两株传染性法氏囊病病毒5‘和3’非编码区结构分析

采用快速扩增cDNA末端（RACE）方法测定了我国超强毒株Harbin-1和经典弱毒株CJ801的非编码区。在两株病毒基因组A节段中，Harbin-1的5‘非编码区包括100个核苷酸，CJ801包括96个核苷酸；两个毒株B节段的5‘非编码区均含有111个核苷酸。Harbin-1毒株A节段的3‘非编码区含95个核苷酸，CJ801含94个核苷酸。Harbin-1毒株B片段的3‘非编码区含79个核苷酸，CJ801含82个核苷酸。结构分析表明：超强毒株Harbin-1与细胞适应株CJ801的5‘和3‘端非编码区在一级和二级结构上均存在差异。Harbin-1与欧洲超强毒株的关系最近，CJ801与欧洲细胞适应株同源性最高。IBDV非编码区结构特征的研究为进一步研究IBDV非编码区的功能，阐明其与病毒复制调控和毒力的关系奠定了基础。     

传染性法氏囊病病毒中国超强毒株Harbin-1基因组A节段全长cDNA的连接

本研究利用单酶切位点Sal消化传染性法氏囊病病毒中国超强毒株Harbin-1基因组A节段的上段和下段克隆pGEM-T-P12和pGEM-T-P34，经过去磷酸化以后，再用连接酶将上下段连接起来。酶切和测序鉴定结果表明，我们得到了全长的IBDV基因组A节段编码区cDNA克隆。本研究结果为以后的工作奠定了基础。     

传染性法氏囊病病毒自然重配株全基因组序列分析

从具有新的流行病学特征的传染性法氏囊病（IBD）发病鸡群中分离到2株传染性法氏囊病病毒（IBDV）,分别命名为QL和ZZ—ll,对4周龄SPF鸡的致死率分别为94％和86％。为分析该毒株的分子生物学特征,对其全基因组序列进行了测定,2个病毒株基因组A节段长度为3260bp、B节段长度2827bp。病毒演化分析结果显示2个病毒基因组A节段的核苷酸序列与已发表的强毒株序列的同源性分别为96．8％～98．1％和96．9％～98．4％,处在IB—DV超强毒株分支上;而B节段与已发表的弱毒株序列的同源性分别为89．7％～90．4％和90．0％～90．7％,位于弱毒株分支上。IBDV超强毒株和弱毒株序列特征氨基酸残基与基序分析表明,QL和ZZ—11两个病毒株的A节段VP2基因的氨基酸残基为222A、249Q、253Q、254G、256I、294I和299S,七肽基序为SWSASGS,均符合超强毒株的分子特征;而B节段777～782位核苷酸序列为GGTGCC,没有KpnI酶切位点,具有弱毒株的序列特点。以上分析结果表明,QL和ZZ—11为IBDV自然重配株,A节段源于超强毒株,而B节段源于弱毒株。     

鸡传染性法氏囊病超强毒Gx及其致弱株基因组B节段的克隆和序列分析

用蛋白酶K法分离提取了鸡传染性法氏囊病病毒强毒株Gx及其致弱株Gt的病毒核酸dsRNA,应用随机引物将RNA反转录成cDNA,以此为模板用长距离一步法扩增出全长基因B节段,将其克隆入PMD18-T载体,进行了测序,并用DNAStar软件进行序列分析.测序结果表明,克隆的Gx株B节段全长为2 827bp,与超强毒参考株UK661的同源性为88.2%,与强毒参考株Harbin-1株的同源性达96.3%;克隆的Gt株B节段全长为2827bp,与弱毒参考株P2的同源性达99.5%.而Gx株与Gt株B节段的核苷酸的同源性只有89.8%;vvIBDV-Gx、IBDV-Gt株B节段全长的获得将为进一步构建感染性分子克隆奠定必要的基础.     

