2,3,7,8-Tetrachlorodibenzo-<Emphasis Type="Italic">p</Emphasis>-dioxin regulates Bovine Herpesvirus type 1 induced apoptosis by modulating Bcl-2 family members

Exposure to environmental contaminants, like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), leads to an increased susceptibility to infectious agents. Infection of bovine cells (MDBK) with Bovine Herpesvirus 1 (BHV-1) anticipates virus-induced apoptosis, suggesting an involvement of TCDD in virus infection. Herein we analyzed the effects of TCDD on apoptotic pathway in MDBK cells infected with BHV-1. After 12 h of infection, TCDD induced a significant increase in apoptotic cells. TCDD caused a dose-dependent up-regulation and anticipated activation of caspases 3, 8 and 9, with respect to unexposed groups. TCDD anticipated cleavage of PARP, compared to controls. Furthermore TCDD increased Bax and Bid levels, and decreased Bcl-2 and Bcl-XL levels. Such events took place earlier in exposed than unexposed cells. These results showed that TCDD influences BHV-1 induced apoptosis through members of Bcl-2 family and up-regulating activation of caspases.     

Expression of iron-related proteins during infection by bovine herpes virus type-1

Bovine herpesvirus 1 (BHV-1), a dsDNA animal virus, is an economically important pathogen of cattle and the aetiological agent of many types of disease. The efficient replication of a DNA virus is strictly dependent on iron since this metal plays a crucial role in the catalytic center of viral ribonucleotide reductase. Consequently, iron metabolism is an important area for virus/host interaction and a large body of evidence suggests that viral infection is potentially influenced by the iron status of the host. The aim of the present study was to address the effects of BHV-1 on iron metabolism in Madin-Darby bovine kidney (MDBK) cells at different times of post-infection. For this purpose, cell viability, iron regulatory proteins (IRPs) activity and levels, transferrin receptor 1 (TfR-1), ferritin expression and LIP were evaluated. Our data demonstrate that a productive BHV-1 infection in MDBK cells determines an overall decrease of IRPs RNA-binding activity without affecting their expression. As consequence of this modulation, an increased ferritin mRNA translation and a decreased TfR-1 mRNA translation were also observed. Moreover, the LIP level was decreased following viral infection. These results are consistent with the hypothesis that by reducing the iron up-take and by enhancing the sequestration of free iron, animal cells will limit the iron availability for virus proliferation. Therefore, the results presented herein support the view that iron metabolism could be critical for the interaction between DNA viruses, such as BHV-1, and mammalian cells. Delineation of the interplay among pathogen and host may provide new antimicrobial agents.     

Coinfection with BHV-1 modulates cell adhesion and invasion by P. multocida and Mannheimia (Pasteurella) haemolytica

Viruses are thought to facilitate bacterial infections of the respiratory tract. The present study shows the effect of BHV-1 on Pasteurella multocida and Mannheimia haemolytica adherence and invasion of MDBK cells. The virus-infected MDBK cells become more susceptible to the adherence of both species of Pasteurella. The observed adherence increase depends on the length of virus pre-incubation time and on virus concentration. When MDBK cells are not infected with virus, they are only invaded by P. multocida, while M. haemolytica is not able to penetrate. The viral infection favours also the invasion by M. haemolytica.     

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.     

Infectious Bovine Viral Diarrhea Virus (Strain NADL) RNA from Stable cDNA Clones: a Cellular Insert Determines NS3 Production and Viral Cytopathogenicity

Bovine viral diarrhea virus (BVDV), strain NADL, was originally isolated from an animal with fatal mucosal disease. This isolate is cytopathic in cell culture and produces two forms of NS3-containing proteins: uncleaved NS2-3 and mature NS3. For BVDV NADL, the production of NS3, a characteristic of cytopathic BVDV strains, is believed to be a consequence of an in-frame insertion of a 270-nucleotide cellular mRNA sequence (called cIns) in the NS2 coding region. In this study, we constructed a stable full-length cDNA copy of BVDV NADL in a low-copy-number plasmid vector. As assayed by transfection of MDBK cells, uncapped RNAs transcribed from this template were highly infectious (>10⁵ PFU/μg). The recovered virus was similar in plaque morphology, growth properties, polyprotein processing, and cytopathogenicity to the BVDV NADL parent. Deletion of cIns abolished processing at the NS2/NS3 site and produced a virus that was no longer cytopathic for MDBK cells. This deletion did not affect the efficiency of infectious virus production or viral protein production, but it reduced the level of virus-specific RNA synthesis and accumulation. Thus, cIns not only modulates NS3 production but also upregulates RNA replication relative to an isogenic noncytopathic derivative lacking the insert. These results raise the possibility of a linkage between enhanced BVDV NADL RNA replication and virus-induced cytopathogenicity.     

