表达牛流行热病毒G基因的牛传染性鼻气管炎病毒转移载体的构建

本试验将克隆到 p BLG中的牛流行热病毒 (BEFV)糖蛋白 G基因亚克隆到含有 CMV启动子和 Poly(A)信号尾的 p CR3- Uni载体上 ,获得 p CR3- G重组体 ,酶切后将含有 CMV、G基因和 Poly(A)的目的片段 (约 3.6 kb)插入通用转移载体 pd TK- CMB中 ,获得 pd TK- CMB- G重组体 ;再将含 SV4 0启动子的 L ac Z报告基因也亚克隆到该载体中获得表达 BEFV G基因的pd TK- L ac Z- G转移载体 ,为开发 BEFV/IBRV二联基因工程疫苗奠定了基础     

BHV-1gG-／tk-／GM-CSF＋重组病毒的构建及其免疫效果的评价

通过引入免疫增强因子GM-CSF,提高重组病毒的免疫原性。将牛GM-CSF克隆到pcDNA3．1载体中,构建GM-CSF基因表达盒;将tk基因上游同源臂、GM-CSF基因表达盒、tk基因下游同源臂克到pcDNA3．1载体中,得到转移栽体pTK-GM-CSF。将亲本病毒BHV-1gG-／TK-／EGFP’基因组与转移载体pTK-GM-csF采用磷酸钙法共转染MDBK~g,得到重组病毒BHV-1gG／TK-／GM-csF’。并对重组病毒的生物学特性和对日本大耳白兔的免疫原性进行了评价。利用PCR和RT-PCR证实GM-CSF基因表达盒已正确插入重组病毒的tk基因位置,并具有转录活性。亲本株和重组株的空斑形态、空斑直径大小无明显差异;两者病毒滴度无明显变化．生长曲线相似;体外遗传稳定性良好。兔体试验表明,在免疫早期重组毒株能产生较亲本株更高水平的IFN-B（P〈0.05）。但中和抗体水平、攻毒后的临床症状及病毒排毒时间等各项指标检测表明,亲本株和重组毒株之间无明显差异。在免疫早期重组株刺激机体产生的IFN-p高于亲本株,具有-定的应用前景。     

伪狂犬病病毒TK-/gI-株PK基因转移载体的构建及LacZ基因表达

伪狂犬病病毒TK-/gI-株基因组经AscⅠ酶切获得含PK基因的8.7 kb片段,将此片段克隆入pPolyⅡ载体的AscⅠ位点,获得中间载体P8-AA,将LacZ表达盒插入其SphⅠ/NdeⅠ位点,构建重组转移载体P8AA-lacZ,将其与伪狂犬病病毒TK-/gI-株基因组共转染PK-15细胞,细胞出现病变后通过覆盖含有X-gal的营养琼脂进行蓝白斑筛选,通过蚀斑克隆方法分离到重组体PRV(rPRV),且经过连续传代的rPRV仍能稳定的表达β-半乳糖苷酶活性.通过对重组病毒PRV-LacZ和亲本株TK-/gI-株TCID50的试验表明,PK基因的缺失对病毒增殖无影响.     

共表达口蹄疫病毒vp1基因和山羊IFN-γ基因的重组山羊痘病毒的构建

将山羊IFN-γ基因插入到合有口蹄疫病毒（FMDV）vp1基因的山羊痘病毒（GPV）转移载体PTK-vp1中,获得合有FMDV vp1基因和山羊IFN-γ基因的重组转移载体pTK-vp-IFNγ,以本实验室构建的表达β-半乳糖苷酶基因（LacZ）的山羊痘重组病毒rAY41-LacZ为亲本毒,与转移载体pTK-vp-IFNγ共同转染犊牛睾丸细胞进行同源重组,通过7轮反向蓝白斑筛选,产生重组山羊痘病毒rAY41-VP-IFNγ,通过PCR和间接免疫荧光实验证实获得了能够稳定表达FMDV vp1和山羊IFN-γ的重组山羊痘病毒。     

