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1.
The proliferative capacity of mammalian cells is regulated by telomerase, an enzyme uniquely specialised for telomeric DNA synthesis. The critical role of telomerase activation in tumor progression and maintenance has been well established in studies of cancer and of oncogenic transformation in cell culture. Experimental data suggest that telomerase activation has an important role in normal somatic cells, and that failure to activate sufficient telomerase also promotes disease. Evidence regarding the role of telomerase in the pathogenesis of several viruses including human immunodeficiency virus has led to an increased interest in the role of telomerase activity in other virus infections. In this research we evaluated the telomerase modulating activity of Bovine herpesvirus 1 (BHV-1) in MDBK cells. MDBK cells were infected at different multiplicity of infection with BHV-1 Cooper strain and telomerase activity at different times post-infection was measured by the TRAP assay. Our data indicate that BHV-1 significantly up-regulates telomerase activity at 3 and 6h post-infection decreasing after the 24h post-infection. Our data, showed that the effect was mediated by an immediate-early or early viral gene, and use of the protein translation inhibitor cycloheximide confirmed that an immediate early gene is primarily responsible.  相似文献   

2.
The detection of virus-specific immunoglobulin M (IgM) antibodies in acute-phase serum samples offers the possibility of making an accurate and rapid serologic diagnosis. We have developed a solid-phase capture assay that uses murine monoclonal antibodies specific for bovine IgM to separate the whole IgM fraction of a bovine serum sample. The IgM specific for bovine herpesvirus-1 (BHV-1) is then detected by the addition of viral antigen, which in turn is detected by BHV-1-specific monoclonal antibodies conjugated to horseradish peroxidase. A BHV-1 IgM antibody response was detected during the early postinfection period (7-40 days PI). Bovine herpesvirus-1 IgM antibody was not detected in sera taken from 3 animals following dexamethasone-induced viral reactivation. This method compares favorably with viral isolation, antigen detection in the clinical samples, and paired serology in the diagnosis of BHV-1 infection at a herd level.  相似文献   

3.
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.  相似文献   

4.
The defective growth of bovine herpesvirus I (BHV-1) was analyzed in non-permissive murine embryo fibroblast, BALB/3T3 A31-1-1 (A31) cells. BHV-1 was able to attach and penetrate into A31 cells at similar levels that were seen in semi-permissive cells. Once penetrated into A31 cells, BHV-1 was efficiently transported to nuclei, but the onset of expression of immediate early (IE) protein and viral DNA replication was not observed. These data suggest that the viral replication of BHV-1 in A31 cells is arrested at the point prior to the expression of IE proteins.  相似文献   

5.
Mannheimia (Pasteurella) haemolytica A1 produces an extracellular leukotoxin (LKT) that is reported to bind the beta(2)-integrin CD11a/CD18 (LEA-1) on ruminant leukocytes. LKT binding induces activation, and subsequent cytolysis, of these cells. It is well known that active viral infection greatly increases the susceptibility of cattle to pasteurellosis. To better understand the mechanism by which this occurs, we investigated the effects of experimental in vivo infection of cattle with bovine herpes virus-1 (BHV-1) on the ex vivo interaction of bovine leukocytes with the M. haemolytica LKT. In this study, we demonstrated that active BHV-1 infection increased the expression of the beta(2)-integrin CD11a/CD18 (as defined by the mAb BAT75) on bovine peripheral blood neutrophils, enhanced the binding of LKT to bronchoalveolar lavage (BAL) leukocytes and peripheral blood neutrophils, and increased the killing of BAL leukocytes and peripheral blood leukocytes by LKT. In addition, BHV-1 greatly increased the number of BAL, resulting in many more LKT-responsive cells being present in the lungs. These findings might explain in part the increased susceptibility of BHV-1 infected cattle to pneumonic pasteurellosis.  相似文献   

