禽痘疫苗病毒中网状内皮组织增生病病毒5′LTR整合位点序列分析

参照国外发表的禽网状内皮组织增生病病毒(REV)5′长末端重复序列(LTR)在禽痘病毒(FPV)疫苗株基因组上的整合位点及相关序列,合成一对来自FPV的引物,从国内5个不同厂家生产的禽痘疫苗中经PCR均扩增到REV-5′LTR。通过序列比较发现,我国5个FPV疫苗毒株中REV-5′LTR整合位点与美国和澳大利亚的天然重组禽痘疫苗完全一致。其中,有3个的REV-5′LTR插入序列也与美国的Vac-3-Am株和澳大利亚的Vac-M3-Au株有100%的同源性。另2个中国疫苗毒株中的REV-LTR插入序列与美国疫苗毒株Vac-1-Am中的REV-LTR插入序列有99.6%的同源性。但是,这5个中国禽痘疫苗毒株中整合的REV LTR与中国近年分离到的REV野毒株HA9901[1]的5′LTR的同源性只有75.4%~92.4%。     

含有禽网状内皮组织增生病病毒基因组片段的天然重组禽痘病毒的研究

将国内5个不同生产厂家来源的禽痘弱毒疫苗株和1株禽痘野毒株在鸡胚成纤维细胞(CEF)上连续传5代,用禽网状内皮组织增生病病毒(Reticuloendotheliosis virus,REV)特异性的单克隆抗体进行间接免疫荧光试验(Immunonuoreseellee assay,IFA),均检测不到传染性REV。但以6株禽痘病毒(FPV)感染的第2代和第5代细胞基因组提取物为模板,通过PCR均能扩增出REV的长末端重复序列(LTR)和囊膜蛋白(env)基因片段。用特异性核酸探针作分子斑点杂交(Dot blot),结果显示所扩增的PCR条带为特异的REV—LTR和REV—env)基因片段。实验结果表明,国内的一些痘病毒疫苗和野毒株基因组中,已稳定地整合进了REV的基因组成分。     

整合进禽反转录病毒基因组片段的鸡马立克氏病病毒重组野毒株的发现

用对禽网状内皮组织增生病病毒(REV)的单抗从广东和广西分离到的二株马立克氏病病毒(MDV)野毒株做间接荧光抗体试验, 用MDV感染细胞的基因组DNA做斑点分子杂交及PCR显示, 这二株MDV基因组中已整合进REV的LTR序列. 根据MDV基因组上易插入REV的LTR的高频位点的序列合成7条引物, 根据REV的LTR合成4条引物, 由此交叉组成28对引物, 分别从这二个MDV野毒株扩增和克隆已整合进MDV的REV-LTR序列及其相连的MDV序列. 测序证明, 二株中的REV-LTR插入序列及在MDV基因组中短独特序列(US)上的插入位点完全相同. 表明这二个毒株很可能是一次重组事件在鸡群中形成的流行毒株.     

网状内皮组织增生症病毒中国分离株HA9901全基因组核苷酸序列的测定与分析

以网状内皮组织增生症病毒(REV)中国分离株HA9901感染的鸡胚成纤维细胞(CEF)基因组DNA作为其前病毒基因组模板, 根据已发表序列设计合成6对引物, 经PCR扩增出6段连续的、相互部分重叠的DNA片段和闭合环形前病毒两末端LTR的连接区段, 并分别连入T载体进行克隆、测序. 用DNAstar软件对测序结果进行剪辑和拼接, 完成了REV第一个中国分离株HA9901前病毒全基因组核苷酸序列. 在从截然不同的地区、不同年份、不同禽类分离到的毒株中, 将该序列与另两个毒株已完成的全基因组序列的比较表明, REV的整个基因组相对保守, 各毒株间对应基因的同源性都在92%以上. 其中, 从我国鸡体分离到的野毒株HA9901与美国鸡源分离株FA在整个基因组上的同源性均显著高于美国的鸭源SNV株.     