牛呼吸道合胞体病毒G蛋白抗原区原核表达及反应原性鉴定

本研究旨在获得重组牛呼吸道合胞体病毒（BRSV） G蛋白并对其进行反应原性鉴定。从NCBI上找出G基因的核苷酸序列，分析其抗原区域，利用Primer Premier 5.0软件设计1对特异性引物，利用RT-PCR方法扩增BRSV G基因片段，将目的片段连接到克隆载体pMD19-T后，对其进行双酶切鉴定和测序。将双酶切后的目的片段进行胶回收，构建重组质粒pET-32a-G，将重组质粒转化大肠杆菌BL21（DE3）感受态细胞，用Ni-IDA亲和层析法纯化经IPTG诱导表达的蛋白，并测定其浓度；通过SDS-PAGE和Western blotting方法对该蛋白进行分析与鉴定。结果显示，本试验成功克隆出大小约为567 bp的G基因，获得分子质量约为40 ku的可溶性重组蛋白，优化诱导条件后用SDS-PAGE对表达产物进行鉴定，在16℃、IPTG浓度1.2 mmol/L、诱导4 h时蛋白表达量最大；通过Western blotting检测发现，重组蛋白可与山羊源BRSV标准阳性血清发生特异性反应，说明该蛋白具有反应原性。综上，本研究成功表达并纯化了BRSV G蛋白，为建立BRSV抗体间接ELISA检测方法和BRSV亚单位疫苗的研制奠定了基础。     

猪伪狂犬病病毒流行株gE/gI双基因缺失突变株的构建及纯化

根据GenBank中公布的猪伪狂犬病病毒(porcine pseudorabies virus,PRV)US区基因序列(KJ789182),设计2对特异性引物,扩增PRV NY株gE/gI基因两侧序列,克隆至pUC-19载体,构建转移质粒pUC-gE/gILR,同时插入绿色荧光蛋白(EGFP)标记基因,构建转移质粒pUC-gE/gILRE,作为重组同源臂。以PRV NY流行株为亲本株,将其基因组与转移质粒pUC-gE/gILRE共转染ST细胞,通过筛选绿色荧光蚀斑,获得含有荧光蛋白的重组病毒rPRV NY-gE~-/gI~--EGFP~+。再以该毒株基因组与转移质粒pUC-gE/gILR进行同源重组,通过筛选无荧光蚀斑,纯化重组病毒,经PCR扩增、测序,证实获得了去除EGFP基因的重组病毒rPRV NY-gE~-/gI~-。该重组病毒在ST细胞上培养时,与亲本株PRV NY具有相似的生长曲线,但该重组病毒的体外生长动力学比亲本株弱。本研究成功构建了1株针对目前PRV流行株的gE/gI双基因缺失病毒,为我国根除PR提供技术支持。     

表达口蹄疫病毒P1基因的重组伪狂犬病病毒TK/gG-/PgG-P1的构建

为了构建口蹄疫与伪狂犬病二价基因工程疫苗株 ,将口蹄疫病毒 (FMDV) P1基因插入到伪狂犬病病毒 (PRV)通用载体 p Pg G- uni中 ,得到 PRV转移载体 p Pg G- P1。将转移载体与 Eco R 线性化后的 PRV弱毒疫苗株 TK- /g G- / lac Z 基因组 DNA共转染 PK- 15细胞 ,转染产物经多次空斑纯化和 PCR鉴定 ,获得了纯化的重组 PRV TK- /g G- / Pg G- P1,重组病毒基因组 DNA经酶切鉴定进一步表明 ,FMDV的 P1基因已成功地整合到 PRV弱毒疫苗株的基因组中。Western blot试验表明 ,FMDV P1基因在重组 PRV中得到表达。该研究为进一步研制口蹄疫与伪狂犬病二价基因工程疫苗奠定了坚实的基础     

