Proteomic Characterization of Bovine Herpesvirus 4 Extracellular Virions

Gammaherpesviruses are important pathogens in human and animal populations. During early events of infection, these viruses manipulate preexisting host cell signaling pathways to allow successful infection. The different proteins that compose viral particles are therefore likely to have critical functions not only in viral structures and in entry into target cell but also in evasion of the host''s antiviral response. In this study, we analyzed the protein composition of bovine herpesvirus 4 (BoHV-4), a close relative of the human Kaposi''s sarcoma-associated herpesvirus. Using mass spectrometry-based approaches, we identified 37 viral proteins associated with extracellular virions, among which 24 were resistant to proteinase K treatment of intact virions. Analysis of proteins associated with purified capsid-tegument preparations allowed us to define protein localization. In parallel, in order to identify some previously undefined open reading frames, we mapped peptides detected in whole virion lysates onto the six frames of the BoHV-4 genome to generate a proteogenomic map of BoHV-4 virions. Furthermore, we detected important glycosylation of three envelope proteins: gB, gH, and gp180. Finally, we identified 38 host proteins associated with BoHV-4 virions; 15 of these proteins were resistant to proteinase K treatment of intact virions. Many of these have important functions in different cellular pathways involved in virus infection. This study extends our knowledge of gammaherpesvirus virions composition and provides new insights for understanding the life cycle of these viruses.     

牛疱疹病毒Ⅳ型(DN-599株)DNA的分子克隆

从感染的MDBK(牛肾)细胞中直接抽提出牛疱疹病毒IV型(BHV-4)DNA。分别经限制性内切酶EcoRⅠ,HindⅢ或BamHI消化后,用鸟枪法和选择法将病毒DNA片段克隆到质粒pBR322和pUC9的相应位点中,构建了三组病毒DNA基因库。阳性克隆是用光生物素标记的病毒DNA和牛胸腺细胞DNA与各重组质粒DNA杂交而筛选的。总共克隆了病毒基因组的94％,其中用EcoRI克隆了83.4％,BamHI克隆了63.4％,HindIlI克隆了45％。     

Detection of a Novel Bovine Lymphotropic Herpesvirus

Degenerate PCR primers which amplify a conserved region of the DNA polymerase genes of the herpesvirus family were used to provide sequence evidence for a new bovine herpesvirus in bovine B-lymphoma cells and peripheral blood mononuclear cells (PBMC). The sequence of the resultant amplicon was found to be distinct from those of known herpesvirus isolates. Alignment of amino acid sequences demonstrated 70% identity with ovine herpesvirus 2, 69% with alcelaphine herpesvirus 1, 65% with bovine herpesvirus 4, and 42% with bovine herpesvirus 1. Phylogenetic analysis placed this putative virus within the tumorigenic Gammaherpesvirinae subfamily, and it is tentatively identified as bovine lymphotropic herpesvirus. This novel agent was expressed in vitro from infected PBMC, and cell-free supernatants were used to transfer infection to a bovine B-cell line, BL3. Analysis, with specific PCR primers, of DNA from bovine PBMC and lymphoma cells identified infection in blood of 91% of adult animals (n = 101), 63% of lymphomas (n = 32), and 38% of juveniles (n = 13). Of the adults, herpesvirus infection was present in 94% of animals that were seropositive for bovine leukemia virus (BLV) (n = 63) and in 87% of BLV-seronegative animals (n = 38). Of the seropositive group, 17 animals exhibited persistent lymphocytosis, and 100% of these were herpesvirus positive by PCR. A role for bovine lymphotropic herpesvirus as a cofactor in BLV pathogenesis is considered.     

Neutralizing Antibodies to Bovine Herpesvirus 1 in Reindeer

Serum samples from cattle and reindeer in Lapland were examined for neutralizing antibodies to the IBR/IPV virus. All the bovine sera tested were negative. The reindeer sera were tested using 2 different virus neutralization methods differing in the serum-virus incubation time prior to inoculation into tissue culture tubes. 12.6 % of the samples tested with a preincubation of 1 h at 37°C were positive, whereas 23 % of those tested with a preincubation time of 24 h at 37°C were positive. The fairly high prevalence of antibodies to IBR/IPV in the reindeer population in Finland indicates the occurrence of the IBR/IPV virus or a closely related cross-reacting herpesvirus.     

Novel Entry Pathway of Bovine Herpesvirus 1 and 5

Herpesviruses enter cells by a yet poorly understood mechanism. We visualized the crucial steps of the entry pathway of bovine herpesvirus 1 (BHV-1) and BHV-5 by transmission and scanning electron microscopy, employing cryotechniques that include time monitoring, ultrarapid freezing, and freeze substitution of cultured cells inoculated with virus. A key step in the entry pathway of both BHV-1 and BHV-5 is a unique fusion of the outer phospholipid layer of the viral envelope with the inner layer of the plasma membrane and vice versa resulting in “crossing” of the fused membranes and in partial insertion of the viral envelope into the plasma membrane. The fusion area is proposed to function as an axis for driving the virus particle into an invagination that is concomitantly formed close to the fusion site. The virus particle enters the cytoplasm through the opened tip of the invagination, and the viral envelope defuses from the plasma membrane. There is strong evidence that the intact virus particle is then transported to the nuclear region.     