不同致病型马立克氏病病毒DNA聚合酶基因序列比较

为了分析马立克氏病病毒(MDV)致病型与其DNA聚合酶基因的关系,本研究比较了9个不同致病型的该基因的同源性关系,这包括四种不同致病型的国际参考株即弱毒疫苗株CV1988／Ripens株、强毒株GA、超剜毒株Md5和特超强毒株648A;中国疫苗株814、中国强毒参考株Jing-1及3个中国野毒株。结果表明：MDV的DNA聚合酶基因非常保守,在比较的9个毒株间,该基因上游约369个碱基的调控序列的同源性在96．7％～100％之间,该基因编码的1220个氨基酸序列的同源性在99．2％～100％之间。尽管不同毒株在一些位点上出现了氨基酸的变异,但这些变异与病毒的致病型或地域分布没有明显的关系。     

猪圆环病毒2型分离毒株全基因组的克隆及序列分析

根据GenBank中猪圆环病毒2型（PCV－2）基因序列,设计两对特异性引物,用PCR方法从接种疑似PMWS仔猪病料的细胞中分段扩增2个分离毒株全基因组。将扩增片段克隆入pGEM-T easy载体,筛选获得含有相应片段的阳性重组质粒。对质粒中的插入片段进行测序拼接,获得2株均为1768bp的全基因组序列。应用DNA star序列分析软件,对所测PCV－2序列与GenBank中的国内外PCV毒株进行同源性比较,并绘制系统发生树,结果发现：所测两毒株之间的核苷酸序列同源性极高,可达99．8％,亲缘关系密切;与PCV－2参考毒株的同源性介于95．3％－99．7％之间,其中与美国的一株PCV－2同源性最高,分别为99．5％和99．7％,亲缘关系很近;与国内两分离株同源性分别为96．4％和98．8％－98．9％;与PCV－1参考毒株同源性仅为77．1％－77．5％。     

两个猪瘟病毒野毒株gp55基因抗原编码区序列的分析及比较

利用反转录－PCR方法扩增了吉林省猪瘟病毒（HCV）两个野毒株gp55基因的主要保护性抗原编码区,并将其克隆到pGEM－T载体中,然后用Sanger双脱氧法测定了其核苷酸序列,并推导了其氨基酸序列。将测定的这两个HCV野毒株的部分序列（350bp）与国内外已知的HCV序列进行比较,结果表明：这两个野毒株的核苷酸序列的同源性为94．9％,氨基酸序列同源性为97．4％,与1985～1992年意大利中部分离4个野毒株的同源性明显高于其它HCV毒株,核苷酸同源性分别为97．2％～98．3％和94.0％～949％,氨基酸同源性分别为98．3％～991％和97.4％～98.3％,而与我国的HCV标准强毒株即石门株的核苷酸同源性仅分别为83.1％和83.1％,氨基酸同源性仅分别为90.6％和91.4％。因此认为吉林省这两个野毒株与意大利中部的4个野毒株具有密切的关系,而与石门株很可能来源不同。     

禽Ⅰ型副粘病毒f基因克隆及序列分析

用RT—PCR一步法对云南省不同禽类(鸡、鸽子)3株禽Ⅰ型副粘病毒F基因进行扩增和克隆,并对其f基因片段核苷酸序列进行分析,结果表明,云南省禽Ⅰ型副粘病毒各毒株同源性为88．1％--94．9％,与疫苗株LaSota和强毒株F48E9的同源性为85．6％。所分离两株新城疫病毒在F蛋白裂解位点区(112—117aa)的氨基酸序列与强毒株在这一区域的序列完全相同,表明为强毒株。鸽Ⅰ型副粘病毒F蛋白裂解位点区的氨基酸序列与PPMVZQ98—1株在这一区域的序列完全相同,揭示为中强毒株。以1662bp核苷酸绘制系统发育树,表明云南地方新城疫病毒届于基因Ⅶ型,鸽Ⅰ型副粘病毒届于基因Ⅵ型。     

