Construction of bovine herpesvirus-1 (BHV-1) recombinants which express pseudorabies virus (PRV) glycoproteins gB, gC, gD, and gE

1. This was a randomized, double-blind comparison of the efficacy and safety of venlafaxine and fluoxetine in outpatients with major depression. 2. Three hundred fourteen patients were randomly assigned to either venlafaxine 37.5 mg twice daily or fluoxetine 20 mg once daily for a maximum of 8 weeks. 3. If the response was inadequate after two weeks of treatment, the dosage of venlafaxine could be increased to 75 mg twice daily. 4. A clinical response, defined as at least a 50% decrease from baseline in the total HAM-D score, was attained at week 6 in 72% of patients on venlafaxine and 60% of patients on fluoxetine (p = 0.023). 5. Among patients who increased their dose at 2 weeks, venlafaxine was significantly (p < 0.05) superior from week 3 onward on the HAM-D. 6. Venlafaxine 75 mg daily is comparable to fluoxetine, but at 150 mg daily, it may be superior to fluoxetine in outpatients with major depression who do not respond early to treatment.     

Protective immunity of bovine herpesvirus-1 (BHV-1) recombinants which express pseudorabies virus (PRV) glycoproteins gB, gC, gD and gE

We have constructed recombinants of bovine herpesvirus-1 (BHV-1) which express pseudorabies virus (PRV) gB, gC, gD or gE either individually or in combination. To test the protective immunity, mice were inoculated with these BHV-1 recombinants and challenged three weeks later with virulent PRV. A BHV-1 recombinant, BHV-1/TF7-1, which express PRV gC, gD, gE and gI but not PRV gB, protected all 7 mice from the challenge with 20 LD50 virulent PRV and 6 out of 7 from the challenge with 100 LD50 PRV, while one dead mice survived for 5 days after the challenge. All the control mice died in 3 days. BHV-1 recombinants which express PRV gB, gC and gD individually also gave some protection but not so effective as BHV/TF7-1. Before challenging with virulent PRV, sera were collected from immunized mice and antibody against PRV was assayed. Western blot analyses indicated that all the recombinants induced antibody in mice against PRV gB, gC or gD individually or in combination. Virus neutralizing (VN) titer against PRV was highest in mice which were inoculated with BHV-1/TF6-1, which expresses PRV gB. BHV-1/TF7-1, which was more effective to protect mice from the challenge of virulent PRV, induced lower VN titer.     

Expression and cellular distribution of baculovirus-expressed bovine herpesvirus 1 (BHV-1) glycoprotein D (gD) sequences

Glycoprotein D (gD) of bovine herpesvirus 1 (BHV-1), a homolog of herpes simplex virus gD, represents a major component of the viral envelope and is a dominant immunogen. To study the antigenic properties of the different regions of gD, we have expressed the full-length gD encoding gene and overlapping fragments spanning various regions of the gD open reading frame in a baculovirus (Autographa californica nuclear polyhedrosis virus)--insect cell (Spodoptera frugiperda, SF-9) system. Maximum levels of expression for all proteins were obtained 48 to 72 h post infection of SF-9 cells by recombinant viruses. Full-length and truncated recombinant gD proteins reacted specifically with anti-gD monospecific serum as determined by immunoprecipitation and immunoblotting, indicating that the proteins retained their antigenicity. However, based on the reactivity with a panel of gD-specific monoclonal antibodies (Mabs), the full-length recombinant gD lacked proper expression for two highly neutralizing linear epitopes identified by Mabs R54 and 9D6. The rest of the epitopes appeared to be preserved and antigenically unaltered. Immunofluorescence studies of recombinant baculovirus infected SF-9 cells using gD monospecific serum, revealed no direct correlation between cellular localization of the expressed proteins and their amino acid sequences.     

Detection of bovine herpesvirus 1 (BHV-1) genomic sequences in bovine semen inoculated with BHV-1 by polymerase chain reaction

Polymerase chain reaction (PCR) technique was applied to detect BHV-1 in bovine semen inoculated with BHV-1. The technique was found to be 10(6) times more sensitive than a non-isotopic dot-blot hybridization method in detecting viral genomic DNA. Of the three primer pairs used, the one chosen from glycoprotein gC appeared to be most sensitive as it could detect up to 0.01 TCID50 of BHV-1 in the semen. The technique could be useful in screening breeding bulls or samples of frozen semen prior to use in artificial insemination.     