传染性法氏囊病病毒自然重配株全基因组序列分析

从具有新的流行病学特征的传染性法氏囊病(IBD)发病鸡群中分离到2株传染性法氏囊病病毒(IBDV),分别命名为QL和ZZ-11,对4周龄SPF鸡的致死率分别为94%和86%。为分析该毒株的分子生物学特征,对其全基因组序列进行了测定,2个病毒株基因组A节段长度为3 260bp、B节段长度2 827bp。病毒演化分析结果显示2个病毒基因组A节段的核苷酸序列与已发表的强毒株序列的同源性分别为96.8%⁹8.1%和96.9%98.1%和96.9%⁹8.4%,处在IBDV超强毒株分支上;而B节段与已发表的弱毒株序列的同源性分别为89.7%98.4%,处在IBDV超强毒株分支上;而B节段与已发表的弱毒株序列的同源性分别为89.7%⁹0.4%和90.0%90.4%和90.0%⁹0.7%,位于弱毒株分支上。IBDV超强毒株和弱毒株序列特征氨基酸残基与基序分析表明,QL和ZZ-11两个病毒株的A节段VP2基因的氨基酸残基为222A、249Q、253Q、254G、256I、294I和299S,七肽基序为SWSASGS,均符合超强毒株的分子特征;而B节段77790.7%,位于弱毒株分支上。IBDV超强毒株和弱毒株序列特征氨基酸残基与基序分析表明,QL和ZZ-11两个病毒株的A节段VP2基因的氨基酸残基为222A、249Q、253Q、254G、256I、294I和299S,七肽基序为SWSASGS,均符合超强毒株的分子特征;而B节段777⁷82位核苷酸序列为GGTGCC,没有KpnⅠ酶切位点,具有弱毒株的序列特点。以上分析结果表明,QL和ZZ-11为IBDV自然重配株,A节段源于超强毒株,而B节段源于弱毒株。     

基因自然重排鸡传染性法氏囊病病毒的分子特征

为了对某鸡群疑似传染性法氏囊病(IBD)的病例进行分子诊断、病毒分离和分子特征分析，本试验通过核酸检测调查该鸡群法氏囊组织和病毒分离物中传染性法氏囊病病毒(IBDV)的感染情况，将分离得到的毒株命名为GL1906,继而对该病毒基因组双节段的VP2高变区(vVP2)序列和VP1-b序列进行分析。结果显示，GL1906 vVP2基因的特征性氨基酸位点在222A、256I、284A、294I和299S上均符合超强毒株(vvIBDV)的特征，但279N符合致弱毒株的特征；VP1-b基因在242D、390L、393E与弱毒株一致，287A与vvIBDV一致，此外第777～782位核苷酸序列为GGTGCC,与弱毒株一致；GL1906 vVP2的核苷酸、氨基酸序列与vvIBDV的同源性最高，在系统进化树中与vvIBDV同为A3分支；GL1906 VP1-b的核苷酸、氨基酸序列与B节段属于独特来源的NN1172同源性最高，在系统进化树中同属于B3独特分支。结果表明，本试验分离株GL1906的基因组双节段vVP2和VP1-b具有不同的来源，是基因型为A3B3的基因自然重排毒株。     

广西乌鸡传染性法氏囊病的诊治

本研究对疑似广西东兰乌鸡感染传染性法氏囊病的病例进行诊治,通过观察临床症状、细菌学检测、病毒检测和分离、序列分析以及病理切片观察,证实该鸡群感染了传染性法氏囊病病毒。分析表明,该毒株与疫苗株间核苷酸同源性为91.0%~93.3%,与超强毒株之间核苷酸同源性为87.0%~96.0%,并且与超强毒株在系统进化树中同处一个分支。说明此次分离于乌鸡群的传染性法氏囊病毒为超强毒株,并且发生了新的变异。     

鸽源传染性法氏囊病病毒的分离鉴定及其A、B节段序列扩增

目的:分离鉴定鸽源传染性法氏囊病病毒(IBDV),扩增其基因组A、B节段cDNA.方法:采用SPF鸡胚尿囊腔接种分离IBDV,RT-PCR鉴定并扩增A、B节段全基因组DNA,进行同源性比较,初步分析分离株序列特征.结果:该分离株经RT-PCR特异性鉴定引物鉴定为强毒株,与强毒株相比,核苷酸序列同源性达97%,氨基酸序列同源性99%.     

