Bovine viral diarrhea virus proteins: relatedness of p175 with p80 and p125 and evidence of glycoprotein processing.

Three monoclonal antibodies, which recognized two nonoverlapping antigenic domains and were reactive to the bovine viral diarrhea virus (BVDV) p80 protein, were found to cross react with the p125 protein of both cytopathic and noncytopathic BVDVs and a molecular weight 175,000 BVDV protein (p175). Results from limited proteolysis and chemical cleavage experiments confirmed the relatedness of these three proteins. In pulse-chase experiments it was apparent that p175 was a transient protein, as it was diminished during the chase, with a half-life of about 30 min. However, both p125 and p80 were also observed in short-pulsed lysates. Furthermore, during the chase, radiolabel was not found to accumulate into p125 or p80. Rather, these two proteins were stable with half-lives greater than 2 h. A fourth nonglycosylated protein, p37, increased during the chase. Processing of several glycoproteins was evident in these experiments. A glycoprotein of molecular weight 75,000 (gp75) diminished during the chase period, while glycoproteins gp62, gp48, and gp25 appeared or increased during the chase period. In contrast, the glycoprotein gp53 was a major protein in pulse-labeled cell lysates and remained constant throughout the chase period. In further experiments two stable forms of p80 differing in intramolecular disulphide bonding were observed.

Secondary in vitro B lymphocyte (antibody) response to microbial antigens: use in appraisal of vaccine immunogenicity and cytokine immunoregulation.

In order to perform preliminary evaluations of subunit vaccine candidates before extensive trials in large food-producing animals, an in vitro B-lymphocyte response assay, based on the principles of an ELISA, was established. The assay was developed in detail for the porcine system using antigens from the Gram-negative bacterium Actinobacillus pleuropneumoniae, but is shown to be applicable to other species and antigens, including viral components. It is further shown that B-cell activity in the assay is dependent on T-helper cells as well as macrophages and/or their secretory products. Thus, in addition to providing a tool for evaluation of T and B memory cell activity, the system also lends itself to dissection of T-B cell collaboration and the regulatory functions of interleukins in secondary (in vitro) antibody responses.

Rotavirus particles function as immunological carriers for the delivery of peptides from infectious agents and endogenous proteins.

A major problem in the development of useful animal subunit vaccines has been the generation of immune responses to weakly immunogenic molecules. For this purpose a new and effective delivery system has been devised. This system is based upon the inner capsid of bovine rotavirus. Under the appropriate conditions, the inner capsid protein, designated BP6, can be made to self-assemble in vitro and form spherical particles. These particles possess an inherent capacity to target to cells of the immune system. Exploitation of these properties has led to the development of technology to couple antigens to the VP6 particles such that the sphere acts as a novel immunological carrier. This is based on a "binding peptide" derived from another rotavirus peptide, VP4, as well as on more traditional techniques of chemical coupling. We have coupled peptides or proteins to this carrier via the binding peptide and have shown that every epitope tested to date gave excellent immune responses. Furthermore, using this carrier, immunity has been developed without the use of adjuvants. This has far-reaching implications for animal and human immunization.

Demonstration of the in vitro antiviral properties of bovine lymphokine-activated killer (LAK) cells.

In cattle, cells with functional characteristics similar to those of natural killer (NK) cells are difficult to detect. However, lymphokine-activated killer (LAK) cells can be detected readily after in vitro activation of peripheral blood mononuclear leukocytes (PBML) with interleukin-2 (IL-2). In the present study, we demonstrated that IL-2-activated PBML preferentially lyse bovine herpesvirus type 1 (BHV-1)-infected cells and that the cell responsible for the lysis copurified with the cell responsible for lysis of K562. The IL-2-activated effector cells were capable of significant reducing virus production. The reduction in virus yield seemed to be by an interferon (IFN)-independent mechanism, as the amount of IFN induced in effector cells by BHV-1 was not altered by the addition of IL-2. Furthermore, enrichment of cytotoxic cells by passage through nylon wool columns removed the capability of PBML to produce IFN in response to the virus. These results suggest that activation of LAK mechanisms in cattle plays a role in controlling virus spread.