光生物素标记DNA探针检测感染细胞的BHV—1 DNA

将七株从不同地区、不同牛种或不同部位分离出的牛Ⅰ型疱疹病毒(BHV-1)培养于MDBK细胞中,从感染的细胞中提取病毒并抽提其DNA将克隆的BHV-1LA株DNA的HindⅢ—Ⅰ片段(11.7Kb)用光生物素标记作为探针,检测上述七株感染细胞的BHV-1 DNA,其中有六株(均属IBR临床型)呈阳性反应,另一株(属IPP临床型)呈阴性反应,这一探针将作为一种有效的检疫工具在本病的防制中起重要作用.     

Transfection of bovine cell culture with bovine herpesvirus 4 DNA obtained by cell nuclear extraction

Bovine herpes virus 4 (BHV-4) is a gamma-herpesvirus not associated with clearly defined clinical entities in cattle. The BHV-4 genome consists of a linear dsDNA of approximately 145 Kbp which is only partially characterized and sequenced. We set up a rapid and practical method to isolate BHV-4 DNA from infected cell culture. Microfuged infected cells after exposure to high salt concentration and detergent allowed viral DNA to be purified. Electrophoretic analysis of the digested DNA showed a complete digestion, corresponding to a classical EcoRI banding pattern of strains Movar 33/63, LVR and DN 599. Moreover the biological integrity of viral DNA here obtained, was demonstrated by transfection experiments. BHV-4 DNA was capable of forming CPE after transfection into BAE-7372 cells. Transfected cells specifically reacted with a BHV-4 infected cow serum demonstrating the presence of viral particles. The possibility of obtaining infectious viral DNA using this method may facilitate the construction of recombinant viruses. Specifically, through the use of cotransfection experiments with deleted or mutated viral DNA sequences, the infectious clones isolated could provide the basis for an increased understanding of BHV-4 viral gene expression, replication and pathogenesis.     

Interference of bovine herpesvirus 1 (BHV-1) in sorbitol-Induced apoptosis

In order to determine the ability of bovine herpesvirus type 1 (BHV-1) to suppress apoptosis, we examined the effects of BHV-1 infection on sorbitol-induced apoptosis on Madin-Darby bovine kidney (MDBK) cells. BHV-1 suppresses sorbitol-induced apoptosis in a manner similar to that of herpes simplex virus type 1 (HSV-1), indicating that BHV-1 has one or more anti-apoptotic genes. To elucidate the molecular mechanisms of apoptosis, expression of some genes encoding apoptosis-inhibiting and -promoting factors were analyzed on BHV-1 infected cells during the process of sorbitol-induced apoptosis. Our results revealed that the expression of bcl-2 and bcl-x(L) decreased after 5 and 3 h p.i., respectively; while bax and procaspase-3 expression increased with respect to control as a function of p.i. times and at 7 h p.i. they were not observed. We further show that the expression of p53 gene was also enhanced, suggesting that this apoptotic mechanism is p53 dependent. From these results, we propose that BHV-1 has one or more genes encoding apoptosis-inhibiting factors which interfere with the involvement of bcl-2 gene family members and apoptotic pathway, depending upon caspase-3, triggered by sorbitol.     

Bovine herpesvirus 1 tegument protein VP22 interacts with histones, and the carboxyl terminus of VP22 is required for nuclear localization

The bovine herpesvirus 1 (BHV-1) UL49 gene encodes a viral tegument protein termed VP22. UL49 homologs are conserved among alphaherpesviruses. Interestingly, the BHV-1 VP22 deletion mutant virus is asymptomatic and avirulent in infected cattle but produces only a slight reduction in titer in vitro. Attenuation of the BHV-1 VP22 deletion mutant virus in vivo suggests that VP22 plays a functional role in BHV-1 replication. In herpes simplex virus type 1, the VP22 homolog was previously shown to interact with another tegument protein,VP16, the alpha-transinducing factor in vitro. In this report, we show that (i) the nuclear targeting of VP22 is independent of other viral factors, (ii) the carboxyl terminus of VP22 is required for its nuclear localization, (iii) VP22 associates with histones and nucleosomes, (iv) an antihistone monoclonal antibody cross-reacts with VP22, and (v) acetylation of histone H4 is decreased in VP22-expressing cells as well as virus-infected cells. Our data suggest that VP22 may have a modulatory function during BHV-1 infection.     