猪瘟病毒糖蛋白E0基因的克隆及其表达研究*

用ＲＴ－ＰＣＲＡ法扩增分别获得了中国猪瘟病毒强毒石门株和免化弱毒株糖蛋白Ｅ０基因ｃＤＮＡ,并克隆到ｐＧＥＭ Ｔ载体中测定其核着酸序列和推导出其对应氨基酸序列;结果表明这两个毒株间的Ｅ０基因核着酸序列同源性为９５．３％,氨基酸序列同源性为９４％,有１４个残基的差异;与几个代表毒株ＡＬＤ株、ＧＰＥ株、Ｂｒｅｓｃｉａ株、Ａｌｆｏｒｔ株和国外测得的免化弱毒Ｃ株相应序列进行比较,所测石门病毒核苷酸序列与上述各株对应序列的同源性分别为９８．０％、９７．１％、９２．７％、８６．８％和９５．４％;氨基酸序列同源性分别为９     

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。     

Reticuloendotheliosis virus sequences within the genomes of field strains of fowlpox virus display variability

Nine field strains of fowlpox virus (FPV) isolated during a 24-year span from geographically diverse outbreaks of fowlpox in the United States were screened for the presence of reticuloendotheliosis virus (REV) sequences in their genomes by PCR. Each isolate appeared to be heterogeneous in that either a nearly intact provirus or just a 248- or 508-nucleotide fusion of portions of the integrated REV 5' and 3' long terminal repeats (LTRs) was exclusively present at the same genomic site. In contrast, four fowlpox vaccines of FPV origin and three originating from pigeonpox virus were genetically homogeneous in having retained only the 248-bp LTR fusion, whereas two other FPV-based vaccines had only the larger one. These remnants of integrated REV presumably arose during homologous recombination at one of the two regions common to both LTRs or during retroviral excision from the FPV genome. Loss of the provirus appeared to be a natural event because the tripartite population could be detected in a field sample (tracheal lesion). Moreover, the provirus was also readily deleted during propagation of FPV in cultured cells, as evidenced by the detection of truncated LTRs after one passage of a plaque-purified FPV recombinant having a "genetically marked" provirus. However, the deletion mutants did not appear to have a substantial replicative advantage in vitro because even after 55 serial passages the original recombinant FPV was still prevalent. As to the in vivo environment, retention of the REV provirus may confer some benefit to FPV for infection of poultry previously vaccinated against fowlpox.     

Sequence analysis for the complete proviral genome of reticuloendotheliosis virus Chinese strain HA9901

Reticuloendotheliosis virus (REV), a member of avian retrovirus, can cause tumor, immune suppression and a runting disease syndrome[1,2]. With several clas-sical reference strains, such as strain SNV from ducks, a replication defective oncogenic strain T …     

Isolation of recombinant field strains of Marek’s disease virus integrated with reticuloendotheliosis virus genome fragments

Two Marek's disease virus (MDV) field strains were isolated from chickens with tumors independently from Guangdong and Guangxi provinces, and it was confirmed that there were no co-infections with reticuloendotheliosis viruses (REV) in chicken embryo fibroblast cells (CEF) in indirect fluorescence antibody test (IFA) with REV-specific monoclonal antibodies. By dot blot hybridization and PCR of genomic DNA of MDV-infected CEF, it was indicated that LTR fragments of REV genome were integrated into genome of these two MDV field strains. To amplify and clone the integrated REV LTR with MDV sequence at the junction, 4 primers from REV LTR and 7 primers from MDV genome fragment with REV LTR insertion hot points were synthesized and 28 (4x7) pairs of primers (one from REV and another from MDV for each pair) were used in PCR while using the genomic DNA of both strains as the templates. The sequence data demonstrated that both recombinant field strains contained the same REV LTR inserted into MDV at the identical sites in US fragment of the genomes. From the above, it was speculated that both recombinant field MDVs were originated from a same recombinant virus and spread among chicken flocks in two provinces.     