Electron microscopy of bovine virus diarrhoea virus.

Bovine virus diarrhoea virus (BVDV) has been (tentatively) identified by electron microscopy in purified virus preparations, in infected cell cultures and in tissues and cells from infected animals. These studies have revealed a spherical membrane-bound particle with a diameter of 40-60 nm. The membrane is smooth, bilaminar and surrounds a dense or semi-dense core of 20-25 nm. The core particle may be isometric or hexagonal. In studies of the morphogenesis of BVDV in infected cell cultures, it was found that assembly and maturation of the viral particles occur via a condensation process within membrane-bound vesicular organelles, in which the virions subsequently accumulate. Release of the virus occurs when the cell finally lyses and/or via exocytosis. Thus, both with regard to morphogenesis and to morphology, BVDV bears close resemblance to the Flaviviridae.

The interaction between bovine herpesvirus type 1 and activated bovine T lymphocytes.

The interaction between activated bovine T lymphocytes (BTLs) and bovine herpesvirus type 1 (BHV-1) was investigated. BHV-1 infection of BTLs reduced the amplitude of recombinant bovine interleukin 2-induced proliferative responses. This decreased proliferation was caused by a virus-induced lymphocytolysis which was dependent on viable virus and was not inhibited by recombinant bovine interferon-alpha I1. Furthermore, lymphocytolysis was not associated with virus replication or with the synthesis of detectable levels of viral proteins. Electron microscopic examination of virus-infected cells revealed that lymphocytolysis was characterized by early nuclear disintegration resembling apoptosis. These observations suggest that activated T cells, localized at the site of BHV-1 infection, may be susceptible to virus-induced cytolysis.

Expression of tumor necrosis factor-alpha receptors on bovine macrophages, lymphocytes and polymorphonuclear leukocytes, internalization of receptor-bound ligand, and some functional effects.

Tumor necrosis factor-alpha (TNF) is a pluripotent protein produced by cells of the monocyte-macrophage lineage. It has important pro-inflammatory functions and is thought to be involved in the pathogenesis of viral-bacterial lung infections of cattle. Binding and internalization of gold-labeled TNF (TNF-G10) by bovine alveolar macrophages as well as by peripheral blood mononuclear leukocytes (PBML) and polymorphonuclear granulocytes (PMN) have therefore been investigated in conjunction with studies of the effect of TNF on some leukocyte functions. TNF-G10 bound to all leukocyte types to varying extents, which however, did not correlate with the influence of TNF on cell functions. Thus, PMN appeared to possess the lowest number of TNF-receptors, but were nevertheless the most sensitive to functional modulation by TNF. The expression of TNF-receptors appeared to be regulated by other cytokines such as IFN-gamma and IL-2, with IFN-gamma down-regulating receptor expression on all cell types, and IL-2 up-regulating receptor expression on lymphocytes. In macrophages internalization of TNF-G10 occurred via clathrin-coated structures, whereas in lymphocytes and PMN the endosomes appeared to lack distinct coating.

A neutrophil-derived antiviral protein: induction requirements and biological properties.