Role of interferon-gamma in inducing cytotoxicity of peripheral blood mononuclear leukocytes to bovine herpesvirus type 1 (BHV-1)-infected cells

In the present study, the possible role of interferon (IFN)-gamma on the induction of cytotoxic activity of peripheral blood mononuclear leukocytes (PBML) from BHV-1-immune cattle was investigated. Supernatants obtained from BHV-1-immune PBML, stimulated under conditions similar to those required to demonstrate cytotoxicity, contained an antiviral substance capable of inducing 2'-5'-oligoadenylate synthetase activity in MDBK cells and MHC class II antigen expression on epithelial cells. These supernatants also contained IFN-alpha, but were devoid of tumor necrosis factor and interleukin-2 biological activities. Further studies during primary infection and hyperimmunization with BHV-1 showed that IFN-gamma production and non-MHC-restricted cytotoxicity against BHV-1-infected targets always occurred concomitantly, suggesting that they represent an important part of the detectable CMI responses mounted against this virus. Furthermore, it was also demonstrated that cytotoxicity of PBML against BHV-1-infected cells was reduced with the addition of antibodies to bovine IFN-gamma to the cytotoxic assay. Bovine recombinant IFN-gamma was able to enhance the in vitro cytotoxic activity of PBML from immune cattle, but not from their nonimmune counterparts. This suggests that other factors, in addition to IFN-gamma, may be essential in the development of non-MHC-restricted cytotoxic responses during BHV-1 infection.     

Cloning and expression of bovine herpesvirus-1 glycoprotein C

Glycoprotein C (gC) of Bovine Herpesvirus-1 (BHV-1) is expressed at high levels on surface of infected cells and on virus envelope. It is relatively immunodominant in antibody response to BHV-1 infection and protective in immunized bovines against BHV-1 challenge. In an attempt to express gC in mammalian cells, the 2.4 kb BamHI-EcoRI fragment, containing complete coding sequence of the gC gene was excised from a recombinant plasmid and cloned under the control of RSV-LTR. The resultant plasmid pRSV-gC was transfected into MDBK cells and expression of gC was detected by indirect immunofluorescence. The non-permeabilized cells revealed surface expression of gC.     

Bovine herpesvirus 1 U(L)3.5 interacts with bovine herpesvirus 1 alpha-transinducing factor

The bovine herpesvirus 1 (BHV-1) U(L)3.5 gene encodes a 126-amino-acid tegument protein. Homologs of U(L)3.5 are present in some alphaherpesviruses and have 20 to 30% overall amino acid homology that is concentrated in the N-terminal 50 amino acids. Mutant pseudorabies virus lacking U(L)3.5 is deficient in viral egress but can be complemented by BHV-1 U(L)3.5 (W. Fuchs, H. Granzow, and T. C. Mettenleiter, J. Virol. 71:8886-8892, 1997). The function of BHV-1 U(L)3.5 in BHV-1 replication is not known. To get a better understanding of its function, we sought to identify the proteins that interact with the BHV-1 U(L)3.5 protein. By using an in vitro pull-down assay and matrix-assisted laser desorption ionization mass spectrometry analysis, we identified BHV-1 alpha-transinducing factor (alphaBTIF) as a BHV-1 U(L)3. 5-interacting protein. The interaction was verified by coimmunoprecipitation from virus-infected cells using an antibody to either protein, by indirect immunofluorescence colocalization in both virus-infected and transfected cells, and by the binding of in vitro-translated proteins. In virus-infected cells, U(L)3.5 and alphaBTIF colocalized in a Golgi-like subcellular compartment late in infection. In transfected cells, they colocalized in the nucleus. Deletion of 20 amino acids from the N terminus of U(L)3.5, but not 40 amino acids from the C terminus, abolished the U(L)3.5-alphaBTIF interaction both in vitro and in vivo. The interaction between U(L)3. 5 and alphaBTIF may be important for BHV-1 maturation and regulation of alphaBTIF transactivation activity.     

Bovine herpesvirus type 4 infection modulates autophagy in a permissive cell line

Bovine herpesvirus type 4 (BoHV‐4), like other herpesviruses, induces a series of alterations in the host cell that modify the intracellular environment in favor of viral replication, survival and spread. This research examined the impact of BoHV‐4 infection on autophagy in BoHV‐4 infected Madin Darby bovine kidney (MDBK) cells. Protein extracts of BoHV‐4 infected and control MDBK cells were subjected to Western blot. The concentrations of the autophagy and apoptosis‐related proteins Beclin 1, p21, PI3 kinase, Akt1/2, mTOR, phospho mTOR, p62 and the light chain three (LC3) were normalized to the actin level and expressed as the densitometric ratio. Western blot analysis of virus‐infected cells revealed that autophagic degradation pathway was induced in the late phase of BoHV‐4 infection. After 48 h post‐infection the protein LC3II, which is essential for autophagy was found to be markedly increased, while infection of MDBK cells with BoHV‐4 resulted in a depletion of p62 levels. Becline 1, PI3 kinase, Akt1/2 and p21 expression increased between 24 and 48 h post‐infection. Surprisingly, mTOR and its phosphorylated form, which are negative regulators of autophagy, also increased after 24 h post‐infection. In conclusion, our findings suggest that BoHV‐4 has developed mechanisms for modulation of autophagy that are probably part of a strategy designed to enhance viral replication and to evade the immune system. Additional studies on the relationship between autophagy and BoHV‐4 replication and survival, in both lytic and latent replication phases, are needed to understand the role of autophagy in BoHV‐4 pathogenesis. J. Cell. Biochem. 114: 1529–1535, 2013. © 2013 Wiley Periodicals, Inc.     

