1型牛疱疹病毒bICP27蛋白在病毒感染中的作用

1型牛疱疹病毒(Bovine herpesvirus type 1,BHV-1)主要感染牛,对牛的呼吸系统、眼结膜、生殖系统和神经系统均有损害。BHV-1编码的牛感染细胞蛋白27(bovine infected cell protein 27,bICP27)是一种可在细胞核和细胞质之间穿梭的立即早期(immediate early, IE)蛋白,可以抑制宿主的先天性免疫应答,能够在病毒感染早期刺激病毒蛋白表达,在感染后期促进病毒复制,调控病毒mRNA转录和宿主基因表达。笔者综述了近年来有关bICP27蛋白在BHV-1感染宿主过程中的作用,以期为BHV-1的深入研究提供参考和借鉴。     

牛传染性鼻气管炎病毒部分糖蛋白的研究进展

牛传染性鼻气管炎又名牛疱疹病毒Ⅰ型感染症,是一种急性、热性、接触性传染病,该病能给养牛业造成重大经济损失.该病毒是双股DNA有囊膜的病毒,病毒囊膜糖蛋白对病毒的吸附、侵入和细胞间扩散是必需的,可刺激机体产生中和抗体.囊膜糖蛋白的研究不仅能了解病毒分子生物学的结构和功能,而且对该病的诊断和预防也具有重要意义.笔者就gB蛋白和gE蛋白的结构和分子生物学特性作一综述,并对其进行了展望.     

牛疱疹病毒型gE基因缺失株的构建和特性研究

牛疱疹病毒 型 (BHV- 1) ,又称传染性牛鼻气管炎病毒 (IBRV) ,主要引起牛的鼻气管炎、结膜炎和生殖器官感染 ,有时还引起流产、肠炎、脑炎和全身感染。 BHV- 1基因组编码约 70种蛋白 ,其中 11种为糖蛋白。由 g E基因编码的糖蛋白 g E并非病毒体外增殖所必需 ,但却是病毒在动物体内潜伏所必需的。目前在欧洲使用一种 IBRV g E缺失标记疫苗根除 IBR,但在美国仍未见此疫苗应用的报道。本研究报告的目的是获得了一株无毒的 g E缺失的IBRV株 ,该毒株含有 β-半乳糖苷酶 (β- gal)标志 ,我们删除了 IBRV Cooper株编码 g E基因的大部…     

牛传染性鼻气管炎病毒gB、gE蛋白的研究概述

牛传染性鼻气管炎病毒（Infectious bovine rhinotracheitis virus，IBRV）又名牛疱疹病毒Ⅰ型（bovine herpesvirus Ⅰ，BHV-1）。BHV-1基因能编码11种糖蛋白，其中gB蛋白和gE蛋白在病毒致病过程和刺激机体产生抗体的过程中都起到较大的作用。笔者就关于gB、gE这2种糖蛋白的研究做一概述。     

表达牛呼吸道合胞体病毒G蛋白基因的重组Ⅰ型牛疱疹病毒的构建与鉴定

为构建表达牛呼吸道合胞体病毒(BRSV)G蛋白基因的牛疱疹病毒Ⅰ型(BHV-1)重组病毒,本研究将人工合成的BRSV全长G蛋白基因编码序列插入到巨细胞病毒(CMV)启动子之下构建TK基因缺失转移载体。利用磷酸钙-DNA沉淀法将该转移载体与亲本病毒BHV-1/TK-/LacZ+的基因组DNA共转染牛鼻甲细胞后收获增殖的病毒。通过反向蚀斑筛选,得到重组病毒BHV-1/TK-/G+。PCR检测结果证实G蛋白基因已经插入到了亲本病毒BHV-1/TK-/LacZ+的基因组中,间接免疫荧光试验和western blot证实BHV-1/TK-/G+中的G蛋白基因在感染的细胞中获得了表达。本研究为研制BRSV及其他重要牛传染病的BHV-1病毒活载体疫苗奠定了基础。     