Establishment of a Bovine Herpesvirus 4 based vector expressing a secreted form of the Bovine Viral Diarrhoea Virus structural glycoprotein E2 for immunization purposes

Background  

The biological characteristics of BoHV-4 make it a good candidate as a gene delivery vector for vaccination purposes. These characteristics include little or no pathogenicity, unlikely oncogenicity, the capability to accommodate large amounts of foreign genetic material, the ability to infect several cell types from different animal species, and the ability to maintain transgene expression in both undifferentiated and differentiated cells.     

Isolation of a Herpesvirus Serologically Related to Bovine Herpesvirus 1 from a Reindeer (Rangifer Tarandus)

Serological evidence of exposure of reindeer (Rangifer tarandus) to a virus related to bovine herpesvirus 1 (BHV1) (Synonym: Infectious bovine rhinotracheitis (IBR) virus) has been reported in Canada (El Azhary 1979) and the USA (Dieterich 1981). A serological survey conducted in Finnish Lapland also detected neutralising antibodies to BHV1 in reindeer sera; 23 % of 300 reindeer had detectable antibodies, whereas none of 300 cattle sera from the same region contained antibodies to BHV1 (Ek-Kommonen et al. 1982). There is currently no evidence of BHV1 infection of cattle in Finland, so the isolation and characterisation of the reindeer herpesvirus was of considerable interest. This short communication describes the isolation and preliminary characterisation of a herpesvirus from a reindeer following the administration of dexamethasone.     

CD4+ Cytotoxic T-Lymphocyte Activity against Macrophages Pulsed with Bovine Herpesvirus 1 Polypeptides

Bovine herpesvirus 1 (BHV-1) induces immune suppression, but the mechanisms for suppression are not well identified. We examined the induction and activity of BHV-1-specific cytolytic CD4⁺ T lymphocytes (CTL) by stimulating peripheral blood mononuclear cells (PBMC) of cattle immunized with attenuated live BHV-1. Cytolytic effector cells were primarily CD4⁺ T lymphocytes and lysed autologous, but not allogeneic, macrophages infected with BHV-1 or pulsed with BHV-1 polypeptides. Apoptosis of BHV-1-expressing target cells was observed in CD4⁺ CTL assays by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) analysis. To determine if apoptosis was mediated by a perforin- or Fas-mediated pathway, EGTA, a known selective inhibitor of the perforin pathway, was used. EGTA did not inhibit CD4⁺-T-cell-mediated cytotoxic activity, but it did limit the NK cell cytotoxicity of virus infected cells. These findings support the concept that CD4⁺ CTL lyse macrophages pulsed with BHV-1 polypeptides through a Fas-mediated lytic pathway by inducing apoptosis in the target cells. The prominent cytotoxicity mediated by CD4⁺ CTL suggests a mechanism of selective removal of viral antigen-associated antigen-presenting cells.     

Bull semen variables after experimental exposure with Bovine Herpesvirus type 5

The influence of Bovine Herpesvirus type 5 (BoHV-5) infection on semen variables and sperm morphology collected from healthy bulls with no reproductive disorder was evaluated in ten ejaculates distributed into two experimental groups: group I, bull semen exposed to 10(2.3) (tissue culture infectious dose) TCID(50)/50 μl of a Brazilian strain of BoHV-5 (US9/BR/2007; GU9457818) and group II, unexposed bull control semen. After experimental infection, the semen was frozen-thawed prior to computerized analysis (CASA) of sperm motility and movement. Also analyzed were sperm phosphatidylserine transposition, acrosomal integrity, mitochondrial function, plasma membrane integrity and Annexin V expression. Viable BoHV-5 particles and their DNA were detected in infected semen after virus isolation and in situ hybridization (ISH) assay. The ISH revealed the BoHV-5 US9 gene in the acrosome and tail of infected spermatozoa. The only remarkable differences between groups I and II were the sperm kinetic variables, whereby infected sperm had a lesser mean velocity (VAP) and curvilinear velocity (VCL) values as compared to controls (P≤0.05). However, the straightness coefficient (STR) and beat cross frequency (BCF) values were higher in infected sperm. These results indicate that BoHV-5 can be found in infected sperm but induces no functional and morphological damage even after freeze-thawing, and, importantly, BoHV-5 can be spread via in vitro and in vivo reproductive biotechnology procedures.     

Bovine serum albumin decreases 4-methyl-2-oxovalerate utilization by isolated rat hepatocytes.

Binding of 4-methyl-2-oxo[1-14C]valerate to defatted bovine serum albumin inhibited the utilization of this 2-oxo acid by fed-rat hepatocytes in vitro. With 0-50g of albumin/l in the presence of 0.05mM 2-oxo acid or on increasing the 2-oxo acid concentration from 0 to 2mM in the presence of 26g of albumin/l, the extent of inhibition was essentially dependent on the change in the free 2-oxo acid concentration. Intrahepatocyte 4-methyl-2-oxo[1-14C]valerate concentrations were similar to extracellular free 2-oxo acid concentrations, suggesting equilibration so that the plasma membrane appears not to be rate-limiting for the utilization of this substrate by the isolated liver cells.     