Susceptibility of zona-intact and zona-free in vitro-produced bovine embryos at different stages of development to infection with bovine herpesvirus-1

The aim of the present study was to determine if BHV-1 is able to replicate within in vitro produced embryos and to investigate the degree to which the zona pellucida (ZP) is able to protect in vitro produced embryos against infection with BHV-1. Both ZP-intact and ZP-free matured oocytes, zygotes (1 d post insemination; 1dpi), 8-cell stage embryos (3 dpi), morulae (6 dpi) were incubated for 1 h in 1 ml of MEM containing 10(7.7) TCID(50)/ml BHV-1 (Cooper strain). Three titers (10(5.7), 10(6.7) and 10(7.7) TCID(50)/ml) of the Cooper strain were used for incubation of hatched blastocysts (9 dpi). Bovine embryonic lung cells (BEL) on microcarriers were inoculated following the same protocol as for the embryos. At 0, 12, 24, 36 and 48 h post inoculation (hpi), groups of embryos and BEL cells were collected for virus titration and for the determination of the percentage of viral antigen positive cells by immunofluorescence. For the 3 developmental stages in ZP-free embryos, similar maximal intracellular virus progeny titers were obtained at 24 to 48 hpi ranging from 10(1.32) to 10(1.43) TCID(50)/ 100 embryonic cells. The intracellular virus titer in the BEL cells peaked at 10(3.08) TCID(50)/ 100 BEL cells. The percentage of cells which expressed viral antigens was 13% in ZP-free hatched blastocysts, 17% in ZP-free morulae and 100% in BEL cells. In ZP-intact embryos, no replication of BHV-1 was detected. These results clearly show that only after removal of the zona pellucida, BHV-1 is able to replicate within the in vitro produced embryos, with only a subset of embryonic cells being fully susceptible.     

Glycoprotein IV of bovine herpesvirus 1-expressing cell line complements and rescues a conditionally lethal viral mutant.

Glycoprotein IV (gIV) of bovine herpesvirus 1 (BHV-1), a homolog of herpes simplex virus glycoprotein D, represents a major component of the viral envelope and a dominant immunogen. To analyze the functional role of gIV during BHV-1 replication, cell line BUIV3-7, which constitutively expresses gIV, was constructed and used for the isolation of gIV- BHV-1 mutant 80-221, in which the gIV gene was replaced by a lacZ expression cassette. On complementing gIV-expressing cells, the gIV- BHV-1 replicated normally but was unable to form plaques and infectious progeny on noncomplementing cells. Further analysis showed that gIV is essential for BHV-1 entry into target cells, whereas viral gene expression, DNA replication, and envelopment appear unchanged in both noncomplementing and complementing cells infected with phenotypically complemented gIV- BHV-1. The block in entry could be overcome by polyethylene glycol-induced membrane fusion. After passaging of gIV- BHV-1 on complementing cells, a rescued variant, BHV-1res, was isolated and shown to underexpress gIV in comparison with its wild-type parent. Comparison of the penetration kinetics of BHV-1 wild type, phenotypically complemented gIV- BHV-1, and BHV-1res indicated that penetration efficiency correlated with the amount of gIV present in virus particles. In conclusion, we show that gIV of BHV-1 is an essential component of the virion involved in virus entry and that the amount of gIV in the viral envelope modulates the penetration efficiency of the virus.     

Apoptosis in bovine herpesvirus-1 infected bovine peripheral blood mononuclear cells

Apoptosis is a process whereby cells die in a controlled manner in response to various stimuli like cytotoxins, viral antigens and normal physiological signals during differentiation and development. Virus induced immunosuppression has been reported for various viral diseases including Bovine Herpesvirus-1 (BHV-1). In the present study, BHV-1 was found to cause apoptosis in ConA stimulated bovine peripheral blood mononuclear cells (PBMCs). Apoptotic index quantified by fluorescent dyes revealed a significant (P < 0.001) increase in percent apoptotic cells at 2, 24 and 48 hr post infection as compared to their respective non-infected controls. Apoptosis specific internucleosomal laddering in DNA from BHV-1 infected PBMCs was seen in agarose gel electrophoresis. No DNA fragmentation was observed in control non-infected PBMCs.