鸡传染性法氏囊病病毒流行株VP1部分序列及VP2高变区序列分析

为了对鸡传染性法氏囊病病毒(IBDV)的13个地方分离株与3个商品疫苗株的VP1-b基因(756 bp～1 522 bp)进行同源性和遗传进化分析,本研究对其进行RT-PCR扩增和序列测定.结果表明,获得的VP1-b基因长度均为767 bp; 13个分离株VP1-b节段核苷酸和氨基酸同源性分别为90.7 ％～100％和72.9％～100％,其中10株超强毒分离株与经典株、弱毒株、疫苗株的同源性较高,核苷酸和氨基酸同源性分别为95.7％～100％和94.5％～100％,所有12个超强毒分离株与超强毒参考病毒株的同源性均较低,核苷酸同源性仅为68.2％～79.2％;在遗传进化分析中,所有病毒株被分为3个分支,与相应病毒株的基于IBDV-VP2高变区(vVP2)序列的遗传进化树比较并结合同源性分析结果表明,除了弱毒株HUN0804之外,其它12个分离株可能均为重组病毒.据此推测,基因重组可能是目前IBDV流行的新趋势.     

传染性法氏囊病病毒A节段编码序列cDNA原克隆和序列分析

对反转录－聚合酶链反应扩增克隆的传染性法氏囊病病毒超强毒Ｈａｒｂｉｎ毒株的Ａ节段编码序列ｃＤＮＡ基因进行了核苷酸序列分析。结果，克隆的Ａ节段基因共３１０ｂｐ，包括两个完整的阅读框架ＯＲＦＡ１和ＯＲＦＡ２分别编码１０１２氨基酸的前体蛋白ＶＰ２－４－３和１４５氨基酸的ＶＰ５，两者有部分重叠。     

Direct evidence of reassortment and mutant spectrum analysis of a very virulent infectious bursal disease virus

The complete genomic sequence of very virulent infectious bursal disease virus (vvIBDV) Gx strain was determined, including the sequences of segment A, encoding the precursor polyprotein, and segment B, encoding the viral RNA polymerase (VP1) and 5'- and 3'-untranslating regions. Alignment of segment A of Gx with the sequences of 12 other vvIBDV strains showed 97.5% to 99.0% amino acid identity, whereas alignment of segment B of Gx with nine other vvIBDV strains revealed high sequence divergence, ranging from 10.3% to 11%. Phylogenetic analysis of segments A and B showed that they were in different branches, indicating that the reassortment occurred in this strain and that segment A and segment B derived from different pathotype strains. The mutant spectrum analysis of quasispecies virus demonstrated that the mean minimum mutation frequency in VP1 was 8.78-fold higher than in the polyprotein. The most frequent mutations were in the first 1986 nucleotides (nonsynonymous mutations) and the last 660 nucleotides (synonymous mutations), indicating that the 219 amino acid residues in the C-terminal of the VP1 form a functional region.     

Differentiation of infectious bursal disease viruses isolated in Croatia

Infectious bursal disease viruses (IBDVs) in 26 IBDV-positive bursa samples collected in Croatia during the period 1996-2000 and in two commercially available vaccines were differentiated by the presence or absence of the CfoI, SacI, SspI, StuI, and TaqI restriction sites in the 422-bp fragment of segment A of the VP2 gene (nt 732-1153). The fragments from 14 (54%) field isolates were TaqI+ StuI+ SspI+ and SacI- CfoI-, indicating their very virulent (vv) character. The presence of CfoI restriction site in 10 (38%) field isolates is uncommon for vvIBDV strains. It was detected in only the 88180 vvIBDV strain. Nevertheless, these isolates can be classified as vv strains according to TaqI+ StuI+ SspI+ SacI- restrictions. Two SacI+ StuI+ CfoI+ TaqI- SspI- field isolates (8%) could be classified as non-vvIBDVs. The StuI+ restriction is common to vvIBDV strains. However, the StuI recognition sequence is present in the F52/70 classic European and 002-73 attenuated strains as well. The SacI+ CfoI+ StuI- SspI- restrictions and the lack of the TaqI restriction at nt position 832 show that the IBDV in GUMBOKAL IM-SPF vaccine corresponds to the attenuated and/or vaccine strains. The TaqI restriction at nt position 875 suggests that the IBDV in GUMBOKAL SPF vaccine could belong to the mild strains.     