Endoproteolytic processing of the ebola virus envelope glycoprotein: cleavage is not required for function

Proteolytic processing is required for the activation of numerous viral glycoproteins. Here we show that the envelope glycoprotein from the Zaire strain of Ebola virus (Ebo-GP) is proteolytically processed into two subunits, GP1 and GP2, that are likely covalently associated through a disulfide linkage. Murine leukemia virions pseudotyped with Ebo-GP contain almost exclusively processed glycoprotein, indicating that this is the mature form of Ebo-GP. Mutational analysis identified a dibasic motif, reminiscent of furin-like protease processing sites, as the Ebo-GP cleavage site. However, analysis of Ebo-GP processing in LoVo cells that lack the proprotein convertase furin demonstrated that furin is not required for processing of Ebo-GP. In sharp contrast to other viral systems, we found that an uncleaved mutant of Ebo-GP was able to mediate infection of various cell lines as efficiently as the wild-type, proteolytically cleaved glycoprotein, indicating that cleavage is not required for the activation of Ebo-GP despite the conservation of a dibasic cleavage site in all filoviral envelope glycoproteins.     

The cytoplasmic domain of bovine herpesvirus 1 glycoprotein B is important for maintaining conformation and the high-affinity binding site of gB

Bovine herpesvirus 1 (BHV-1) glycoprotein B (gB) has been shown to interact with two types of receptor on Madin Darby bovine kidney cells. The first receptor is heparan sulfate proteoglycan, whereas the second high-affinity receptor remains unknown. In order to study the structural requirement for gB's high-affinity binding activity, different forms of the gB ectodomain were expressed and compared with authentic gB. By using chemical cross-linking and sucrose gradient centrifugation, we found that BHV-1 gB was able to form dimers. A region between the cleavage site and the transmembrane anchor region, residues 506 to 763, was found to be required for gB oligomerization. Although the purified gBt and gBtM, two truncated forms of gB, formed oligomers, they did not block the high-affinity cellular receptor, suggesting that oligomerization was not the reason for the loss of the high-affinity binding site on gB. However, an N-terminal juxtamembrane region-located epitope recognized by a monoclonal antibody, designated epitope I, was lost from gBt and gBtM, indicating that both truncated gBs are conformationally changed. Therefore, the structure around this particular region may be required for the existence of the gB high-affinity binding site.     

Attachment but not penetration of bovine herpesvirus 1 is necessary to induce apoptosis in target cells

Bovine herpesvirus 1 (BHV-1) induces apoptotic cell death in bovine peripheral blood mononuclear cells and B-lymphoma cells. Using a BHV-1 glycoprotein H null mutant, we have demonstrated that although penetration of BHV-1 is not required, attachment of BHV-1 viral particles is essential for the induction of apoptosis.     

N-linked oligosaccharide processing is not necessary for glycoprotein secretion in plants

Mitochondria contain a potassium specific channel (mitoKATP channel) sensitive to ATP and antidiabetic sulfonylureas. The mitochondrial KATP channel plays an important role in the mitochondrial volume control and in regulation of the components of protonmotive force. This minireview describes the properties and current hypotheses concerning the function of mitoKATP channel.     

Functional complementation of UL3.5-negative pseudorabies virus by the bovine herpesvirus 1 UL3.5 homolog

The UL3.5 gene is positionally conserved but highly variable in size and sequence in different members of the Alphaherpesvirinae and is absent from herpes simplex virus genomes. We have shown previously that the pseudorabies virus (PrV) UL3.5 gene encodes a nonstructural protein which is required for secondary envelopment of intracytoplasmic virus particles in the trans-Golgi region. In the absence of UL3.5 protein, naked nucleocapsids accumulate in the cytoplasm, release of infectious virions is drastically reduced, and plaque formation in cell culture is inhibited (W. Fuchs, B. G. Klupp, H. Granzow, H.-J. Rziha, and T. C. Mettenleiter, J. Virol. 70:3517-3527, 1996). To assay functional complementation by a heterologous herpesviral UL3.5 protein, the UL3.5 gene of bovine herpesvirus 1 (BHV-1) was inserted at two different sites within the genome of UL3.5-negative PrV. In cells infected with the PrV recombinants the BHV-1 UL3.5 gene product was identified as a 17-kDa protein which was identical in size to the UL3.5 protein detected in BHV-1-infected cells. Expression of BHV-1 UL3.5 compensated for the lack of PrV UL3.5, resulting in a ca. 1,000-fold increase in virus titer and restoration of plaque formation in cell culture. Also, the intracellular block in viral egress was resolved by the BHV-1 UL3.5 gene. We conclude that the UL3.5 proteins of PrV and BHV-1 are functionally related and are involved in a common step in the egress of alphaherpesviruses.     