伪狂犬病病毒gE/TK基因缺失突变株的构建

在伪狂犬病病毒转移载体pBdTK-Uni的多克隆位点中插入由SV40启动子控制下的IacZ基因表达盒,同时在右侧同源臂下游插入一个1.7kb的KpnI片段,构建成一个新的转移载体pUhi-LacZ.用该载体与Bartha-K61株基因组通过脂质体法共转染Vero细胞,经过10代蓝斑筛选纯化和PCR鉴定获得了一株稳定表达LacZ基因的伪狂犬病病毒gE/TK基因缺失突变株,命名为rPrV-LacZ.在不同的细胞(PK-15、IBRS-2、Vero和CEF)上,对该重组病毒与亲本病毒的增殖滴度和细胞病变进行比较,未见显著差异.结果表明转移载体pBdTK-Uni具有实用性,可用于构建伪狂犬病病毒基因工程活载体疫苗.     

共表达与牛疱疹病毒1型VP22融合的猪繁殖与呼吸综合征病毒E及M蛋白的重组伪狂犬病毒的构建及其免疫原性观察

为了研制猪繁殖与呼吸综合征病毒（PRRSV）基因工程疫苗，以伪狂犬病毒（PRV）gE基因缺失标志疫苗株TK^-／gE^-／LacZ^＋为病毒载体，通过同源重组，构建了共表达与牛疱疹病毒1型VP22（BHV-1 VP22）融合的PRRSV E及M蛋白的重组伪狂犬病毒（rPRV）TK^-／gE^-／VP22E^＋／VP22M^＋。经PCR、Southern blot、Western blot证实rPRV构建正确，并能表达与BHV-1 VP22融合的PRRSV E及M蛋白。rPRV在IBRS-2、PK-15细胞中的增殖滴度与PRV亲本株相比无显著差异，表明外源基因的插入不影响rPRV增殖。用该rPRV免疫BALB／c小鼠,检测免疫小鼠抗PRRSV中和抗体及脾淋巴细胞增殖反应，并与未融合VP22的单表达PRRSV E蛋白及共表达E及M蛋白的rPRV TK^-／gE^-／E^＋与TK^-／gE^-／E^＋／M^＋进行比较。结果显示TK^-／gE^-／VP22E^＋／VP22M^＋可诱导小鼠产生更好的体液与细胞免疫反应，BHV-1 VP22发挥了佐剂效应。本研究为研制安全、有效的猪繁殖与呼吸综合征-伪狂犬病二价基因工程疫苗奠定了基础。     

牛疱疹病毒I型gB基因的原核表达

根据GenBank中BHV-1 Colorado 1毒株的全基因组序列,针对gB基因的主要B细胞免疫区域设计一对引物。提取BHV-1 Colorado 1毒株的基因组DNA,PCR扩增大小为585 bp的gB片段,将其克隆至pET-22b（+）原核表达载体并测序分析,同时用Nde I和Not I酶切鉴定。SDS-PAGE和Western blot试验结果证明,获得的可溶性gB重组蛋白能与牛传染性鼻气管炎病毒标准阳性WEI血清发生特异性反应,为ELISA等免疫诊断方法的建立奠定了物质基础。     

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.     

Glycoprotein M of bovine herpesvirus 1 (BHV-1) is nonessential for replication in cell culture and is involved in inhibition of bovine respiratory syncytial virus F protein induced syncytium formation in recombinant BHV-1 infected cells

Cell cultures infected with BHV-1/F(syn), a recombinant bovine herpesvirus 1 (BHV-1) which expresses a synthetic open reading frame encoding the fusion (F) protein of the bovine respiratory syncytial virus (BRSV), showed a cytopathic effect (CPE) indistinguishable from that induced by wildtype BHV-1 although transient transfection experiments demonstrated that expression of the F protein leads to formation of large syncytia. Since it has been shown that glycoprotein M (gM) of pseudorabies virus inhibits BRSV F-induced syncytium formation in transient plasmid transfection experiments [Pseudorbies virus glycoprotein M inhibits membrane fusion. J. Virol. 74 (2000) 6760], the gM ORF of wtBHV-1 and BHV-1/F(syn) was interrupted. Infection of cell cultures with the resulting gM(-) mutant of BHV-1/F(syn) led to formation of syncytia, whereas the CPE in gM(-)BHV-1 infected cells was comparable to the CPE in wtBHV-1 infected cultures. Our results demonstrate that gM is not essential for BHV-1 replication in cell culture and that gM is involved in inhibition of the cell fusion activity of the BHV-1 expressed BRSV F protein.     

