共查询到20条相似文献，搜索用时 15 毫秒
1.
2.
Najat Chbab Danièle Chabanne-Vautherot Annick Francineau Nikolaus Osterrieder Caroline Denesvre Jean-Fran?ois Vautherot 《Veterinary research》2009,40(4)
Marek’s disease virus type 1 (MDV-1) shows a strict dependency on the direct cell-to-cell spread for its propagation in cell culture. As MDV-1 shows an impaired nuclear egress in cell culture, we wished to address the characterization of capsid/tegument genes which may intervene in the maturation of intranuclear capsids. Orthologs of UL17 are present in all herpesviruses and, in all reported case, were shown to be essential for viral growth, playing a role in capsid maturation and DNA packaging. As only HSV-1 and PrV UL17 proteins have been characterized so far, we wished to examine the role of MDV-1 pUL17 in virus replication. To analyze MDV-1 UL17 gene function, we created deletion mutants or point mutated the open reading frame (ORF) to interrupt its coding phase. We established that a functional ORF UL17 is indispensable for MDV-1 growth. We chose to characterize the virally encoded protein by tagging the 729 amino-acid long protein with a repeat of the HA peptide that was fused to its C-terminus. Protein pUL17 was identified in infected cell extracts as an 82 kDa protein which localized to the nucleus, colocalizing with VP5, the major capsid protein, and VP13/14, a major tegument protein. By using green fluorescent protein fusion and HA tagged proteins expressed under the cytomegalovirus IE gene enhancer/promoter (PCMV IE), we showed that MDV-1 pUL17 nuclear distribution in infected cells is not an intrinsic property. Although our results strongly suggest that another viral protein retains (or relocate) pUL17 to the nucleus, we report that none of the tegument protein tested so far were able to mediate pUL17 relocation to the nucleus. 相似文献
3.
新城疫病毒M蛋白细胞核定位的机制与功能研究进展 总被引：1，自引：1，他引：0
新城疫病毒（Newcastle disease virus,NDV）M蛋白一种非糖基化膜相关蛋白,主要位于病毒囊膜内表面,构成了病毒囊膜和核衣壳连接的支架。研究表明,M蛋白是一种细胞核-细胞质穿梭蛋白。在NDV感染早期,M蛋白可通过自身携带的核定位信号进入细胞核。根据已报道的相关RNA病毒M蛋白功能的研究结果,推测NDV M蛋白早期的细胞核定位有利于细胞质中病毒基因组的复制和转录,并且可能会抑制细胞基因的转录和蛋白质合成。目前,国内外对M蛋白的研究主要集中在M蛋白与NDV毒力和复制的关系以及以M蛋白为核心的病毒样颗粒形成机制和利用方面,而对M蛋白细胞核定位的机制和功能研究相对较少。鉴于M蛋白细胞核定位在NDV复制和致病过程中的重要作用,本文主要从NDV M蛋白的细胞定位特征、M蛋白细胞核定位的分子机制和功能方面进行阐述,以期为NDV M蛋白细胞核定位的功能及其作用机制研究提供理论参考。 相似文献
4.
猪圆环病毒2型ORF3编码蛋白的体外表达 总被引：1，自引：0，他引：1
设计特异引物,以猪圆环病毒2型(PCV-2)杭州株HZ0201的基因组DNA为模板,PCR扩增出ORF3基因,构建了pGEX-4T-1-ORF3原核表达载体和pEGFP-C2-ORF3真核表达载体。ORF3基因全长315 bp,编码105个氨基酸。SDS-PAGE、Western blot分析及真核PK15细胞转染结果显示:ORF3蛋白在大肠杆菌中以包涵体形式存在,分子量大小约为37.7 ku;ORF3重组蛋白在真核PK15细胞的细胞核和细胞浆都有表达,尤其在细胞核中表达量较高,且对细胞有一定的毒性。 相似文献
5.
