A polymerase chain reaction (PCR) assay for the detection of bovine herpesvirus 1 (BHV1) in selectively digested whole bovine semen

A PCR assay for the detection of bovine herpesvirus type 1 (BHV1) DNA in selectively digested whole bovine semen was developed and evaluated. A brief treatment with proteinase-K was used to lyse free virus, virus present in non-sperm cells and virus adhering to the spermatozoa. Genomic bovine DNA was not released by this treatment. Primers and probes were based on the nucleotide sequence of the gD gene. BHV1 virus-spiked split samples were used as positive controls and the PCR products were detected by eye in ethidium bromide-stained agarose gels. Sequentially collected non-extended semen samples from experimentally infected bulls were used to compare this assay with virus isolation. Of a total of 162 ejaculates, 51 were found positive by virus isolation, whereas PCR detected BHV1 DNA in 73. PCR detected BHV1 DNA for a longer period after infection and reaction. Apart from its superior sensitivity, this PCR assay also has the advantage of being a relatively simple procedure, providing results within 24 h.Abbreviations AI artificial insemination - BHV1 bovine herpesvirus type 1 - PCR polymerase chain reaction - TCID₅₀ tissue culture infective dose, 50%     

利用RNAi抑制口蹄疫病毒的复制

根据口蹄疫病毒 IRES和 L 串联序列两侧的保守区域设计了 2个引物 ,利用 RT- PCR和 PCR方法扩增出该串联序列 ,并进行了测序。测序结果表明 ,扩增产物与 Gen Bank上相应的序列具有很高的序列同源性 (大于 99% )。在测序的基础上 ,选择了 L 基因上的 1个靶位点 (位于启始密码子下游第 2 2 9nt后 2 1 nt长的序列 ) ,合成了 si RNA表达盒SEC- L2 2 9。细胞单层长成 5 0 %～ 70 %时 ,将纯化的 SEC- L2 2 9转染到 BHK细胞中 ,转染 4 h后用高感染复数 FMDV接种 ,2 4 h后用间接免疫荧光方法对口蹄疫病毒在 BHK细胞中的复制进行检测。研究结果表明 ,SEC- L 2 2 9极大地抑制了口蹄疫病毒在 BHK细胞中的复制 ,且该抑制作用具有序列特异性 ,并降低了 BHK细胞的死亡率。另外 ,2 5 ng和5 0 ng SEC- L2 2 9处理组间对病毒复制的抑制作用差异不明显 ,可能是病毒基因组发生了突变。本试验表明 ,利用 PCR方法合成的 SEC在 BHK细胞中能特异性地抑制 FMDV的复制 ,RNAi技术可能为防治口蹄疫提供一个新的途径     

The effectiveness of trypsin treatment to remove Sendai virus adhering to the zona pellucida of mouse preimplantation embryos.

The standard washing and trypsin treatment procedures to remove viruses adhering to the zona pellucida (ZP) were evaluated. Mouse embryos at the early blastocyst stage were exposed to Sendai virus, and then washed or treated with trypsin. Even after washing or trypsin treatment, Sendai virus was detected in the twelfth and final wash. The virus was still shown to adhere to the ZP by immunofluorescence assay. The embryos developed into expanded blastocysts following 24 hours of in vitro culture. Viral antigen was clearly demonstrated in the cells forming the expanded blastocysts, indicating that viral replication occurred in these cells. The present results suggest that the standard washing or trypsin treatment are not sufficient to remove Sendai virus adhering to the ZP of mouse embryos.     

The effect of general anesthesia on canine lymphocyte function

The in vitro response of canine peripheral blood lymphocytes to mitogenic stimulation was evaluated following general anesthesia for a relatively minor diagnostic procedure. A marked suppression in the blastogenic response to phytohemagglutinin and concanavalin A was observed 4 hours postinduction which persisted through the first 24 hours and was normal by 4 days. A mild suppression to stimulation with pokeweed mitogen was observed at 4 hours postinduction but the response was back to normal by 24 hours. These results suggest general anesthesia has a transient immunosuppressive effect on canine lymphocytes and T cells are more susceptible to the immunosuppressive effect than B cells.     