Duration of immunity of a quadrivalent vaccine against respiratory diseases caused by BHV-1, PI3V, BVDV, and BRSV in experimentally infected calves

Several laboratory studies assessed the duration of immunity of a quadrivalent vaccine (Rispoval™4, Pfizer Animal Health) against bovine respiratory diseases (BRD) caused by bovine herpes-virus type-1 (BHV-1), parainfluenza type-3 virus (PI₃V), bovine viral-diarrhoea virus type 1 (BVDV), or bovine respiratory syncytial virus (BRSV). Calves between 7 weeks and 6 months of age were allocated to treatment and then were injected with two doses of either the vaccine or the placebo 3 weeks apart. Six to 12 months after the second injection, animals were challenged with BHV-1 (n = 16), PI₃V (n = 31), BVDV (n = 16), or BRSV (n = 20) and the course of viral infection was monitored by serological, haematological (in the BVDV study only), clinical, and virological means for ≥2 weeks. Infection induced mild clinical signs of respiratory disease and elevated rectal temperature in both vaccinated and control animals and was followed by a dramatic rise in neutralising antibodies in all treatment groups. Titres reached higher levels in vaccinated calves than in control calves after challenge with BHV-1, BVDV, or BRSV. On day 3 after PI₃V challenge, virus shedding was reduced from 3.64 log₁₀ TCID₅₀ in control animals to 2.59 log₁₀ TCID₅₀ in vaccinated animals. On days 6 and 8 after BRSV challenge, there were fewer vaccinated animals (n = 2/10 and 0/10, respectively) shedding the virus than control animals (n = 8/10 and 3/10, respectively). Moreover, after challenge, the mean duration of virus shedding was reduced from 3.8 days in control animals to 1 day in vaccinated animals in the BVDV study and from 3.4 days in control animals to 1.2 days in vaccinated animals in the BRSV study. The duration of immunity of ≥6 months for PI₃V, BHV-1 and BVDV, and 12 months for BRSV, after vaccination with Rispoval™4, was associated mainly with enhanced post-challenge antibody response to all four viruses and reduction of the amount or duration of virus shedding or both.     

Association of herd BRSV and BHV-1 seroprevalence with respiratory disease and reproductive performance in adult dairy cattle

Background

The aim of this study was to detect the associations between bovine herpesvirus 1 (BHV-1) status of a herd and respiratory disease (BRD) occurrence and reproductive performance in pregnant heifers and cows. The association between management-related factors and higher BRD occurrence was also estimated.

Methods

Serum samples, collected from cows and youngstock from 103 dairy cattle herds, were analyzed for antibodies against BHV-1, bovine respiratory syncytial virus (BRSV), bovine viral diarrhoea virus (BVDV), and Mycoplasma bovis. A questionnaire was used to collect data concerning herd management factors and reproductive performance, as well as the occurrence of clinical signs of respiratory disease in the last two years, as evaluated by the veterinarian or farm manager. Multiple correspondence analysis (MCA) and logistic regression analysis were performed to identify and quantify the risk factors.

Results

A low to moderate prevalence (1-49%) of BRSV antibodies among youngstock was associated with a high occurrence of respiratory disease (OR = 6.2, p = 0.010) in cows and in-calf heifers. Employees of the farm may participate in the spread of such disease. Larger herd size, loose-housing of cows, housing youngstock separately from cows until pregnancy, and purchasing new animals were factors possibly related to a high occurrence of respiratory disease symptoms in pregnant heifers and cows. The highest risk of abortions (> 1.3%) and increased insemination index (number of inseminations per pregnancy) (> 1.9) occurred in herds with a moderate prevalence of BHV-1 antibodies (1-49%) in cows.

Conclusions

BHV-1 was not associated with acute respiratory disease in adult dairy cattle, however was significantly related to reproductive performance. BRSV possesses the main role in respiratory disease complex in adult dairy cattle.     