Li H Cunha CW Gailbreath KL O'Toole D White SN Vanderplasschen A Dewals B Knowles DP Taus NS 《Veterinary microbiology》2011,150(3-4):270-277
Malignant catarrhal fever (MCF) is a frequently fatal lymphoproliferative disease syndrome primarily of ruminant species, caused by gammaherpesviruses in the genus Macavirus. Ovine herpesvirus 2 (OvHV-2), carried by sheep, causes sheep-associated MCF worldwide, while Alcelaphine herpesvirus 1 (AlHV-1), carried by wildebeest, causes wildebeest-associated MCF, mainly in Africa. Diseases in rabbits can be induced by both viruses, which are clinically and pathologically similar; however, recent studies revealed different expression of viral genes associated with latency or lytic replication during clinical disease between the two viruses. In this study, we further characterized experimentally induced MCF in rabbits by nebulization with OvHV-2 from sheep nasal secretions to elucidate the course of viral replication, along with in vivo incorporation of 5-Bromo-2'-Deoxyuridine (BrdU), to evaluate lymphoproliferation. All six rabbits nebulized with OvHV-2 developed MCF between 24 and 29 days post infection. OvHV-2 DNA levels in peripheral blood leukocytes (PBL) remained undetectable during the incubation period and increased dramatically a few days before onset of clinical signs. During the clinical stage, we found that predominantly lytic gene expression was detected in PBL and tissues, and both T and B cells were proliferating. The data showed that the viral gene expression profile and lymphoproliferation in rabbits with OvHV-2 induced MCF were different from that in rabbits with AlHV-1 induced MCF, suggesting that OvHV-2 and AlHV-1 may play a different role in MCF pathogenesis. 相似文献
6.
Dewals B Gillet L Gerdes T Taracha EL Thiry E Vanderplasschen A 《Veterinary microbiology》2005,110(3-4):209-220
Bovine herpesvirus 4 (BoHV-4) has been isolated from cattle throughout the world. Interestingly, a survey of wild African buffaloes mainly from the Maasai Mara Game Reserve in Kenya revealed that 94% of the animals tested had anti-BoHV-4 antibodies [Rossiter, P.B., Gumm, I.D., Stagg, D.A., Conrad, P.A., Mukolwe, S., Davies, F.G., White, H., 1989. Isolation of bovine herpesvirus-3 from African buffaloes (Syncerus caffer). Res. Vet. Sci. 46, 337–343]. These authors also proposed that the serological antigenic relationship existing between BoHV-4 and alcelaphine herpesvirus 1 (AlHV-1) could confer to BoHV-4 infected buffaloes a protective immune response against lethal AlHV-1 infection. In the present study, we addressed two questions related to Rossiter et al. paper. Firstly, to investigate the role of the African buffalo as a natural host species of BoHV-4, the seroprevalence of anti-BoHV-4 antibodies was analysed in wild African buffaloes throughout eastern and southern Africa. A total of 400 sera was analysed using two complementary immunofluorescent assays. These analyses revealed that independently of their geographical origin, wild African buffaloes exhibit a seroprevalence of anti-BoHV-4 antibodies higher than 68%. This result is by far above the seroprevalence generally observed in cattle. Our data are discussed in the light of our recent phylogenetic study demonstrating that the BoHV-4 Bo17 gene has been acquired from a recent ancestor of the African buffalo. Secondly, we investigated the humoral antigenic relationship existing between BoHV-4 and AlHV-1. Our results demonstrate that among the antigens expressed in AlHV-1 infected cells, epitope(s) recognised by anti-BoHV-4 antibodies are exclusively nuclear, suggesting that the putative property of BoHV-4 to confer an immune protection against AlHV-1 relies on a cellular rather than on a humoral immune response. 相似文献
7.
斜纹夜蛾核型多角体病毒Ⅱ型(SpltMNPVⅡ)分离株是繁殖率和毒力极强的新型病毒株,ORF63是该病毒分离株的一个功能未知基因。从SpltMNPVⅡ分离株基因组中克隆了ORF63。序列分析表明,该基因读码框为1 071 bp,编码356个氨基酸,蛋白质分子质量为41.3 kD;起始密码子ATG上游存在一个早期和晚期启动子基序,编码蛋白序列含有CNX、MutH等多种结构域。启动子活性和转录时相分析表明ORF63是一个早、晚期表达的基因,在病毒感染4 h和18 h时有2个转录峰,24 h以后转录水平略有下降,但总体趋于稳定。构建该基因的原核表达载体pET-28a-ORF63,并转化大肠杆菌BL21(DE3),表达并纯化融合蛋白后制备多克隆抗体,Western blot检测制备的多克隆抗体特异性较好,效价可达1∶3 200以上。由以上结果推测,SpltMNPVⅡ分离株的ORF63基因是一个早期和晚期均有表达的病毒基因,可能与SpltMNPVⅡ感染宿主细胞后自身DNA的复制有关,参与早期芽生病毒(BV)的发生和晚期包涵体衍生病毒(ODV)的成熟2个过程。 相似文献
8.