牛疱疹病毒1型诱发牛呼吸道综合征的研究进展

牛疱疹病毒1型(BHV-1)感染牛可导致上呼吸道疾病、结膜炎、生殖疾病和免疫抑制。BHV-1诱导的免疫抑制引起了牛呼吸系统疾病(BRDC)。BHV-1至少编码3个可以抑制免疫系统的特定蛋白:1.hICP0抑制干扰素依赖性转录。2.UL41.5蛋白通过防止病毒多肽转运到细胞表面抑制CD8~+T细胞识别的感染细胞。3.糖蛋白G是一种化学激酶结合蛋白,可以防止淋巴细胞回到感染的部位。在犊牛急性感染后,BHV-1也可感染和诱导CD4~+T细胞的高水平凋亡。因此,BHV-1损害免疫反应的能力可导致BRDC。急性感染后,BHV-1在三叉神经节感觉神经元(TG)和咽扁桃体生发中心建立潜伏期。BHV-1从潜伏期周期性地重新激活,病毒脱落,从而发生病毒传播。潜伏期相关基因和ORF-E两种病毒基因在潜伏期大量表达,说明它们调控着潜伏期再活化周期。BHV-1能够进入受纳细胞,感染感觉神经元,促进病毒从感觉神经元向黏膜表面传播,潜伏期的再激活也受几种病毒糖蛋白的调控。本文综述了BHV-1的生物学特性及其与BRDC的关系。     

BHV-1在牛胚气管细胞中的增殖规律研究

为研究牛疱疹病毒Ⅰ型(BHV-1)在牛胚气管细胞(EBTr)中的生长特性和增殖规律,参考文献设计合成特异性引物和探针,建立了TaqMan实时荧光定量PCR方法,检测BHV-1感染EBTr细胞6、12、24、48、72、96、120、144h后病毒的增殖规律,并观察对应时间点的细胞病变(CPE)。结果显示,用100TCID_(50)的BHV-1感染EBTr细胞,48h后开始出现细胞病变,72h细胞病变明显,120h细胞大部分变圆,开始脱落,144h细胞大面积脱落、崩解。实时荧光定量PCR检测结果表明,BHV-1感染EBTr在6h~24h内,病毒缓慢增殖,48h~96h,病毒增殖速度加快,拷贝数呈对数增长,120h~144h,BHV-1的含量仍然呈现升高的趋势,但增长速度变慢。结果证明,BHV-1能够在EBTr中产生CPE,而且CPE程度与病毒DNA增殖规律一致,该结果可以为深入研究BHV-1对EBTr细胞的致病机理提供基础资料。     

牛传染性鼻气管炎

<正>牛传染性鼻气管炎是由牛传染性鼻气管炎病毒引起牛的一种急性接触性传染病。又称牛疱疹病毒I型感染、红鼻病或牛传染性坏死性鼻炎。临床特征为呼吸困难和发热,有鼻炎、鼻窦炎、喉炎和气管炎。1病毒牛传染性鼻气管炎病毒又称牛疱疹病毒I型(BHV-1),属于疱疹病毒科疱疹病毒甲亚科成员。病毒在pH6.9～9.0时稳定,在pH4.5～5.0下可被灭活。病毒在4℃可保存1个月,37℃存活10天左右,多种消毒剂均可使病毒灭活,如0.5%氢氧化钠、0.01%氯化汞、1%漂白粉、1%酚     