Seroprevalence of OHV-2, BVDV, BHV-1, and BRSV in ranch-raised bison (Bison bison).

Serum samples were collected at slaughter from 226 24-30-month-old American bison (Bison bison) bulls from Kansas, Minnesota, North Dakota, and Manitoba and assayed for antibodies to ovine herpesvirus type-2 (OHV-2), bovine viral diarrhea virus (BVDV), bovine herpesvirus type-1 (BHV-1), and bovine respiratory syncytial virus (BRSV). Antibodies were detected by serum neutralization for BVDV, BHV-1, and BRSV, while antibodies to OHV-2 were detected by competitive inhibition-ELISA (CI-ELISA). Detectable antibodies were found against all viruses: 10 of 226 (4.40%) against OHV-2, 125 of 226 (55.3%) against BVDV, 99 of 226 (43.8%) against BHV-1, and 208 of 226 (92.0%) against BRSV. Titers from 93.6% of the BVDV-positive animals, 79.8% of the BHV-1-positive animals, and 98.1% of the BRSV-positive animals were > or = 1.25. These data indicate that a low percentage of clinically normal bison are seropositive for OHV-2 while a high percentage of bison sampled are seropositive for BVDV, BHV-1, and BRSV.     

Release of tumor necrosis factor-alpha from bovine alveolar macrophages stimulated with bovine respiratory viruses and bacterial endotoxins

The release of tumor necrosis factor-alpha (TNF-) from cultured bovine alveolar macrophages (BAM) was evaluated following stimulation of BAM with bovine herpesvirus-1 (BHV-1), parainfluenza-3 (PI-3) virus, bovine respiratory syncytial virus (BRSV), Escherichia coli 0111:B4 endotoxin, Pasteurella haemolytica type 1 endotoxin, Pasteurella multocida endotoxin, and virus/endotoxin combinations. A cytotoxic assay system using Georgia bovine kidney cells as targets was used to measure TNF- activity. The cytotoxic activity was neutralized by an anti-human TNF- monoclonal antibody.

Stimulation of BAM with 1 median tissue culture infectious dose (TCID₅₀) of live or ultraviolet (UV)-inactivated PI-3 virus/cell resulted in release of TNF- in significantly (P<0.05) higher amounts than sham-induced BAM. The quantities of TNF- released after live or UV-inactivated BHV-1 or BRSV induction were not significantly higher than sham-induced BAM. E. coli 0111:B4, P. haemolytica type 1 and P. multocida endotoxins stimulated TNF- release in a dose-dependent manner. Sequential exposure of BAM to 1 TCID₅₀ per cell of either live BHV-1, PI-3 virus or BRSV and then 5 μg ml⁻¹ of either E. coli 0111:B4, P. haemolytica type 1 or P. multocida endotoxin caused a significant (P<0.05) reduction in detectable TNF- in seven of nine virus/endotoxin combinations tested, when compared with 5 μg ml⁻¹ of endotoxin alone. Parainfluenza-3 virus/endotoxin combinations stimulated higher TNF- release when compared with other virus/endotoxin combinations. Five out of six test animals had serum-neutralizing antibodies to PI-3 virus, one out of six had serum-neutralizing antibodies to BHV-1, and two out of six had serum-neutralizing antibodies to BRSV, suggesting a possible relationship between serum neutralizing antibodies and TNF- release from in vitro cultivated BAM.     