6.
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7.
OBJECTIVE: To determine whether a combination viral vaccine containing modified-live bovine herpesvirus-1 (BHV-1) would protect calves from infection with a recent field isolate of BHV-1. DESIGN: Randomized controlled trial. ANIMALS: Sixty 4- to 6-month-old beef calves. PROCEDURE: Calves were inoculated with a placebo 42 and 20 days prior to challenge (group 1; n = 10) or with the combination vaccine 42 and 20 days prior to challenge (group 2; 10), 146 and 126 days prior to challenge (group 3; 10), 117 and 96 days prior to challenge (group 4; 10), 86 and 65 days prior to challenge (group 5; 10), or 126 days prior to challenge (group 6; 10). All calves were challenged with BHV-1 via aerosol. Clinical signs, immune responses, and nasal shedding of virus were monitored for 14 days after challenge. RESULTS: Vaccination elicited increases in BHV-1-specific IgG antibody titers. Challenge with BHV-1 resulted in mild respiratory tract disease in all groups, but vaccinated calves had less severe signs of clinical disease. Extent and duration of nasal BHV-1 shedding following challenge was significantly lower in vaccinated calves than in control calves. In calves that received 2 doses of the vaccine, the degree of protection varied with the interval between the last vaccination and challenge, as evidenced by increases in risk of clinical signs and extent and duration of viral shedding. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that this combination vaccine provided protection from infection with virulent BHV-1 and significantly reduced nasal shedding of the virus for at least 126 days after vaccination.  相似文献   

8.
OBJECTIVE: To study the antiviral activity of genistein, a soya isoflavone, on in vitro replication of bovine herpesvirus type 1 (BHV-1). SAMPLE POPULATION: Madin-Darby bovine kidney (MDBK) cells. PROCEDURE: Effects of genistein on the magnitude and kinetics of inhibition of BHV-1 phosphorylation of glycoprotein E (gE) and in vitro replication of BHV-1 in MDBK cells were evaluated. Antiviral activity of genistein was compared with 2 compounds, estradiol-17beta (EST) and tamoxifen (TAM), that have estrogenic and antiestrogenic activity, respectively. High-performance liquid chromatography (HPLC) was used to determine the concentration of genistein in medium from infected and uninfected MDBK cultures. RESULTS: Genistein reduced BHV-1, but not gE-deleted BHV-1 (BHV-1gEdelta3.1), replication by 90% at 18 hours after inoculation. This inhibition was not sustained through 24 hours after inoculation. The genistein concentration in media from MDBK cells was decreased by 40% during BHV-1 infection, compared with 16% for uninfected cells, at 24 hours after inoculation. Genistein inhibited gE phosphorylation and BHV-1 replication in a dose-dependent manner. Dosing with 25 microM genistein at 0 and 12 hours after inoculation of BHV-1 was optimal for decreasing BHV-1 replication. Estradiol-17beta EST and TAM did not affect BHV-1 replication. CONCLUSIONS AND CLINICAL RELEVANCE: The decrease in genistein concentration was a viral infection-dependent event. Genistein is an inhibitor of BHV-1 replication because of its ability to inhibit tyrosine kinase activity. A possible application may be for the control of BHV-1 infection in cattle by feeding soya products rich in genistein prior to or during periods of stress.  相似文献   

9.
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.  相似文献   

10.
OBJECTIVE: To construct and characterize a recombinant glycoprotein (g)E gene-deleted bovine herpesvirus (BHV) type 1 (BHV-1). PROCEDURE: The BHV-1 gEgene-coding region and the flanking upstream and downstream sequences were cloned. The aforementioned cloned DNA was digested with suitable enzymes to release the amino terminal two thirds of that region, and was ligated to the beta-galactosidase (beta-gal) gene. The resulting plasmid DNA was cotransfected with DNA from full-length, wild-type (WT), BHV-1 Cooper strain of the virus. Recombinant viruses expressing beta-gal (blue plaques) were plaque purified and assayed further by blot hybridization for genetic characterization and by immunoblotting for reactivity against BHV-1 gE peptide-specific rabbit polyclonal antibody. One recombinant virus, gEdelta3.1IBR, was characterized in vitro and in vivo. The ability of the recombinant virus to induce BHV-1 neutralizing antibodies in infected calves was investigated by plaque-reduction tests. RESULTS AND CONCLUSIONS: The gEdelta3.1IBR virus contained a deletion in the viral gE gene-coding sequences where a stable chimeric reporter (beta-gal) gene was inserted. One-step growth kinetics and virus yield of the recombinant and parent viruses were similar, but early after infection, the recombinant virus yield was comparatively less. After intranasal inoculation, the recombinant gEdelta3.1IBR virus replicated in the upper respiratory tract of calves, but the amount of progeny viruses produced was hundredsfold reduced, and duration of virus shedding was shorter. Results of in vivo calf experiments and serum neutralization tests indicated that deleting the gE gene has little effect on inducing neutralizing antibodies against BHV-1, but is sufficient to reduce BHV-1 virulence in calves.  相似文献   