牛疱疹病毒I型二重LUX荧光PCR鉴别检测方法的建立

采用LUX新型荧光PCR技术原理，建立了快速检测牛疱疹病毒I型（BHV-1）以及鉴别野毒感染和基因缺失疫苗免疫动物的二重实时荧光PCR方法。结果显示，该方法对多株BHV-1病毒均呈典型gE、gC双基因阳性反应，对其他常见动物疱疹病毒以及健康牛基因组DNA等均呈阴性反应；对细胞增殖病毒液的gE、gC双基因鉴别的检测敏感性可达0．4～O．04TCID50比常规PCR方法高100倍以上；对带毒牛血清、抗凝全血、牛新鲜精液和冷冻精液基因鉴别的检测敏感性分别达0．04TCID50、0．4TCID50、0．4TCID50和4TCID50。采用该方法从临床牛血样、鼻拭子样品中检出IBRV阳性样品，检测全程仅需约2h。对单基因克隆质粒的检测进一步证实该方法能特异地鉴定gE、gC基因，检测灵敏度分别达90、30拷贝。结果表明，该方法可应用于临床快速诊断BHV-1病毒感染，鉴别BHV-1病毒感染与对应的基因缺失疫苗免疫动物。     

1型牛疱疹病毒(BHV-1)感染最新研究进展

1 病毒 1型牛疱疹病毒(BHV-1)可以引起传染性牛鼻气管炎(IBR)。IBR是一种在世界各地都有发生的地方性疾病,常常引起大规模饲养场的暴发,引起轻微到严重的呼吸道和泌尿生殖道疾病,被列为OIE的B类疾病名单中,对社会经济和(或)公共卫生有重要的影响,对动物和动物产品的国际贸易有明显的影响。     

Persistent infection of bovine herpesvirus type 4 in bovine endothelial cell cultures

Herpesviruses can establish a persistent infection in the cells and tissues of their natural hosts and thus may produce diseases due to cytolytic infections. We have isolated a herpesvirus from a bovine vascular endothelial cell culture after continuous subculturing. Typical cytopathic changes were observed in bovine endothelial cell cultures 2 days after inoculation of the virus. The virus had an icosahedral nucleocapsid of 100-150 nm in diameter and an envelope. The sequences of some DNA fragments of the virus were highly homologous to those of the bovine herpesvirus type 4 (BHV-4) strains. The DNA restriction maps of the virus and the reference strains of BHV-4, DN 599 and Movar 33/63 were very similar but not identical. Therefore, the newly isolated virus has been designated Taiwan strain. The presence of BHV-4 DNA in apparently normal bovine endothelial cell cultures was shown by Southern blot hybridization with the BamHI fragment of the newly isolated BHV-4 and was further confirmed by digestion of the DNA with BamHI plus AccI. In conclusion, we have demonstrated that BHV-4 persisted in the bovine endothelial cell cultures and continuous subcultures could lead to the production of infectious viral particles.     

Immunogenic properties of a bovine herpesvirus-1 (BHV-1) recombinant expressing major pseudorabies virus (PrV) glycoproteins in combination

A recombinant bovine herpesvirus type 1 (BHV-1), designated BHV-1/TF17-1, which expresses pseudorabies virus (PrV) glycoproteins gB, gC, gD, gE and gI in combination was constructed. To test the protective immunity, 10 mice were inoculated with BHV-1/TF17-1 and three weeks later 10 mice were intraperitoneally (i.p.) challenged with 20 LD50 virulent PrV (YS-81). BHV-1/TF17-1 protected all the mice from the PrV lethal challenge while all the control mice died in around 3 days. Mice vaccinated with BHV-1/TF17-1 acquired high PrV-neutralizing antibody titers and demonstrated strong delayed type hypersensitivity responses and moderate in vitro lymphocyte proliferative responses to PrV antigen. Since the major PrV glycoproteins were integrated into virions (probably into viral envelope), BHV-1/17-1 was neutralized with anti-PrV antiserum. However, the susceptibility of BHV-1/TF17-1 to anti-PrV antiserum is 2- to 4-fold lower than that of PrV vaccine lines. Our results demonstrated the possibility of BHV-1/17-1 as a vaccine to protect piglets from Audjesky's disease where maternal antibodies against PrV interfere attenuated live PrV vaccines.     

ISCOM of BHV-1 envelope glycoproteins protected calves against both disease and infection.