Dongge Li Jing Wang Shangen Xu Shanxia Cai Chaojie Ao Liurong Fang Shaobo Xiao Huanchun Chen Yunbo Jiang 《Veterinary research communications》2018,42(1):1-10
In the present study, the function of a novel ORF6 gene in the PCV2 genome was determined and functionally analyzed in vitro. ORF6 expression was demonstrated by indirect immunofluorescence in PCV2-infected cells. The antibody against ORF6 was detected in PCV2-infected pigs. The start codon of ORF6 was mutated and an infectious clone was used to create an ORF6-deficient mutant virus. Viral DNA replication curves and immunofluorescence analysis indicated that ORF6 is unnecessary for viral replication and ORF6 deletion reduces viral DNA replication in PK-15 cells. The activities of caspases 3 and 8 in ORF6-deficient virus-infected cells were significantly different from those in wild-type virus-infected cells. The ORF6 protein can increase the expression of IFN-β, TNF-α, IL-1b, IL-10, and IL-12p40. These results demonstrated that the newly discovered ORF6 protein may be involved in caspases regulation and the expression of multiple cytokines in PCV2-infected cells. The functions of this gene in viral pathogenesis remain to be further elucidated. 相似文献
9.
PRRSV核衣壳蛋白基因在杆状病毒中的表达 总被引：2，自引：0，他引：2
根据已发表的猪生殖 -呼吸道综合征病毒 ( PRRSV) CH-1 a分离株核衣壳 ( N)蛋白基因核苷酸序列和杆状病毒转移载体 p Blue-Bac-His B多角体蛋白阅读框架 ,设计合成了 1对特异性引物 P7S1 /P7R1。应用PCR对重组质粒 p UC1 8-ORF7扩增 ,获得了 CH-1 a株 N基因的片段。经 Hind 和 Bgl 双酶切 ,将其定向克隆到同样双酶切的 p Blue-Bac-His B的 PH启动子下游 ,获得转移载体 p Blue-Bac-His B-ORF7。将转移载体p Blue-Bac His B-ORF7与苜蓿银蚊夜蛾多核型多角体病毒 ( Ac MNPV,简称杆状病毒 )线性化 DNA ( Bac-N-Blue TMDNA)共转染 Sf9细胞 ,经过蓝斑筛选和蚀斑纯化 ,获得重组病毒 r Bac7。经用引物 P7S1 /P7R1和杆状病毒多角体蛋白基因通用引物 PCR鉴定 ,证明目的基因已插入到杆状病毒中。将重组病毒接种于对数生长期的 Sf9细胞 ,分别于感染后 2 4、4 8、72、96、1 2 0 h收集感染细胞 ,经 SDS-PAGE和 Western blot分析 ,结果表明 ,细胞接种重组病毒 2 4 h即开始表达重组蛋白 ,至 96h达到峰值 ,占整个细胞蛋白的 8.4 % ,此后开始下降。表达产物为融合蛋白 ,大小约 2 0 0 0 0。将重组病毒感染的 Sf9细胞用抗 PRRSV N蛋白的单克隆抗体SDOW-1 7进行间接免疫荧光试验 ,结果在细胞浆和细胞核中观察到了特异性的亮绿 相似文献
10.
Xiang QW Wang X Xie ZJ Sun YN Zhu YL Wang SJ Liu HJ Jiang SJ 《Veterinary microbiology》2012,159(1-2):251-256
Duck circovirus (DuCV) is classified in the genus Circovirus of the Circoviridae family. Two major open reading frames (ORFs), encoding the replicase (ORF1/rep) and the capsid protein (ORF2/cap), have been recognized for DuCV. Sequence analysis show that another major conserved ORF (named ORF3) is located in the complementary strand of ORF1/rep of DuCV, and its function remains to be investigated. In this study, the ORF3 of DuCV was expressed in recombinant baculovirus-infected Sf9 cells. By IFA and Western blot analysis, the ORF3 protein was positive for the sera from ducks infected with DuCV. The percentages of apoptotic cells of the Sf9 cells infected with the recombinant baculovirus encoding ORF3 of DuCV were significantly higher than (P<0.05) that of the Sf9 cells infected with wild-type baculovirus at 24, 48 and 72 h postinfection. Based on our knowledge, we deduced that the ORF3 protein of DuCV might play an important role in viral pathogenesis via its apoptotic activity. 相似文献
11.
