regions are conserved among the symptomatic rotaviruses, while a different set of sequences are conserved among the asymptomatic rotaviruses. It is possible that some or all of these regions of sequence dimorphism may be responsible for the difference in virulence of these two groups of human rotaviruses. There are 13 regions in the VP3 protein sequence which exhibit the greatest variability; the majority of these variable regions are observed between amino acids 106 to 192. These regions may represent potential antigenic sites related to heterotypic rotavirus neutralization.     

Genetic variation of the VP1 gene of the virulent duck hepatitis A virus type 1 (DHAV-1) isolates in Shandong province of China

To investigate the relationship of the variation of virulence and the external capsid proteins of the pandemic duck hepatitis A virus type 1 (DHAV-1) isolates, the virulence, cross neutralization assays and the complete sequence of the virion protein 1 (VP1) gene of nine virulent DHAV-1 strains, which were isolated from infected ducklings with clinical symptoms in Shandong province of China in 2007–2008, were tested. The fifth generation duck embryo allantoic liquids of the 9 isolates were tested on 12-day-old duck embryos and on 7-day-old ducklings for the median embryonal lethal doses (ELD₅₀s) and the median lethal doses (LD₅₀s), respectively. The results showed that the ELD₅₀s of embryonic duck eggs of the 9 DHAV-1 isolates were between 1.9 × 10⁶/mL to 1.44 × 10⁷/mL, while the LD₅₀s were 2.39 × 10⁵/mL to 6.15 × 10⁶/mL. Cross-neutralization tests revealed that the 9 DHAV-1 isolates were completely neutralized by the standard serum and the hyperimmune sera against the 9 DHAV-1 isolates, respectively. Compared with other virulent, moderate virulent, attenuated vaccine and mild strains, the VP1 genes of the 9 strains shared 89.8%–99.7% similarity at the nucleotide level and 92.4%–99.6% at amino acid level with other DHAV-1 strains. There were three hypervariable regions at the C-terminus (aa 158–160, 180–193 and 205–219) and other variable points in VP1 protein, but which didn’t cause virulence of DHAV-1 change.     

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.     

Molecular characteristics and evolutionary analysis of a very virulent infectious bursal disease virus

Infectious bursal disease virus(IBDV) poses a significant threat to the poultry industry. Viral protein 2(VP2), the major structural protein of IBDV, has been subjected to frequent mutations that have imparted tremendous genetic diversity to the virus. To determine how amino acid mutations may affect the virulence of IBDV, we built a structural model of VP2 of a very virulent strain of IBDV identified in China, vv IBDV Gx, and performed a molecular dynamics simulation of the interaction between virulence sites. The study showed that the amino acid substitutions that distinguish vv IBDV from attenuated IBDV(H253Q and T284A) favor a hydrophobic and flexible conformation of ?-barrel loops in VP2, which could promote interactions between the virus and potential IBDV-specific receptors. Population sequence analysis revealed that the IBDV strains prevalent in East Asia show a significant signal of positive selection at virulence sites 253 and 284. In addition, a signal of co-evolution between sites 253 and 284 was identified. These results suggest that changes in the virulence of IBDV may result from both the interaction and the co-evolution of multiple amino acid substitutions at virulence sites.     

Determinants of bluetongue virus virulence in murine models of disease

Bluetongue is a major infectious disease of ruminants that is caused by bluetongue virus (BTV). In this study, we analyzed virulence and genetic differences of (i) three BTV field strains from Italy maintained at either a low (L strains) or high (H strains) passage number in cell culture and (ii) three South African "reference" wild-type strains and their corresponding live attenuated vaccine strains. The Italian BTV L strains, in general, were lethal for both newborn NIH-Swiss mice inoculated intracerebrally and adult type I interferon receptor-deficient (IFNAR(-/-)) mice, while the virulence of the H strains was attenuated significantly in both experimental models. Similarly, the South African vaccine strains were not pathogenic for IFNAR(-/-) mice, while the corresponding wild-type strains were virulent. Thus, attenuation of the virulence of the BTV strains used in this study is not mediated by the presence of an intact interferon system. No clear distinction in virulence was observed for the South African BTV strains in newborn NIH-Swiss mice. Full genomic sequencing revealed relatively few amino acid substitutions, scattered in several different viral proteins, for the strains found to be attenuated in mice compared to the pathogenic related strains. However, only the genome segments encoding VP1, VP2, and NS2 consistently showed nonsynonymous changes between all virulent and attenuated strain pairs. This study established an experimental platform for investigating the determinants of BTV virulence. Future studies using reverse genetics will allow researchers to precisely map and "weight" the relative influences of the various genome segments and viral proteins on BTV virulence.     

人A组轮状病毒北京地方株VP4血清型特异片段编码基因的序列分析

轮状病毒是引起婴幼儿严重腹泻的重要病原,其第四基因编码主要中和抗原VP4,而VP4可裂解为VP8和VP5两个片段。VP8为抗原型特异性片段。克隆并测定了具有代表性的三个轮状病毒北京株VP4编码基因5′端（VPS＋VPS一部分）887个核苷酸序列并据此推导出其氨基酸序列。结果表明,相同血清型的地方株和标准株之间具有高度同源性（92％～966％）,不同血清型间则变异较大（70．5％～71％）。氨基酸最大变异处位于aa84～172,并对胰酶作用位点在致病性中的可能性进行了讨论。     

Comparative analysis of the VP3 gene of divergent strains of the rotaviruses simian SA11 and bovine Nebraska calf diarrhea virus.

The gene encoding outer capsid protein VP3 of subpopulations of two animal rotaviruses, simian SA11 and Nebraska calf diarrhea virus (NCDV), was analyzed. Two laboratory strains of simian SA11 rotavirus (SA11-SEM and SA11-FEM) differed with respect to VP3. This dimorphism was indicated by a difference in electrophoretic mobility and a difference in reactivity with anti-VP3 monoclonal antibodies. The overall VP3 amino acid homology between the two SA11 VP3 proteins was 82.7%, whereas the VP3 protein of SA11-FEM was 98.5% homologous in amino acid sequence to NCDV VP3, suggesting that SA11-FEM VP3 was derived by gene reassortment in the laboratory during contamination with a bovine rotavirus. A comparison of the deduced amino acid sequence of the VP3 of two virulent NCDV strains and an attenuated NCDV strain (RIT 4237), revealed only five amino acid differences which were scattered throughout the protein but did not involve the trypsin cleavage sites. Of interest, the VP3 of the standard strain of NCDV which is virulent for cows differed in only one amino acid (position 23, Gln to Lys) from the VP3 of an NCDV mutant which was attenuated both for cows and for children.     

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.