J亚群禽白血病病毒的免疫荧光检测效果

应用特异性抗J亚群禽白血病病毒（ALV－J）的单克隆抗体JE9建立了免疫荧光检测ALV－J抗原和抗体的方法。结果表明，在ALV－JADOL－Hcl感染鸡胚成纤维细胞（CFF）24小时后，可以用1FA检测出阳性细胞，感染72小时后，可以检测出最小病毒感染量≥1．25TCID50／孔。对人工感染ALV－J鸡肾脏冰冻切片检测结果证明，免疫荧光法可以检测出组织中的ALV－J抗原，表现强阳性反应。用重组病毒rBac4817－env感染的Sf9细胞作抗原，可以检测出鸡血清中抗ALV－J的特异性抗体。该方法在ALV－J的控制中必将产生重要作用。     

Comparison of a tissue-culture virus-neutralization test and the enzyme-linked immunosorbent assay for measurement of antibodies t infectious bronchitis

Two serological tests, the virus-neutralization (VN) test in tissue culture using a tissue-cell-adapted virus and the enzyme-linked immunosorbent assay (ELISA), were compared to detect antibodies against Massachusetts 41 and Connecticut 46 strains of Infectious Bronchitis Virus (IBV). The VN test was conducted in wells of microplates by the usual procedure. The two strains of IBV were adapted after 20 serial passages to induce CPE in 24 hours in chickens embryos kidney cells (CEKC). The ELISA test was carried out using partially virus following ultracentrifugation of each stain of IBV as antigen. The ELISA test detected higher geometric mean antibody titers (GMT) against both strains of IBV than did the VN test. One hundred four serum samples taken at 1, 3, 5, 9, 22, 24, and 26 weeks of age from a flock of chickens vaccinated with the Mass strain three times and the Conn strain of IBV two times during the growing period showed higher antibody titer responses to the Conn 46 than to the Mass 41 strain. Maternal antibodies in chicks one week of age were readily detected by the ELISA test, whereas low or insignificant titers were found by the VN test. Sera of vaccinated chickens collected following challenge with Mass 41 or Conn 46 strain of IBV showed that the ELISA was more sensitive and showed higher titers than did the VN test. Although the VN test showed no rise in GMT in the same sera tested with the heterologous virus, the ELISA showed a slight increase or cross-reaction. The serum samples from the unchallenged control group showed no change in GMT with either test or IBV strain.     

抗禽流感病毒H5和H9亚型血凝素特异性单克隆抗体的研制及应用

以禽流感题H5和H9亚型病毒分别免疫Balh／c小鼠，取其脾细胞与SP2／0的骨髓瘤细胞融合，用血凝抑制试验(HI）检测细胞培养上清，结果获得了6株特异性单克隆抗体，其中抗禽流感题亚型病毒血凝素特异性单克隆抗体细胞株3株，分别命名为4B6、4A3、3H1；抗H9亚型病毒血凝素单克隆抗体细胞株3株，命名为6E6、6B6和5B4。这些单克隆抗体小鼠腹水HI效价为2^13-15，细胞培养上清抗体HI效价为2^7-8。研究结果表明，所有这些单克隆抗体仅与试验的相应题或ID亚型病毒株发生特异性反应，而不能与鸡新城疫病毒、鹅新城疫病毒、鹅腺病毒和鸡产蛋下降综禽征病毒(EDS76)等反应。实验室检测结果证明，应用这些单克隆抗体能在24h内迅速检测出相应的禽流感病毒。所有这些单克隆抗体将在禽流感的预警预报工作中发挥重要作用。     

Investigations of the efficacy of European H1N1- and H3N2-based swine influenza vaccines against the novel H1N2 subtype