A subunit vaccine in the form of immunostimulating complex (iscom) was prepared to contain the envelope glycoproteins of bovine herpesvirus type 1 (BHV-1). This iscom preparation was tested in a vaccination experiment on 4-month-old calves seronegative to BHV-1. In this experiment, four groups with three animals per group were used. Two groups were vaccinated with the iscom preparation twice, four weeks apart, one group with 50 micrograms and the other with 100 micrograms per calf. The third group received a commercial inactivated whole-virus vaccine applying the same vaccination program. The fourth group served as control. Two weeks after the second vaccination, all the animals were challenge-infected intranasally with a virulent BHV-1 strain and four days later with a virulent Pasteurella multocida--this in order to mimic hard field conditions. When exposed to challenge infection, all the animals vaccinated with the iscom were fully protected, i.e., no virus could be recovered from their nasal secretions and no clinical symptoms were recorded. In contrast, the animals vaccinated with the commercial vaccine, responded to challenge with moderate fever and loss of appetite, and virus was isolated from the nasal secretions. The animals in the control group developed severe clinical symptoms. In the sera of iscom-vaccinated animals, the virus neutralization titers reached levels of 1/3500 or higher.     

Seroprevalence to bovine virus diarrhoea virus and other viruses of the bovine respiratory complex in Venezuela (Apure State)

Six hundred and fifteen serum samples obtained from cows in five districts of Apure State, Venezuela, were tested by ELISA for antibodies to bovine virus diarrhoea virus (BVDV). The same samples were also ELISA-tested for antibodies to bovine herpesvirus type 1 (BHV-1) and bovine respiratory syncytial virus (BRSV). Additionally, the haemagglutination-inhibition (HI) test was used for detecting antibodies to parainfluenza virus type 3 (PIV-3). Overall, seroprevalence to BVDV was 36±7% (SE); seroprevalence varied by district (19–42%). BHV-1 seroprevalence was 67±4%; variation by district was similar to that of BVDV. However, the first 80 serum samples tested by BHV-1 ELISA all had a strong background reaction with the control antigen. Therefore, these sera were adsorbed to a homogenate of non-infected bovine kidney cell line (MDBK) and re-tested by ELISA. The non-specific reactivity was significantly reduced (p < 0.001 by Wilcoxon's signed-rank test). Compared to the virus-neutralisation (VN) test, the adsorbed BHV-1 ELISA showed 94% agreement and gave a κ value of 0.84, indicating that the adsorption did not interfere with test accuracy. Seroprevalence against BRSV was 85±3%, and showed differences across districts. Most of the cows (94±2%) were seropositive to PIV-3, and there were no significant differences among districts.     

Effect of lymphocytes and broncho-alveolar washing cells on the replication of bovine herpesvirus type 1 in tracheal organ cultures

The daily addition of lymphocytes collected from a calf between 7 and 11 days after experimental infection with bovine herpesvirus type 1 (BHV-1) to bovine fetal tracheal organ cultures after infection with BHV-1 did not inhibit virus replication. The daily addition of normal lymphocytes, together with a low concentration of serum antibody against BHV-1, had a slight viral inhibitory effect which was believed to be due to antibody-dependent cell-mediated cytotoxicity. The addition of broncho-alveolar washing (BAW) cells, collected before infection or 30 days after infection of a calf with BHV-1, together with lymphocyte culture supernatant, to tracheal organ cultures immediately after infection with BHV-1 produced some inhibition of virus replication. Virus replication was markedly inhibited when BAW cells collected from the calf 18 days after infection were used in a similar manner.     

Bovine herpes virus expressing envelope protein (E2) of bovine viral diarrhea virus as a vaccine candidate.