传染性法氏囊病病毒NN1107广西株的分离鉴定及遗传进化分析

为研究从广西南宁市某鸡场疑似患传染性法氏囊病的鸡群中分离鉴定出的一株传染性法氏囊病病毒NN1107株的分子特征,通过反转录-聚合酶链反应特异扩增后进行克隆、序列分析。NN1107株VP2基因高变区(vVP2)的克隆测序和序列比较结果显示,序列符合超强毒传染性法氏囊病病毒(vvIBDV)的分子特征;其与广西vvIBDV毒株的核苷酸同源性在96%～99.6%之间。根据vVP2核苷酸序列同源性绘制的遗传进化树结果显示,NN1107株属于vvIBDV毒株群,与2004年、2005年、2007年和2010年流行毒株BH09、NNTZ(3)、NN07122、HP1001的亲缘关系最近,与疫苗株B87(in)、Bursine-2、FW2512株的亲缘关系较远。     

Prokaryotic Expression and Bioinformatics Analysis of VP2 Gene of Infectious Bursal Disease Virus C4 Strain

This study was aimed to investigate the relationship between the virulence characteristics of infectious bursal disease virus(IBDV) C4 strain and its VP2 amino acid sequence. The RNA of IBDV C4 strain was extracted,and its VP2 gene was amplified by RT-PCR.VP2 nucleotide sequences and deduced amino acids of different virulent IBDV strains were compared. At the same time, prokaryotic expression vector pET-32a(+) was used to express the VP2 gene. The expression of recombinant VP2 protein was detected by SDS-PAGE and Western blotting. The results showed that the VP2 gene of IBDV C4 strain belonged to the very virulent infectious bursal disease virus (vvIBDV) in evolutionary relationship, the VP2 nucleotides homology between IBDV C4 strain and other vvIBDV strains were 98.1% to 98.7%, and there were no mutations in S-W-S-A-S-G-S (326-332 amino acids) and 222(A), 256(I), 294(I) and 299(S). The VP2 amino acid sequence of IBDV C4 strain was consistent with the characteristics of other vvIBDV strains. However, there were three differences amino acids sites at 201(D/G), 281(G/R) and 313(V/A) between the amino acids of the C4 strain and the very virulent strain UK661. And the change of 281(R) was in the small hydrophilic region of 279 to 290, which was related to the antigenicity of the virus; The recombinant VP2 protein molecular weight expressed in Escherichia coli BL21 was about 67 ku. This study provided a basis for further research on antigenic changes resulting from amino acid variation of 201(G), 281 (R) and 313(A). These results indicated that the VP2 gene of the IBDV C4 strain was consistent with the major characteristics of the vvIBDV strain VP2 gene. The difference of three amino acid sites in the vvIBDV strain C4 might be related to the evolution of virulence of IBDV strain in China.     

传染性法氏囊病病毒C4毒株VP2基因的原核表达及生物信息学分析

试验旨在研究一株传染性法氏囊病病毒（IBDV）河南分离株的毒力特征及其与VP2氨基酸序列特征的关系。通过提取IBDV C4株RNA,利用RT-PCR扩增其VP2基因,与其他不同毒力IBDV毒株进行核苷酸及推导的氨基酸序列比对分析,同时使用pET-32a（+）原核表达载体表达VP2基因,用SDS-PAGE和Western blotting检测重组VP2蛋白的表达。结果显示,扩增的IBDV C4株的VP2基因序列在进化关系上属于超强毒力IBDV（vvIBDV）分类,与选取的vvIBDV毒株代表毒株核苷酸序列同源性在98.1%~98.7%之间,其七肽区为S-W-S-A-S-G-S（第326-332位氨基酸）符合超强毒株特征,且222（A）、256（I）、294（I）和299（S）位氨基酸与超强毒力毒株的4个特征性氨基酸一致;但IBDV C4毒株的VP2蛋白氨基酸序列与超强毒力毒株代表毒株UK661相比,201（D/G）、281（G/R）、313（V/A）位氨基酸不同,其中281位氨基酸的改变处于279-290的小亲水区内,与病毒抗原性有关;构建的pET-32a（+）-VP2原核表达载体在大肠杆菌BL21感受态细胞上表达出分子质量约67 ku的重组VP2蛋白,为进一步比较201（G）、281（R）、313（A）位氨基酸差异导致的抗原特性改变提供了研究基础。本试验结果表明,IBDV C4株VP2基因与vvIBDV毒株VP2基因的主要特性一致,但也有3处氨基酸与代表毒株UK661存在差异,这些改变可能与中国IBDV毒株毒力的进化有关。