牛疱疹病毒1型作为活病毒载体的研究进展

牛疱疹病毒1型（BHV1）是牛的易感病毒,作为大分子量DNA病毒,它具有插入并表达外源基因成为活病毒载体的潜力。随着对BHV1分子生物学的深入研究,BHV1已被广泛用于研究宿主范围狭窄而且安全的活病毒载体。     

Bovine brain ketimine reductase

We report the purification from bovine brain of an NAD(P)H-dependent reductase which actively reduces a new class of cyclic unsaturated compounds, named ketimines. Ketimines arise from the transamination of some sulphur-containing amino acids, such as L-cystathionine, S-aminoethyl-L-cysteine and L-lanthionine. The enzyme also reduces delta 1-piperidine 2-carboxylate, the carbon analog of aminoethylcysteine ketimine. Some kinetic and molecular properties of this enzyme have been determined. Subcellular localization and regional brain distribution have also been studied. The ketimine reductase activity was found to be associated with the soluble fraction, and was located prevalently in the cerebellum and cerebral cortices. Cyclothionine and 1,4-thiomorpholine-3,5-dicarboxylic acid, the enzymatic reduction products of cystathionine ketimine and lanthionine ketimine, respectively, have been detected in bovine brain, thus suggesting a role of this enzyme in their biosynthesis.     

Serum-free produced Bovine Herpesvirus type 1 and Bovine Parainfluenza type 3 virus vaccines are efficacious and safe

The studies described in this report were performed to determine, whether it is possible to produce live virus vaccines without serum or fractions thereof used during any cell or virus passage, thus completely serum-free. Two viruses were included in the experiments: Bovine Herpesvirus 1 (BHV-1) and Bovine Parainfluenza type 3 virus (PI3). Both viruses were found to grow to satisfactory titers, and to be stable after freeze-drying and subsequent storage at temperatures of +4 °C and −20 °C for at least one year. Moreover, a vaccine containing serum free produced BHV-1 was tested in a vaccination-challenge experiment. For comparison, a vaccine batch with BHV-1 grown in serum-containing cell culture medium was included in the study. Both vaccine preparations performed equally well and both met the strict requirements as laid down in the European Phamacopeia. Moreover, in two separate experiments the safety of serum-free produced BHV-1 and PI3 after overdose and repeated administration even in very young calves and even after four administrations has been demonstrated. This report is the first, which to our knowledge demonstrates the safety and efficacy of serum-free produced live vaccines in the target animal as well as the stability of these products. This revised version was published online in September 2006 with corrections to the Cover Date.     

Bovine Herpesvirus 1 Glycoprotein M Forms a Disulfide-Linked Heterodimer with the UL49.5 Protein

Nine glycoproteins (gB, gC, gD, gE, gG, gH, gI, gK, and gL) have been identified in bovine herpesvirus 1 (BHV-1). gM has been identified in many other alpha-, beta-, and gammaherpesviruses, in which it appears to play a role in membrane penetration and cell-to-cell fusion. We sought to express BHV-1 open reading frame U_L10, which encodes gM, and specifically identify the glycoprotein. We corrected a frameshift error in the published sequence and used the corrected sequence to design coterminal peptides from the C terminus. These were expressed as glutathione S-transferase fusion proteins in Escherichia coli. The fusion protein containing the 63 C-terminal amino acids from the corrected gM sequence engendered antibodies that immunoprecipitated a 30-kDa protein from in vitro translation reactions programmed with the U_L10 gene. Proteins immunoprecipitated by this antibody from virus-infected cells ran at 36 and 43 kDa in reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 43 and 48 kDa in nonreducing SDS-PAGE. Only the larger of the pair was present in virions. A 7-kDa protein was released from gM by reducing agents. The 7-kDa protein was not recognized in Western blots probed with the anti-gM antibody but reacted specifically with antibodies prepared against BHV-1 U_L49.5, previously reported to be a 9-kDa protein associated with an unidentified 39-kDa protein (X. Liang, B. Chow, C. Raggo, and L. A. Babiuk, J. Virol. 70:1448–1454, 1996). This is the first report of a small protein covalently bound to any herpesvirus gM. Similar patterns of hydrophobic domains and cysteines in all known gM and U_L49.5 homologs suggest that these two proteins may be linked by disulfide bonds in all herpesviruses.     

Microarray Chip Based Identification of a Mixed Infection of Bovine Herpesvirus 1 and Bovine Viral Diarrhea 2 From Indian Cattle

Bovine herpesvirus 1 (BHV1) and bovine viral diarrhea virus 2 (BVD2) are endemic in India although no mixed infection with these viruses has been reported from India. We report first mixed infection of these viruses in cattle during routine screening with a microarray chip. 62 of the 69 probes of BHV1 and 42 of the 57 BVD2 probes in the chip gave positive signals for the virus. The virus infections were subsequently confirmed by RT-PCR. We also discuss the implications of these findings.