11.
A bovine herpesvirus-1 (BHV-1) isolate (FI) from an aborted fetus was used to infect 9 heifers at various stages of gestation. Two heifers were inoculated IV on postbreeding day (PBD) 1, 7, or 14, and 3 heifers were inoculated in the sixth month of pregnancy. Plasma progesterone assays were used to monitor corpus luteum function in heifers inoculated during early pregnancy. Low progesterone values and infertility were seen in the 2 heifers inoculated on PBD 1. Luteal function remained normal in heifers inoculated on PBD 7 or 14. These 4 heifers inoculated on PBD 7 or 14 carried their fetuses to term, and their calves were free of BHV-1 infection at birth. Three heifers inoculated during the sixth month of pregnancy also carried their fetuses to term. Two calves were born alive, and BHV-1 was not isolated from nasal swab samples of either calf; the third calf was stillborn. Virus was not isolated from the stillborn calf's tissues, but BHV-1 was isolated from the placenta. Lesions were not detected in several tissues examined by light microscopy, and BHV-1 antigen was not detected by immunohistochemical examination of paraffin sections. Restriction endonuclease analysis of viral DNA was used to compare the FI virus to other BHV-1 isolates (Colorado-1, Iowa, and K22). On the basis of restriction endonuclease analysis, the FI isolate should be classified as a type-2 (infectious pustular vulvovaginitis) virus, specifically subtype a.  相似文献   

12.
Restriction endonuclease analysis was used, in conjunction with viral neutralization and growth-curve experiments, to compare a bovine herpesvirus type 1 (BHV-1) isolate, originally obtained from bovine mammary gland lesions, with a standard BHV-1 strain, infectious bovine rhinotracheitis virus. Although differences were not detected by viral neutralization or growth-curve experiments, restriction fragment patterns generated by Bam HI, Eco RI, Hind III, and Hpa I, revealed definite differences between the isolate and the prototype strain. Additionally, Eco RI, Hind III, and Hpa I patterns revealed that the mammary gland isolate had DNA-fragment patterns characteristic of infectious pustular vulvovaginitis strain of BHV-1, type 2b. Seemingly, type-2b isolates, similar to types 1 and 2a, may be capable of causing divergent types of infection of variable severity in cattle.  相似文献   

13.
This study was performed to investigate the presence of bovine herpesvirus-1 (BHV-1), bovine leukemia virus (BLV) and bovine viral diarrhea virus (BVDV) infections in dromedary camels (Camelus dromaderius) kept in mixed herds with sheep and goats in Algeria, since the prevalence of BHV-1, BVDV, and BLV infections among dromedary camels in Algeria is unknown. Totally, 111 camel sera were collected from two provinces (Laghouat and Ghardaia) in Algeria. The sera were analyzed for BHV-1 specific antibodies, BVDV specific antibodies and BVDV antigen using the ELISA, and BLV nucleic acid using PCR. The seropositivity rate was 9.0% for BVDV-specific antibody, although 41.4% of camels tested were positive for BVDV antigen. Moreover, there was no evidence of BHV-1 and BLV infections. The results indicated that camels might represent an important source for BVDV infection in all ruminants, including cattle, sheep, and goats bred in mixed herds in Algeria, since they had a higher BVDV prevalence rates. Therefore, the prevention and control measures for BVDV infection should put in place in camel populations to limit the spread of BVDV infection to ruminant populations in Algeria.  相似文献   