Isolation of recombinant field strains of Marek's disease virus integrated with reticuloendotheliosis virus genome fragments

Two Marek's disease virus (MDV) field strains were isolated from chickens with tumors independently from Guangdong and Guangxi provinces, and it was confirmed that there were no co-infections with reticuloendotheliosis viruses (REV) in chicken embryo fibroblast cells (CEF) in indirect fluorescence antibody test (IFA) with REV-specific monoclonal antibodies. By dot blot hybridization and PCR of genomic DNA of MDV-infected CEF, it was indicated that LTR fragments of REV genome were integrated into genome of these two MDV field strains. To amplify and clone the integrated REV LTR with MDV sequence at the junction, 4 primers from REV LTR and 7 primers from MDV genome fragment with REV LTR insertion hot points were synthesized and 28 (4x7) pairs of primers (one from REV and another from MDV for each pair) were used in PCR while using the genomic DNA of both strains as the templates. The sequence data demonstrated that both recombinant field strains contained the same REV LTR inserted into MDV at the identical sites in US fragment of the genomes. From the above, it was speculated that both recombinant field MDVs were originated from a same recombinant virus and spread among chicken flocks in two provinces.     

Sequence analysis for the complete proviral genome of reticuloendotheliosis virus Chinese strain HA9901

The genomic DNA extracted from chicken embryo fibroblast (CEF) infected with a Chinese field isolate HA9901 of reticuloendotheliosis virus (REV) was used as the template to amplify the REV proviral genomic cDNA by PCR with 6 pairs of primers according to published sequences. Six overlapping fragments were amplified, cloned into the TA vector and sequenced, including a fragment which was amplified from the circular proviral cDNA and covering both 5′-and 3′-ends. The complete sequence of the whole genome was established and analyzed with a DNAstar software. Comparisons of the sequence with two other strains demonstrated that the genomes of REV were relatively conservative, the homogenecity for all genes or LTR fragments of the 3 strains was over 92%, no matter whether they were isolated from different species and regions in different years. But, the homology of Chinese strain HA9901 to a fowl pox virus-associated strain from Chickens was higher than that to strain SNV isolated from ducks.     

Complete Genome Sequence of a Recombinant Marek's Disease Virus Field Strain with One Reticuloendotheliosis Virus Long Terminal Repeat Insert

Marek''s disease virus (MDV) Chinese strain GX0101, isolated in 2001 from a vaccinated flock of layer chickens with severe tumors, was the first reported recombinant MDV field strain with one reticuloendotheliosis virus (REV) long terminal repeat (LTR) insert. GX0101 belongs to very virulent MDV (vvMDV) but has higher horizontal transmission ability than the vvMDV strain Md5. The complete genome sequence of GX0101 is 178,101 nucleotides (nt) and contains only one REV-LTR insert at a site 267 nt upstream of the sorf2 gene. Moreover, GX0101 has 5 repeats of a 217-nt fragment in its terminal repeat short (TRS) region and 3 repeats in internal repeat short (IRS) region, compared to the other 10 strains with only 1 or 2 repeats in both TRS and IRS.     

A comparison of complete genome sequences of a rabies virus chinese isolate SH06 with the vaccine strains

In this study, we determined the complete nucleotide and deduced amino acid sequence of a primary isolate of rabies virus (SH06) obtained from the brain of a rabid dog. The overall length of the genome was 11 924 nucleotides. Comparison of the genomic sequence showed the homology of SH06 at nucleotide level with full-length genomes of reference vaccine strains ranged from 82.2% with the PV strain to 86.9% with the CTN strain. A full-length genome-based phylogenetic analysis was performed with sequences available from GenBank. Phylogenetic analysis of the complete genome sequences indicated that the SH06 exhibited the highest homology with rabies street virus BD06 and CTN vaccine strain originated from China.