Neuropathology of bovine herpesvirus type 5 (BHV-5) meningo-encephalitis in a rabbit seizure model

CI-994 (acetyldinaline) is an orally active anticancer drug currently in Phase 1 clinical trials. To assess its preclinical toxicity, CI-994 was administered orally as suspensions to Wistar rats (10/sex/dose) and in capsules to beagle dogs (3/sex/dose) once daily for two weeks. Doses were 1.5, 5, and 15 mg/kg for rats (9, 30, and 90 mg/m2, respectively), and 0.5, 2, and 5 mg/kg for dogs (10, 40, and 100 mg/m2, respectively). Systemic exposure was dose-proportional based on toxicokinetic analysis in dogs. Severe clinical signs and mortality occurred at the highest dose in both species beginning on Day 10. Neutropenia, lymphocytopenia, thrombocytopenia, lymphoid depletion, bone marrow hypocellularity, and testicular degeneration were observed in both species, primarily at the mid- and high-doses. Despite continued treatment, neutrophil counts in dogs returned to control levels in Week 2. Other microscopic findings in rats included splenic hematopoietic depletion at all doses and epithelial cell necrosis in various tissues at 15 mg/kg. Additional bone marrow changes in dogs involved myeloid and megakaryocyte hyperplasia at 2 mg/kg and abnormal myeloid and megakaryocyte maturation at 2 and 5 mg/kg. Except for the testicular effects in both species, all changes were reversible within a 4-week (rat) or 9-week (dog) recovery period. The results of these studies show that target organ effects of CI-994 principally involve tissues with rapidly dividing cell populations and that bone marrow suppression is the dose-limiting toxicity. CI-994 also seems to interfere with the release and/or maturation of cells in the bone marrow.     

Lymphocyte proliferative responses to recombinant bovine herpes virus type 1 (BHV-1) glycoprotein gD (gIV) in immune cattle: identification of a T cell epitope

Effects of the antipsychotics risperidone and clozapine on 5-HT2 and D2-dopamine receptor binding were examined using [3H]N-methylspiperone ([3H]NMSP) and in vitro receptor autoradiography on human whole hemisphere cryosections. The 5-HT2 receptor antagonist ketanserin and the D2-dopamine receptor antagonist raclopride were used as references. [3H]NMSP binding was observed in caudate nucleus, putamen, and cerebral cortex indicating binding to D2-dopamine and 5-HT2 receptors. Risperidone and clozapine counteracted the binding to both receptor types. This was in contrast to raclopride, which selectively blocked the D2-dopamine receptors in the basal ganglia, and ketanserin, which selectively blocked the 5-HT2 receptors in the cerebral cortex. Risperidone (100 nM and 10 microM) blocked up to 90% of [3H]NMSP binding to both receptor types, whereas the blocking capacity of clozapine (10 microM) was lower (approximately 60%). The lack of total blockade of D2-dopamine receptors is in line with results obtained in with [11C]raclopride and positron emission tomography studies of clozapine treated human subjects. However, autoradiographic studies of clozapine competition of [3H]raclopride binding show total displacement of the binding at high clozapine concentrations, thus contradicting the PET results with [11C]raclopride, as well as the autoradiographic results obtained with [3H]NMSP. In conclusion it can be stated that pharmacological concentrations of the two drugs clozapine and risperidone block a large proportion of D2-dopamine receptors and 5-HT2 receptors in the human brain. Moreover, the study shows the usefulness of human whole hemisphere autoradiography for the study of interaction of drugs with different central neurotransmitter receptors.     

The glycoprotein D (US6) homolog is not essential for oncogenicity or horizontal transmission of Marek's disease virus

RB1BUS6lacgpt, a Marek's disease virus (MDV) mutant having a disrupted glycoprotein D (gD) homolog gene, established infection and induced tumors in chickens exposed to it by inoculation or by contact. Lymphoblastoid cell lines derived from RB1BUS6lacgpt-induced tumors harbored only the mutant virus. These results provide strong evidence that an intact gD homolog gene is not essential for oncogenicity or horizontal transmission of MDV.     