The efficacy of a commercial swine influenza vaccine based on A/New Jersey/8/76 (H1N1) and A/Port Chalmers/1/73 (H3N2) strains was tested against challenge with an H1N2 swine influenza virus. Influenza virus-seronegative pigs were vaccinated twice with the vaccine when they were four and eight weeks old, or with the same vaccine supplemented with an H1N2 component. Control pigs were left unvaccinated. Three weeks after the second vaccination, all the pigs were challenged intratracheally with the swine influenza strain Sw/Gent/7625/99 (H1N2). The commercial vaccine induced cross-reactive antibodies to H1N2, as detected by the virus neutralisation (VN) assay, but VN antibody titres were 18 times lower than in the pigs vaccinated with the H1N2-supplemented vaccine. The challenge produced severe respiratory signs in nine of 10 unvaccinated control pigs, which developed high H1N2 virus titres in the lungs 24 and 72 hours after the challenge. Vaccination with the commercial vaccine resulted in milder respiratory signs, but H1N2 virus replication was not prevented. Mean virus titres in the pigs vaccinated with the commercial vaccine were 1-5 log10 lower than in the controls at 24 hours but no different at 72 hours. In contrast, the H1N2-supplemented vaccine prevented respiratory disease in most pigs. There was a 4-5 log10 reduction in the mean virus titre at 24 hours in the pigs vaccinated with this vaccine, and no detectable virus replication at 72 hours. These data indicate that the commercial swine influenza vaccine did not confer adequate protection against the H1N2 subtype.     

Use of the indirect fluorescent antibody test in the detection of bovine respiratory syncytial virus antibodies in bovine serum.

The indirect fluorescent antibody test was adapted for identifying bovine respiratory syncytial virus and its specific antibody, using goat turbinate (GTU) cells. The virus caused maximal cytopathic effects in GTU cells 4 to 8 days postinfection, but fluorescence was not readily detected during this period. Fluorescence was maximal in infected GTU cells at 24 to 36 hours postinfection, but could be detected 48 hours postinfection. Bovine serums (331) which had been submitted to the Oklahoma Animal Disease Diagnostic Laboratory were tested for antibodies to this virus, and 73.6% were found to be positive.     

鸭肠炎病毒gL蛋白在鸡胚成纤维细胞中动态表达的检测

为研究鸭肠炎病毒(DEV)gL蛋白在感染鸡胚成纤维细胞(CEF)过程中的表达情况,本研究以DEVClone-03基因组为模板,应用PCR方法分别扩增得到截短的(gLt,181 bp～711 bp)和全长的gL(1 bp～711 bp)两个基因片段。将gLt基因片段克隆至pET-30a原核表达载体,转化E.coli BL21(DE3),经IPTG诱导表达并对表达产物进行纯化复性,免疫BALB/c小鼠,制备鼠抗gL蛋白多克隆抗体。同时将全长gL基因克隆至真核表达载体pcDNA3.1(+),构建真核表达重组质粒pcDNA-gL,转染293T细胞。采用获得的抗gL蛋白抗体检测DEV感染CEF后及真核表达质粒pcDNA-gL转染293T细胞后gL蛋白在不同时间点的表达情况。结果表明,在pcDNA-gL转染293T细胞后12 h应用western blot方法能够检测到gL蛋白的表达,其表达量随着转染时间增加而增加;在病毒感染CEF后24 h应用间接免疫荧光方法能够检测到gL蛋白少量的表达,western blot方法在病毒感染CEF48 h后检测到gL蛋白的表达,其表达量随着病毒感染时间增加而增加。上述结果提示,编码gL蛋白基因可能是病毒复制的晚期表达基因。     

Transmissible gastroenteritis in neonatal dogs: experimental intestinal infection with transmissible gastroenteritis virus.