14.
A total of 457 nasal swab specimens from cases of respiratory disease in 2 feed lots were evaluated for the detection of bovine herpesvirus Type 1 (BHV-1) by ELISA. Thirty-three were found to be positive for BHV-1 by the recovery of infectious virus and 21 of these were positive by ELISA, yielding a sensitivity of 64%. Fifteen other virus isolations were made and included bovine viral diarrhea viruses, rhinoviruses and parainfluenza Type 3 viruses; none of these cases were positive with the BHV-1 ELISA. Specificity of the ELISA was 100%. Eighty percent of the specimens with BHV-1 titers greater than 10(5) TCID50 were detected by ELISA; the median amount of virus in positive specimens that were detected by ELISA was 7 X 10(5) TCID50 and the median amount of virus in specimens not detected was 1.5 X 10(4) TCID50. BHV-1 infection was most frequently diagnosed in feedlot cattle that had been in the feedlot for 40-80 days. Approximately half of the infected cattle were carrying virus-neutralizing antibodies in their serum.  相似文献   

15.
Active infection with bovine herpesvirus-1 (BHV-1) increases the susceptibility of cattle to secondary bacterial pneumonia with Mannheimia (Pasteurella) haemolytica A1. In the present study we found that bovine PMNs incubated with conditioned media from BHV-1 infected peripheral blood mononuclear cells (PBMCs) exhibited increased LFA-1 expression, enhanced LKT binding and increased LKT cytotoxicity. These effects were abrogated when the conditioned medium was pre-incubated with an anti-IL-1beta Mab before being added to the PMNs. These findings suggest that BHV-1 infection, and the resulting release of IL-1beta and perhaps other inflammatory cytokines, can stimulate activation of LFA-1 in bystander bovine PMNs, thus enhancing the binding and biological effects of LKT.  相似文献   

16.
Venereal infection of bulls with bovine herpesvirus type 1.2 (BHV-1.2) may result in acute balanoposthitis followed by the establishment of latent infection, presumably in dorsal root nerve ganglia. We herein report the characterization of the acute and latent infection of young bulls with a Brazilian BHV-1.2 isolate and the investigation of neural and non-neural sites in which viral DNA persists during latent infection, i.e. 110 days after inoculation and 50 days after experimental reactivation. Intrapreputial inoculation of BHV-1.2 isolate SV-56/90 (10(6.5)pfu per animal) resulted in severe balanoposthitis, characterized by redness of the penis and preputial mucosa, coalescent vesicles and fibrinous exsudate in all four infected bulls. Virus shedding was detected in preputial secretions and semen up to days 14 and 13 pi, respectively. Dexamethasone administration at day 60 pi led to reactivation of the infection in all animals, resulting in virus shedding in preputial secretions and/or in semen. At day 50 post-reactivation (pr), the animals were euthanized and regional tissues were collected for PCR and virus isolation. Viral DNA was consistently detected in the dorsal root ganglia of nerves genito-femoral (4/4) and obturator (4/4); frequently in the pudendal (3/4), sciatic (3/4) and rectal caudal nerve ganglia (2/3). In addition, viral DNA was detected in the pelvic sympathetic plexus of one bull and in regional lymph nodes (deep inguinal (2/4); sacral (1/4); medial iliac (1/4)) of two bulls. No infectious virus could be recovered from homogenates of DNA positive tissues, indicating the absence of actively replicating virus. These results demonstrate that BHV-1.2 DNA may persist in several sacral nerve ganglia and in regional lymph nodes as well during latent infection, i.e. 50 days after experimental reactivation. These findings may help in understanding the pathogenesis of acute and latent genital infection by BHV-1.2.  相似文献   

17.
ABSTRACT: A variety of mechanisms contribute to the viral-bacterial synergy which results in fatal secondary bacterial respiratory infections. Epidemiological investigations have implicated physical and psychological stressors as factors contributing to the incidence and severity of respiratory infections and psychological stress alters host responses to experimental viral respiratory infections. The effect of stress on secondary bacterial respiratory infections has not, however, been investigated. A natural model of secondary bacterial respiratory infection in naive calves was used to determine if weaning and maternal separation (WMS) significantly altered mortality when compared to calves pre-adapted (PA) to this psychological stressor. Following weaning, calves were challenged with Mannheimia haemolytica four days after a primary bovine herpesvirus-1 (BHV-1) respiratory infection. Mortality doubled in WMS calves when compared to calves pre-adapted to weaning for two weeks prior to the viral respiratory infection. Similar results were observed in two independent experiments and fatal viral-bacterial synergy did not extend beyond the time of viral shedding. Virus shedding did not differ significantly between treatment groups but innate immune responses during viral infection, including IFN-γ secretion, the acute-phase inflammatory response, CD14 expression, and LPS-induced TNFα production, were significantly greater in WMS versus PA calves. These observations demonstrate that weaning and maternal separation at the time of a primary BHV-1 respiratory infection increased innate immune responses that correlated significantly with mortality following a secondary bacterial respiratory infection.  相似文献   