Polymorphonuclear neutrophilic granulocytes (PMN) have been implicated as playing a role in antiviral defense. In addition to having phagocytic and cytotoxic activities, PMN may produce an antiviral substance with interferon (IFN)-like activity. The product, for which the name polyferon (PF) has been coined, is produced upon direct encounter of PMN with bovine herpesvirus 1 (BHV-1)-infected bovine cells or membranes thereof. Exposure to purified virus only does not induce PF. The intimate interaction between PMN and the membranes was also revealed by electron microscopy studies. Bovine cells infected with herpes simplex virus type 1 could also induce PF production by bovine PMN, whereas cells infected with BHV-2, herpes simplex virus type 2, equine herpesvirus 1, bovine respiratory syncytial virus, bovine viral diarrhea virus, or parainfluenza virus 3 were unable to do so. Results obtained in experiments using transfected cells expressing BHV-1 glycoproteins as well as blocking experiments using BHV-1 glycoprotein-monospecific antibodies suggested that a combination of both viral product(s) and host cell factor(s) unique to bovine cells is required for induction of PF production by PMN. PF, which appeared in detectable amounts 12 to 18 h after exposure of PMN to the appropriate inducer, could not be neutralized by antibodies to bovine IFN-alpha, -beta, and -gamma. PF may nevertheless belong to the IFN family of proteins, as indicated by its ability to induce 2',5'-oligoadenyl synthetase in various cell types that are responsive to bovine IFNs and by its antiviral spectrum. It does, however, differ from the other cytokines in most immunological characteristics tested so far, including major histocompatibility complex class II antigen induction, cell migration, and cytotoxicity.

2'-5' oligo-A-synthetase activity in bovine peripheral blood leukocytes and alveolar macrophages exposed to recombinant interferons and tumor necrosis factor-alpha.

In vitro treatment of bovine peripheral blood mononuclear leukocytes, polymorphonuclear neutrophilic granulocytes and alveolar macrophages with recombinant bovine interferons -alpha 1 1, -beta 2 or -gamma induced an immediate increase in the intracellular level of 2'-5' oligoadenylate synthetase activity. The induction was dose-dependent, with interferon -alpha 1 1 and -beta 2 being more potent than interferon-gamma. Maximal levels were reached within 10-12 h with IFN-alpha 1 1, which corresponded well with findings in vivo. In contrast to what has been found in nonlymphoid bovine cells, tumour necrosis factor-alpha did not potentiate the induction of 2-5A synthetase by interferons, neither did it by itself induce the enzyme.

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.

Studies on the in vitro biological activities of recombinant bovine tumor necrosis factor (rBoTNF) alpha. I. Synergistic antiviral efficacy of rBoTNF alpha, recombinant bovine interferon gamma (rBoIFN gamma) and their combination.

The recent demonstration of the antiviral activity of recombinant human TNF has launched an interest in the use of TNF alpha for antiviral therapy in veterinary medicine. In the precent report, we demonstrate that pretreatment of bovine cells with rBoTNF alpha reduces the yield of bovine herpesvirus type-1 (BHV-1) from infected cells. Reduction in yield was similar to that observed in the presence of rBoIFN gamma. Similarly, rBoTNF alpha was able to protect bovine cells from virus-induced cytopathology. Enhanced antiviral activity was demonstrated when rBoTNF alpha was administered in combination with rBoIFN gamma. Studies on the induction of 2',5'-oligoadenylate synthetase (2'-5' AS) production by cytokine-treated cells showed that although rBoTNF alpha by itself did not induce 2'-5' AS activity it was capable of enhancing the production of 2'-5' AS by rBoIFN gamma-treated cells. Combination of these two cytokines was also evident in the inhibition of proliferation of treated cells. In contrast, the cytotoxic effect of rBoTNF alpha towards actinomycin D-treated cells was not affected by the combination of rBoTNF alpha with rBoIFN gamma.

Induction of MHC class II antigens on bovine cells of nonlymphoid origin by recombinant bovine interferon-gamma and tumor necrosis factor-alpha.