Fourteen neonatal dogs (4 through 11 days of age) were exposed orally to the Purdue strain of transmissible gastroenteritis (TGE) virus, and six dogs of similar age were noninoculated controls. Clinical signs of enteric disease did not develop. Both exposed and control dogs had normal fecal passages and appetite throughout the experiment. Jejunal epithelium from dogs euthanatized at 12, 24, 48, and 96 hours and at 10 days after exposure did not exhibit morphologic alterations detectable by light microscopy. Electron microscopic examination indicated that jejunal epithelial cells contained TGE viral particles as early as 12 hours after dogs were exposed. There were no apparent morphologic alterations or signs of desquamation of virus-infected cells, however. Results of pig transmission studies indicated that viable TGE virus was in jejunal tissue of the dogs as early as 12 hours and as late as 10 days after exposure to the virus.     

猪血凝性脑脊髓炎病毒在PK-15细胞中的增殖特性

利用光学显微镜观察、间接免疫荧光（IFA）、Real-time PCR和病毒感染滴度（TCID50）等测定方法,分别从病毒抗原分布、病毒基因组复制水平以及病毒感染滴度变化等方面对猪血凝性脑脊髓炎病毒（HEV-67N）在猪肾上皮传代细胞系（PK-15细胞）上的增殖特性进行了研究。使用HEV-67N株感染24孔细胞培养板内的PK-15细胞,接种剂量为400个TCID50（104.37）/孔,间接免疫荧光检测结果显示,在感染后8h即可检测到被荧光抗体标记的感染细胞,且随着感染时间的延长,出现荧光的细胞数量逐渐增多,至感染后32h,几乎所有的细胞均出现有荧光。Real-time PCR检测结果显示,病毒基因组RNA的复制在感染后32～48h呈快速上升趋势,其基因组拷贝数在感染后48h达到最高值,之后增殖速度减慢,至感染后56h细胞出现CPE。TCID50的测定结果显示,HEV感染滴度的变化趋势与基因组RNA含量的变化相一致,在感染后32～48h增殖速度最快,之后逐渐减缓,至72h病毒感染滴度达到最高。     

Cultivation of a porcine adenovirus in porcine thyroid cell cultures

The porcine adenovirus type 4 was adapted to grow in porcine thyroid cell cultures. A readily recognizable cytopathic effect appeared in these cells as soon as the first passage of the virus and complete degeneration of the monolayers was obtained after only 72 hours post-infection at the fourth passage. A viral yield of 10(6.0) TCID50/ml was calculated after the third passage. The virus was purified by CsCl density gradient centrifugation and was shown to possess a buoyant density of 1.33 g/ml. A specific antiserum was prepared from two specific-pathogen-free piglets and used for indirect immunofluorescent staining. The fluorescence was observed in the nucleus of infected cells at 24 to 72 hours post-inoculation. The use of TP cells is suggested for routine porcine adenovirus diagnosis.     

Evaluation of bovine viral diarrhea virus uptake by preimplantation embryos

Bovine embryos were exposed to bovine viral diarrhea (BVD) virus in vitro. An uptake of BVD virus by the embryos could not be detected by several assay systems. A significant decrease in the titer of BVD virus was found to occur when the virus was incubated in saline solution + 5% goat serum or minimal essential medium + 5% goat serum for 24 hours at 37 C. Since there was significant inactivation of the BVD virus during the incubation period, lack of viral infectivity of the embryos may have been due to adverse effects of the experimental environmental conditions on the virus or the embryos or upon viral-embryo interaction.     

Bovine respiratory syncytial virus infection of bovine embryonic lung cultures: a kinetic study by the fluorescent antibody technique.