18.
Apoptosis seems to play an important role in the pathogenic profile of bovine herpesvirus 1 (BHV-1) infection. Nitric oxide (NO) is also important as a signal molecule. In this study, apoptosis was selectively induced in HEp-2 cells in the early stage [1-3 h postinfection (PI)] of BHV-1 multiplication, and this apoptotic process was realised through the caspase-8, and partially through the caspase-3, pathway. BHV-1 infection inhibited staurosporine- (SS-) induced apoptosis only if the SS was added at 6 h PI. The results of this study showed that the 'NO-apoptosis' relation was realised through the caspase-8 pathway ('outer membrane receptor' pathway) at a later stage of infection in apoptosis induced by BHV-1 + SS. Our previous report (Yazici et al., 2004) and this study together showed that BHV-1 might induce and inhibit cell-type-specific pathways of apoptosis.  相似文献   

19.
Viruses have evolved different strategies to interfere with apoptotic pathways in order to halt cellular responses to infection. One previous study showed that transient transfection of bovine herpesvirus type-1 (BHV-1) UL14 protein is efficient in protecting Madin Darby kidney (MDBK) and human chronic myelogenous leukemia (K562) cells from sorbitol-induced apoptosis. This protein corresponds to a putative protein of BHV-1, which shares aminoacid sequence with a part of the peptide-binding domain conserved in human heat shock protein (HSP70) family. The pBK-CMV-UL14 plasmid transfected MDBK cells treated with sorbitol did not show caspase-3 and caspase-9 activation with respect to non-transfected MDBK cells (UL14 negative). Furthermore, we report that the expression of the full length sequence of BHV-1 UL14 is evident after 7 h of infection of BHV-1 on MDBK cells which were then treated with sorbitol. These results indicate that UL14 gene product has important implications to enhance cell survival in response to apoptotic stimuli.  相似文献   

20.
A nested polymerase chain reaction (PCR) assay was developed for the detection of bovine herpesvirus 1 (BHV-1) in bovine semen and compared with the virus isolation method. When extended semen, commonly used in the bovine artificial insemination industry, was inoculated with BHV-1, the PCR assay detected BHV-1 DNA in semen inoculated at 0.25-2.5 TCID50 per 0.5 mL. In contrast, the lower limit of detection for virus isolation was 250 TCID50 of BHV-1 inoculated in 0.5 mL of extended semen. These methods were also used to detect BHV-1 in the semen of four bulls which were experimentally infected with BHV-1. All infected bulls demonstrated balanitis at 3 d post-inoculation (DPI) and severe balanoposthitis at 4 DPI. BHV-1 was detected in raw semen by virus isolation and PCR at 2 DPI, before balanitis was evident. For virus isolation, the last day that BHV-1 was detected during primary infection was 7 DPI for two bulls and 9 and 11 DPI for the other two bulls. In contrast, PCR detected BHV-1 in the bulls' semen until 14 or 18 DPI. For individual animals, PCR detected BHV-1 during primary infection for at least 1-10 d longer than virus isolation. Reactivation of BHV-1 from latency without the presence of visible lesions was promoted twice by two series of 5 d dexamethasone injections. For the first series of dexamethasone treatments, a positive virus isolation result was obtained on the 5th d of treatment for only one bull. In contrast, two bulls demonstrated evidence of viral reactivation on this day by PCR. All bulls shed BHV-1 in semen on d 4 after dexamethasone treatment, as evidenced by positive virus isolation and PCR results. One bull was still PCR positive 13 d later. For the second series of dexamethasone treatments, a small amount of virus was isolated from semen collected on d 3 or 4 after treatment for two bulls but not from the other two bulls. In contrast, semen samples from all bulls were PCR positive for either or both of these 2 d. In total, from 80 semen samples, 45 were PCR positive and 26 were virus isolation positive. Thus, the PCR assay detected BHV-1 shedding in bulls earlier, more often, and for a longer duration, than did the virus isolation method.  相似文献   

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