Immunogenicity of a recombinant pseudorabies virus expressing ORF1-ORF2 fusion protein of porcine circovirus type 2

Porcine circovirus type 2 (PCV2) is associated with post-weaning multisystemic wasting syndrome (PMWS). Pseudorabies (PR) is also an important infectious disease in swine and sometimes co-infect with PCV2. An attenuated pseudorabies virus (PRV) has been successfully used as a vector for live viral vaccines. In this study, a recombinant PRV expressing ORF1-ORF2 fusion protein of PCV2 was constructed and its immunogenicity was tested in mice and pigs. The ORF1 and partial ORF2 gene of PCV2 Yu-A strain were amplified by PCR and inserted into a transfer vector. The recombinant transfer plasmid was co-transfected with the EcoRI digested genome of vector virus (PRV TK-/gE-/LacZ+) into IBRS-2 cells. The recombinant pseudorabies virus PRV-PCV2 was purified by plaque purification and identified by PCR and Southern blotting. Expression of the ORF1-ORF2 fusion protein by the recombinant PRV-PCV2 virus was demonstrated by Western blotting analysis. The growth properties of the recombinant virus in cells were similar to that of the parent vector virus. In animal experiments, PRV-PCV2 elicited strong anti-PRV and anti-PCV2 antibodies in Balb/c mice as indicated by PRV-neutralizing assay, anti-PCV2 ELISA and PCV2 specific lymphocyte proliferation assay, respectively. And PRV-PCV2 immunization protected mice against a lethal challenge of a virulent PRV Ea strain. In pigs, PRV-PCV2 elicited significant immune response towards PRV and PCV2 as indicated by PRV-ELISA, PRV neutralizing assay and PCV2 specific lymphocyte proliferation assay, respectively. This is a first step toward the development of a potential candidate divalent vaccine against PRV and PCV2 infections.     

Seroprevalence of bovine respiratory viruses in North-Western Turkey

Bovine respiratory disease complex is a very important health problem around the world. Present study describes serological distribution of bovine major respiratory viruses in non -vaccinated cattle population of Marmara region in north-western Turkey. Neutralising antibodies specific to bovine viral diarrhoea virus (BVDV), bovine herpesvirus 1 (BHV-1), bovine respiratory syncytial virus (BRSV), bovine parainfluenza virus 3 (PI-3), bovine adenovirus serotype 1 (BAV-1) and serotype 3 (BAV-3) were investigated. Among 584 serum samples collected from 39 establishments in 7 provinces, 41.4% were positive for BVDV, 17.1% for BHV-1, 73.0% for BRSV, 43.0% for PI-3, 89.5% for BAV-1 and 92.3% for BAV-3. There were significant differences observed between seroprevalence rates detected in neighbouring provinces. Serological prevalence of BVDV, BHV-1 and BRSV were extremely higher in large capacity dairy farms than of small capacity farms (p < 0.0001). This study demonstrates that herd capacity is a very important risk factor for respiratory viruses and, on the other hand bovine adenoviruses and BRSV are the common reason of respiratory diseases in the region.     

A seroepidemiological study of the importance in cow-calf pairs of respiratory and enteric viruses in beef operations from northwestern Quebec.

Serum antibody analyses for bovine herpesvirus type 1 (BHV-1), bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), bovine coronavirus (BCV), and bovine rotavirus (BRV) were performed on 527 randomly selected cows, before calving, and on 407 three-week-old calves. In cows and calves, BCV and BRV were the most seroprevalent viruses (80% to 100% according to virus and vaccination status). Bovine respiratory syncytial virus was the least seroprevalent in the cows, independent of the vaccination status. In nonvaccinated cows the seroprevalence to BRSV was 36.7%, and 53.5% in cows vaccinated less than two weeks prior to collecting blood, and 67.6% in cows vaccinated two weeks or more prior to blood collection. In their calves, BHV-1 was the least seroprevalent, independent of the vaccination status. The serological status and antibody titers in calves were generally associated with those of the dam. The occurrence of respiratory diseases in the calves was associated with cow and calf serological profiles (BHV-1, BRSV and BCV in the nonvaccinated group, BHV-1, BVDV and BCV in the vaccinated group). The occurrence of diarrhea was not associated with cow and calf serological profiles but was negatively associated with high level calf serum IgG in the nonvaccinated group (odds ratio = 0.73). Bovine coronavirus and BRV were shed by 1.4% and 4.9% of calves in the nonvaccinated group, and by 0% and 9.9% of calves in the vaccinated group, respectively. Bovine rotavirus shedding was associated with fecal diarrheic consistency at the moment of fecal sampling but not with previous occurrence of diarrhea.