Proteolytic cleavage of urokinase-type plasminogen activator by stromelysin-1 (MMP-3)

Matrix metalloproteinase-3 (MMP-3, or stromelysin-1) specifically hydrolyzes the Glu143-Leu144 peptide bond in 45-kDa single-chain urokinase-type plasminogen activator (scu-PA) and in its two-chain (tcu-PA) derivative, yielding a 17-kDa NH2-terminal domain comprising the u-PA receptor (u-PAR) binding site and a 32-kDa COOH-terminal moiety containing the serine proteinase domain of u-PA. The conversion is completely abolished in the presence of the MMP inhibitors EDTA or 1,10-phenanthroline. Biospecific interaction analysis indicates that binding of MMP-3 occurs through the 32-kDa fragment. The 32-kDa fragment derived from scu-PA (scu-PA-32k) has a specific activity of 相似文献   

Virion incorporation of human immunodeficiency virus type-1 Vif is determined by intracellular expression level and may not be necessary for function

OBJECTIVE: To define the role of laparoscopic ultrasound (LUS) in the staging of pancreatic tumors. SUMMARY BACKGROUND DATA: Laparoscopy has recently been established as a valuable tool in the staging of pancreatic cancer. It has been suggested that the addition of LUS to standard laparoscopy could improve the accuracy of this procedure. METHODS: A prospective evaluation of 90 patients with pancreatic tumors undergoing laparoscopy and LUS was performed over a 27-month period. LUS equipped with an articulated curved and linear array transducer (6 to 10 MHz) was used. All patients underwent rigorous laparoscopic examination. Clinical, surgical, and pathologic data were collected. RESULTS: The median age was 65 years (range 43 to 85 years). Sixty-four patients had tumors in the head, 19 in the body, and 3 in the tail of the pancreas. Four patients had ampullary tumors. LUS was able to image the primary tumor (98%), portal vein (97%), superior mesenteric vein (94%), hepatic artery (93%), and superior mesenteric artery (93%) in these patients. LUS was particularly helpful in determining venous involvement (42%) and arterial involvement (38%) by the tumor. This resulted in a change in surgical treatment for 13 (14%) of the 90 patients in whom standard laparoscopic examination was equivocal. CONCLUSIONS: LUS is useful in evaluating the primary tumor and peripancreatic vascular anatomy. When standard laparoscopic findings are equivocal, LUS allowed accurate determination of resectability. Supplementing laparoscopy with LUS offers improved assessment and preoperative staging of pancreatic cancer.     

Palmitylation of the influenza virus hemagglutinin (H3) is not essential for virus assembly or infectivity

The C terminus of the influenza virus hemagglutinin (HA) contains three cysteine residues that are highly conserved among HA subtypes, two in the cytoplasmic tail and one in the transmembrane domain. All of these C-terminal cysteine residues are modified by the covalent addition of palmitic acid through a thio-ether linkage. To investigate the role of HA palmitylation in virus assembly, we used reverse genetics technique to introduce substitutions and deletions that affected the three conserved cysteine residues into the H3 subtype HA. The rescued viruses contained the HA of subtype H3 (A/Udorn/72) in a subtype H1 helper virus (A/WSN/33) background. Rescued viruses which do not contain a site for palmitylation (by residue substitution or substitution combined with deletion of the cytoplasmic tail) were obtained. Rescued virions had a normal polypeptide composition. Analysis of the kinetics of HA low-pH-induced fusion of the mutants showed no major change from that of virus with wild-type (wt) HA. The PFU/HA ratio of the rescued viruses grown in eggs ranged from that of virus with wt HA to 16-fold lower levels, whereas the PFU/HA ratio of the rescued viruses grown in MDCK cells varied only 2-fold from that of virus with wt HA. However, except for one rescued mutant virus (CAC), the mutant viruses were attenuated in mice, as indicated by a > or = 400-fold increase in the 50% lethal dose. Interestingly, except for one mutant virus (CAC), all of the rescued mutant viruses were restricted for replication in the upper respiratory tract but much less restricted in the lungs. Thus, the HA cytoplasmic tail may play a very important role in the generation of virus that can replicate in multiple cell types.     

Sublingual plicae (anterior processes) are not necessary for garter snake vomeronasal function.

The anterior processes of snakes may transfer odorants from the tongue to the vomeronasal (VN) organ. To test whether the anterior processes are required for a vomeronasally mediated behavior, the authors tested garter snakes (Thamnophis sirtalis) preoperatively and after cauterization of the anterior processes or control cauterization with artificial earthworms covered with earthworm wash (EWW) or distilled water. Snakes in both groups attacked EWW-covered artificial worms but not controls both pre- and postoperatively. In addition, snakes with anterior processes cauterization or control cauterization tongue flicked 3H-proline. Radioautographs of the VN organs of snakes with and without anterior processes were indistinguishable: Snakes in both groups had reduced silver grains over the VN sensory epithelium as had been reported previously with intact snakes. These findings indicate that the anterior processes are unnecessary both for a behavior known to require a functional VN system and for delivery of odorants to the VN organ. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Herpes simplex virus glycoprotein D can bind to poliovirus receptor-related protein 1 or herpesvirus entry mediator, two structurally unrelated mediators of virus entry