Franceschi V Capocefalo A Ravanetti L Vanderplasschen A Gillet L Cavirani S van Santen VL Donofrio G 《Veterinary microbiology》2011,148(2-4):219-231
The ORF50/Rta gene has been shown to be an essential gene for many gammaherpesviruses. Although the BoHV-4 ORF50/Rta homolog, immediate early gene 2 (IE2), has been shown to activate several BoHV-4 early and late promoters in cotransfection assays, there is no direct proof of its indispensability for progression of the virus to the lytic replication cycle in the context of the viral genome. In the present communication, replication defective BoHV-4-V.test IE2 mutants were efficiently rescued, with respect to production of infectious virus and DNA replication, upon the expression of BoHV-4 ORF50/Rta in trans. Surprisingly, in the course of our studies, we discovered that the IE2 gene is duplicated in the genome of BoHV-4-U. 相似文献
12.
Thirteen infectious laryngotracheitis virus (ILTV)-specific monoclonal antibodies (MAbs) were isolated after immunization of mice with purified infectious laryngotracheitis virions. On the basis of their reactions in western blot analyses of ILTV-infected cells, the MAbs were assigned to five different virus proteins or protein groups. Two of the viral target proteins could be identified after transient expression of cloned ILTV genes in eucaryotic cells. The MAbs of group II detected a 60-kD protein that was shown to be the ILTV homologue of herpes simplex virus type 1 (HSV-1) glycoprotein (g)C. The MAbs of group I reacted with the positional homologue of HSV-1 gJ, which is encoded by the open reading frame (ORF) 5 gene within the unique short genome region of ILTV. The ORF 5 gene product of ILTV was previously described as a 60-kD glycoprotein (gp60), whereas multiple protein bands with apparent molecular masses of 85, 115, 160, and 200 kD were identified in the present study. Immunoelectron microscopy revealed that both gC and gJ of ILTV are localized in the envelope of virus particles, whereas the 15-kD protein detected by the MAbs of group III presumably represents a tegument component. Immunofluorescence analyses of infected cells demonstrated that the epitopes of the gC- and gJ-specific MAbs are conserved in all tested ILTV isolates originating from different parts of the world and that these MAbs are also suitable for in situ antigen detection in tissues of ILTV-infected chickens. The remaining ILTV-specific MAbs recognized viral proteins of 22 kD (group IV) and 38 kD (group V) that were not further characterized up to now. 相似文献
13.
Expression or subcellular targeting of virus capsid proteins with cloning genome of a canine parvovirus from China 总被引：1，自引：0，他引：1
A strain of canine parvovirus (CPV), designated B2004, was isolated from the stool of a sick dog in Beijing. The partial genome (4623 bp) was cloned, sequenced with sequence showing B2004 to be a member of the widely distributed CPV-2a subclade. A completed VP2 or 11-residue N-terminal peptide (MAPPAKRARRG) of VP1 from B2004 was also tested for its ability to mediate nuclear transport of a heterologous protein, in this case enhanced green fluorescence protein (EGFP). EGFP was detected in the nucleus when it fused with the VP1 peptide; it was distributed primarily in the nucleus and also in the cytoplasm either when it fused with VP2, or in the cytoplasm when expressed on its own. In common with other parvoviruses the CPV VP1 N-terminal peptide contributes to the nuclear localization of the gene product. 相似文献
14.
根据GenBank中猪圆环病毒2型(PCV2)的核酸序列,设计了1对引物,采用PCR方法从疑似断奶仔猪多系统衰竭综合征(PMWS)的死亡仔猪病料中扩增出了ORF3基因,将其克隆到高效真核表达载体pEGFP- N2中,筛选出含有ORF3基因的重组质粒,命名为pEGFP-N2-ORF3.对此重组质粒进行测序分析,结果表明,克隆的ORF3基因与其他PCV2的ORF3核苷酸序列相似性为96.2％-99.0％,氨基酸序列同源性为90.5 ％～98.1％.将重组质粒纯化后转染COS-7细胞,用PCR方法扩增出了特征性的基因片段,并采用特异性单抗进行间接免疫荧光,结果转染pEGFP- N2-ORF3重组蛋白在细胞核和细胞浆中均有表达,尤其在细胞核中表达量较高,且对细胞有一定的毒性.本研究为进一步探讨ORF3的结构和功能提供理论依据. 相似文献
15.