The gene encoding the envelope protein (E2) of bovine viral diarrhea virus (BVDV) was expressed under the thymidine kinase (TK) promoter of Korean bovine herpesvirus 1 (BHV-1) isolate. Thymidine kinase negative (TK-) BHV-1 recombinants expressing E2 of BVDV were constructed and the expression of E2 was identified by immunofluorescence and Western blotting. Compared to wild type BHV-1, the recombinant BHV-1 had a delayed cytopathogenic effect in cells. The immunogenicity of the recombinant BHV-1 was examined in guinea pigs and cattle. Although an increase in body temperature was detected for a few days, the inoculated cattle returned to normal temperature with the development of neutralizing antibodies to BVDV.     

Effects of hypervaccination with bovine herpesvirus type 1 gE-deleted marker vaccines on the serological response and virological status of calves challenged with wild-type virus

Twenty-four calves were immunised four times with gE-deleted infectious bovine rhinotracheitis marker vaccines before being challenged with small doses of wild-type bovine herpesvirus type 1 (BHV-1). The repeated vaccinations induced strong immunity that prevented detectable virus replication and gE-seroconversion after the challenge infection in most of the calves. The hypervaccinated calves that shed virus after the challenge infection showed no delay in gE-seroconversion compared with unvaccinated control calves. Using a sensitive nested PCR, BHV-1 gE sequences could be detected in the trigeminal ganglia of several of the gE-seronegative, challenge-infected calves, possibly indicating the presence of wild-type BHV-1 DNA.     

Effects of some components of the humoral immune response on the replication of bovine herpesvirus type I in tracheal organ cultures

The addition of high concentrations of serum neutralizing antibody against bovine herpesvirus type I (BHV-1) to bovine fetal tracheal organ cultures before and after infection with a minimal infectious dose of BHV-1 completely inhibited virus replication. The daily addition of serum antibody from day 0 to day 2 after infection markedly reduced virus yields but failed to cure the infection. The antiviral effect of nasal antibody was not superior to that of an equivalent concentration of serum antibody. Treatment of infected organ cultures with complement sometimes enhanced the antiviral effect of antibody. Peripheral blood lymphocytes from an experimentally infected calf were cultivated in the presence of BHV-1 antigen, and the culture supernatants were shown to possess interferon activity. Pretreatment of organ cultures with this material failed to inhibit BHV-1 replication, but when the interferon treatment was continued daily after infection, there was a transient reduction in BHV-1 replication.     

Bovine herpesvirus type 1 infection of bovine bronchial epithelial cells increases neutrophil adhesion and activation

Respiratory infection of cattle with bovine herpesvirus type 1 (BHV-1) predisposes cattle to secondary pneumonia with Mannheimia haemolytica as part of the bovine respiratory disease complex (BRD). One cell type that has received limited investigation for its role in the inflammation that accompanies BRD is the respiratory epithelial cell. In the present study we investigated mechanisms by which BHV-1 infection of respiratory epithelial cells contributes to the recruitment and activation of bovine polymorphonuclear neutrophils (PMNs) in vitro. Primary cultures of bovine bronchial epithelial (BBE) cells were infected with BHV-1 and assessed for cytokine expression by real-time PCR. We found that BHV-1 infection elicits a rapid IL-1, IL-8 and TNF-α mRNA response by BBE cells. Bovine PMNs exhibited greater adherence to BHV-1 infected BBE cells than uninfected cells. The increased adherence was significantly reduced by the addition of an anti-IL-1β antibody or human soluble TNF-α receptor (sTNF-αR). Pre-incubation of bovine PMNs with conditioned media from BHV-1 infected BBE cells increased PMN migration, which was inhibited by addition of an anti-IL-1β antibody, sTNF-αR, or an IL-8 peptide inhibitor. Conditioned media from BHV-1 infected BBE cells activated bovine PMNs in vitro as demonstrated by PMN shape change, production of reactive oxygen species and degranulation. PMNs also exhibited increased LFA-1 expression and susceptibility to M. haemolytica LKT following incubation with BHV-1 infected BBE cell conditioned media. Our results suggest that BHV-1 infection of BBE cells triggers cytokine expression that contributes to the recruitment and activation of neutrophils, and amplifies the detrimental effects of M. haemolytica LKT.