Several cell membrane proteins have been identified as herpes simplex virus (HSV) entry mediators (Hve). HveA (formerly HVEM) is a member of the tumor necrosis factor receptor family, whereas the poliovirus receptor-related proteins 1 and 2 (PRR1 and PRR2, renamed HveC and HveB) belong to the immunoglobulin superfamily. Here we show that a truncated form of HveC directly binds to HSV glycoprotein D (gD) in solution and at the surface of virions. This interaction is dependent on the native conformation of gD but independent of its N-linked glycosylation. Complex formation between soluble gD and HveC appears to involve one or two gD molecules for one HveC protein. Since HveA also mediates HSV entry by interacting with gD, we compared both structurally unrelated receptors for their binding to gD. Analyses of several gD variants indicated that structure and accessibility of the N-terminal domain of gD, essential for HveA binding, was not necessary for HveC interaction. Mutations in functional regions II, III, and IV of gD had similar effects on binding to either HveC or HveA. Competition assays with neutralizing anti-gD monoclonal antibodies (MAbs) showed that MAbs from group Ib prevented HveC and HveA binding to virions. However, group Ia MAbs blocked HveC but not HveA binding, and conversely, group VII MAbs blocked HveA but not HveC binding. Thus, we propose that HSV entry can be mediated by two structurally unrelated gD receptors through related but not identical binding with gD.     

Immune responses and protective efficacy of recombinant baculovirus-expressed glycoproteins of equine herpesvirus 1 (EHV-1) gB, gC and gD alone or in combinations in BALB/c mice

Baculovirus-expressed glycoproteins of EHV-1 gB, gC and gD alone or in combination evoked antibody responses and protected vaccinated mice against a challenge with EHV-1. gB, gD, gB + gC, gB + gD and gC + gD elicited very high levels of ELISA antibodies while gC and gC + gD elicited high levels of virus neutralising antibodies. Western blotting demonstrated that the antibodies produced were not only specific for the baculovirus-expressed glycoproteins gB, gC and gD, but also highly specific for each EHV-1 glycoprotein. Vaccination of mice with gB or gD prevented clinical signs of infection in mice challenged with EHV-1 and all vaccinated groups of mice except controls showed a rapid clearance of virus from the lungs and a reduction in lesions characteristic of herpesviruses in the lungs post-challenge. Notably, the lungs of mice vaccinated with gB, gD or gB + gD and challenged with EHV-1 showed prominent peribronchiolar and perivascular aggregations of mononuclear cells, predominantly lymphocytes. Immunocytochemical staining of these sections showed large numbers of T cells, suggesting an active role for these cells at the site of virus replication post-challenge.     

Longitudinal studies of immune function in cattle experimentally infected with bovine immunodeficiency-like virus and/or bovine leukemia virus

The effects of single or dual infection with bovine immunodeficiency-like virus (BIV) and/or, bovine leukemia virus (BLV) on bovine immune function were examined over a 4 year period. Holstein calves were infected with BIV (four calves), BLV (five calves), BIV and BLV (five calves), or sham inoculated (three calves). Lymphocyte blastogenesis to mitogens, seven tests of neutrophil function, and mononuclear cell subset analysis by flow cytometry (BoCD4, BoCD8, BoCD2, BoWC1, sIgM+, and monocytes) were performed at regular intervals to 49 months post-infection. These data were analyzed for main effects of each virus and interaction as a 2 x 2 factorial. BIV infected cattle had lower neutrophil antibody-dependent cell-mediated cytotoxicity and iodination responses during 2 of the 4 years post-infection (P < 0.05). BIV infection was not associated with any long-term significant changes in lymphocyte blastogenesis to mitogens or changes in mononuclear cell subset numbers in blood. There was a tendency for animals infected with BIV alone to have decreased lymphocyte blastogenic responses to mitogens, but this was not statistically significant. BLV infection caused an increase in total mononuclear cells with no dramatic shift in the relative proportions of the various subsets. Co-infection with BIV and BLV did not consistently cause a different response than either virus did individually. One BIV infected animal died of non-BLV lymphosarcoma 7 months after infection. All other animals had no unusual clinical signs. In summary, infection with BIV caused a significant, temporary decrease in neutrophil function with no consistent statistically significant alteration in lymphocyte blastogenesis or mononuclear cell numbers during the first 4 years after infection. BLV infection caused an increase in lymphocyte numbers, and there appeared to be no synergism between the viruses.