Development of fluorescence in bovine embryonic lung cells infected with bovine respiratory syncytial virus (BRSV) was studied by the fluorescent antibody (FA) test. Similar patterns of fluorescence were seen with the direct FA test, in which the immunoglobulin G fraction of antiserum to BRSV was conjugated with fluorescein isothiocyanate and used; and the indirect test, in which antiserum to the Long strain of respiratory syncytial virus and fluorescein isothiocyanate-conjugated anti-rabbit immunoglobulin G were used. In different trials, fluorescence was first detected between 16 and 18 hours after inoculation with BRSV. Fluorescence always was confined to the cytoplasm. Before 24 hours, fluorescence consisted of fine fibrils, usually parallel to the long axis of the cell, and cytoplasmic granules. After 24 hours, coincident with rounding of the cells, fluorescence slowly moved to the periphery of the cytoplasm. Under the growth conditions used, syncytia did not develop. By the FA test and as determined by the release of BRSV into the supernatant fluid, the minimal time for a single cycle of infection was between 24 and 26 hours.     

Mechanical transmission of capripox virus and African swine fever virus by Stomoxys calcitrans

Stomoxys calcitrans can act as an efficient mechanical vector of capripox virus and African swine fever virus. Capripox virus was transmitted to a susceptible goat by flies infected 24 hours previously and the virus survived in some flies for at least four days. African swine fever virus was transmitted to susceptible pigs by flies infected one hour and 24 hours previously and the virus survived in these flies for at least two days without apparent loss of titre.     

Inhibition of natural killer activity in porcine mononuclear cells by African swine fever virus

The coincubation at 37 degrees C for 24 hours of swine peripheral blood mononuclear cells with African swine fever virus inhibited in part the natural killer activity shown by cells incubated without the virus. This inhibition depended on the dose of the virus and on the time that cells were incubated with it. When the virus preparation was fractionated by ultracentrifugation, most of the inhibitory activity was found in the sedimented fraction, where viral particles were present; however, the loss of inhibitory activity in respect to the whole virus preparation indicated that some inhibitory activity was present in the supernatant fraction, probably as factors released by infected cells. Most of the inhibitory activity shown by the sedimented fraction was lost when the virus was inactivated by ultraviolet radiation, indicating an active role of virus infectivity in the inhibition.     

Detection of equine immunoglobulin-secreting cells by a plaque assay.

A protein A-hemolytic plaque assay was applied to detect immunoglobulin (Ig)-producing cells in horse peripheral blood, using pokeweed mitogen as a B lymphocyte activator. A maximum number of Ig-secreting cells was obtained when horse peripheral blood lymphocytes were cultured in a medium containing horse serum. The number of Ig-secreting cells in young horses (2 years old) was lower than that in adult horses (6 to 23 years old). In addition, the plaque formation was unchanged from blood samples kept at 4 degrees C for 24 hours, while blood samples kept for 72 hours did not yield plaques. These results indicate that the plaque assay is a reliable and useful method for detecting Ig-secreting cells in the peripheral blood of the horse.     

Detection of infectious laryngotracheitis virus infected cells with cloned DNA probes.

A genomic library of infectious laryngotracheitis virus (ILTV) DNA BamH1 fragments was prepared and two cloned fragments were evaluated for their potential as probes for the detection of ILTV infected cells. The virus was purified by a modified sucrose density gradient procedure for the isolation of pure ILTV DNA. A genomic library was constructed using BamH1-digested ILTV DNA and pGEM7 as a vector. A 1.1 kb cloned BamH1 fragment of ILTV DNA was tested in a slot or dot blot assay for the detection of ILTV infected cells. The limit of detection for this probe was at least 0.12 ng of pure ILTV DNA. The probe was able to identify both chicken embryo liver (CELi) cells and choriallantoic membranes infected with ILTV. Chicken embryo liver cells infected with several field isolates and a vaccine strain of ILTV were positive by dot blot analysis using this probe. Some qualitative differences in the degree of hybridization to cells infected by different ILTV isolates were observed. Uninfected cells and cells infected with fowlpox virus, turkey herpesvirus, Marek's disease virus or Newcastle disease virus were negative by the same assay. Compared with the 1.1 kb fragment, a larger 6 kb cloned BamH1 fragment of ILTV DNA showed a stronger hybridization signal to DNA from ILTV infected cells.