猪圆环病毒2型广西株的分离和全基因组序列分析 总被引：1，自引：1，他引：0
从广西表现为断奶仔猪多系统衰竭综合征的猪群中分离到1株猪圆环病毒2型(PCV-2),命名为GXB株.对GXB株的全基因组进行PCR扩增,扩增产物克隆至PMD18-T载体.测序结果表明,全基因组为1 767 bp,与GenBank上已知的8株PCV-2参考株序列的同源性在95.0%～99.6%之间.序列分析表明,GXB株基因组包含11个读码框(ORF),其中ORF1和ORF2是最主要的读码框,分别编码314个和233个氨基酸,与其他PCV-2毒株的ORF1、ORF2氨基酸的同源性分别为97.8%～100%、91.5%～98.7%.对GXB株ORF2编码的Cap蛋白基因进行功能分析,表明含有1个潜在的糖基化位点,3个明显的亲水区,有较强的抗原性和亲水性,为作为主要的免疫原性蛋白基因提供了依据. 相似文献
16.
猪繁殖与呼吸综合征病毒的分子生物学研究进展 总被引：1，自引：0，他引：1
猪繁殖与呼吸综合征是目前养猪业中一种严重的病毒性传染病,引起猪严重繁殖障碍和呼吸道疾病。该病的病原体属于动脉炎病毒属,是一种不分节段的单股正链RNA病毒,含有8 个开放阅读框(ORFs),ORF1 编码非结构蛋白,ORF2~ORF7 编码结构蛋白。其中ORF7 编码的核衣壳(N)蛋白和ORF6编码的非糖基化基质(M)蛋白为优势结构蛋白。猪繁殖与呼吸综合征病毒基因组存在广泛的遗传变异性,M蛋白和N蛋白在所有的毒株之间相对比较保守,可作为血清学诊断的靶抗原。文章就猪繁殖与呼吸综合征病毒在分子生物学方面的研究情况做作一综述。 相似文献
17.
猪传染性胃肠炎病毒核衣壳(N)蛋白基因的克隆与表达 总被引：5，自引：1，他引：5
以猪传染性胃肠炎病毒亚基因组mRNA为模板根据文献设计一对引物，通过RT－PCR技术，扩增其核衣壳（N）蛋白基因的cDNA；将其按正确的阅读框架定向克隆到表达载体pProEXHTb中特异酶切位点；将重组质粒PHN转化进大肠杆菌TG1株，在浓度为1.0mM IPTG和37℃条件下诱导，PHN基因融合蛋白获得了表达；经SDS－PAGE，Western-blot试验，确定其表达的融合蛋白产物大小为预期的47kD。试验结果证明，在大肠杆菌中表达的TGEV N基因的融合蛋白产物的确具有天然蛋白的抗原性。 相似文献
18.
Equine herpesvirus type 1 (EHV-1) is associated with abortions, respiratory distress, and neurological disturbances in horses. The ORF37 of EHV-1 encodes a protein homolog to UL24 gene product of human herpesvirus that has been associated with neurovirulence. In the present work, ORF37 PCR fragments derived from two Brazilian EHV-1 isolates, a German isolate and an American reference strain were sequenced and characterized by molecular phylogenetic analysis. This genomic region is highly conserved an allowed to infer genetic distances between EHV-1 strains and other animal herpesvirus. 相似文献
19.
Porcine circovirus type 2 (PCV2), a single-stranded DNA virus, is associated with postweaning multisystemic wasting syndrome
(PMWS). ORF2 protein (capsid) of PCV2 was recently demonstrated to be a major immunogenable to induce protection in pigs with
a prime–boost protocol. In this study, the ORF2 gene of PCV2 was expressed in insect cells. The product self-assembled into
particles that were structurally and antigenically indistinguishable from regular PCV2 capsids. To evaluated the immunogenicity
of these virus-like particles, PCV2-free piglets were vaccinated with the crude lysate from recombinant baculovirus (Ac.ORF2)-infected
insect cells, at doses of 0.1 ml (106 cells), 0.5 ml (5 × 106 cells) or 1.0 ml (107 cells). The immune response was monitored by an indirect enzyme-linked immunosorbent assay (ELISA) for PCV2 antibody and
lymphocyte proliferation assay. The ELISA results indicated that primary immune response was elicited with 0.5 ml or 1.0 ml
of crude lysate from Ac.ORF2. After boost immunization, relatively higher levels of PCV2 antibody were elicited in 0.5-ml
or 1.0-ml vaccinated groups, compared to the 0.1-ml group. In addition, higher PCV2 specific lymphocyte proliferation response
was developed in piglets vaccinated with 0.5 ml or 1.0 ml of crude lysate, especially in those vaccinated with with 1.0 ml
of crude lysate. Thus, the expressed ORF2 protein has significant potential as a subunit vaccine against PCV2 infection. 相似文献