To test a hypothesis that immune mechanisms may be involved in the disease process in cattle persistently infected with and immunotolerant to bovine viral diarrhea virus, the effect of interferon-alpha I 1 (IFN-alpha I 1) and -gamma (IFN-gamma) and tumor necrosis factor (TNF-alpha) on bovine endothelial, epithelial, and fibroblast cell class II major histocompatibility complex (MHC) gene product expression was investigated. Untreated control cultures did not express MHC class II antigens. However, following exposure to either recombinant bovine IFN-gamma or TNF-alpha (rBoIFN-gamma, rBoTNF-alpha) MHC class II (Ia) antigen expression was induced on these nonlymphoid cell types. rBoIFN-alpha I 1 did not induce class II antigens, but suppressed their induction by rBoIFN-gamma and TNF-alpha. Induction of Ia-antigen expression, which was dependent on de novo protein synthesis, showed dose- and time-dependency, but once induced, Ia-antigen expression appeared to be fairly stable. Bovine viral diarrhea virus, which, at least in vivo, can induce IFN-alpha, neither induced Ia-antigen expression in the nonlymphoid cell types, nor did the virus interfere with cytokine induction of Ia-antigen expression.

Induction of receptors for complement and immunoglobulins by herpesviruses of various species.

Cells infected with herpes simplex virus type 1 (HSV-1) express a viral glycoprotein on the cell surface, which can function as a receptor for a cleavage product of complement factor 3 (C3b), and it has been suggested that this has biological relevance in the infected host (Smiley et al., 1985, J. Virol. 19, 217). As herpesviruses of different species share common determinants on their glycoproteins, a possible conservation of biological function was investigated for bovine herpesviruses type 1 and 2 (BHV-1 and -2), equine herpesvirus type 1 (EHV-1) and HSV-1 and -2, respectively. Only HSV-1 and EHV-1 induced C3b-receptors on infected cells. Nevertheless, BHV-1 infected cells could be killed by complement-dependent neutrophil mediated cytotoxicity (CDNC) as could EHV-1-infected cells. HSV-1-infected cells were not killed by this mechanism, but were highly susceptible to direct C-lysis. Four different scenarios for interaction between herpesvirus-infected cells and the nonspecific host defense system are presented.

Viral-bacterial synergistic interaction in respiratory disease.

In the present review we have identified how viruses can alter the host's susceptibility to bacterial infections by altering both environmental conditions in the lung which favor bacterial replication as well as by suppressing the host's defense mechanisms which prevent clearance of the bacteria. In many instances, these interactions are extremely complex but similar for many viruses. If the virus can overcome the initial host defense mechanisms, which include local antibody and mucus, the virus initiates tissue damage as a result of direct replication within the epithelial cells lining the mucosal surfaces of the respiratory tract. As a result of virus infection, the host cells respond by producing a variety of mediators including various types of interferons, which can alter both virus replication and host response. Replication also produces by-products of virus infection capable of initiating an inflammatory process, which in turn, through release of other mediators, can further modify lung defense mechanisms and encourage bacterial adherence and growth. The bacterium, in turn, releases chemotactic factors which encourage infiltration of specific effector cells into the lung. These effector cells can cause tissue damage and immunopathology, which encourage rapid bacterial growth and may result in death of the animal. In order to be able to control this complicated scenario, it is important either to prevent the initial infection with viruses or to reduce the degree of immunosuppression, so that bacterial clearance can occur rapidly before microcolony formation and extensive lung damage occur. Once a large amount of bacterial replication and lung damage is present, the use of antibiotics is generally of limited value. A schematic illustration of the complexity of the various interactions and counteractions occurring during virus--bacterial synergistic interactions is presented in Fig. 1.

Effect of dexamethasone on bovine leukocyte functions and bovine herpesvirus type-1 replication.

In an attempt to elucidate the mechanism whereby dexamethasone could reactivate bovine herpesvirus type-1 the effect of dexamethasone on virus replication and leukocyte functions was assessed. No effect was detectable on either virus yield or in vitro replication kinetics. In contrast, dexamethasone influenced several leukocyte functions thought to be of importance in antiviral defense and maintenance of latency. In vitro exposure of peripheral blood polymorphonuclear neutrophilic granulocytes of normal animals to dexamethasone depressed their migratory and cytotoxic activities, but had no effect on Fc- and complement receptor expression. Dexamethasone also depressed lectin-induced lymphocyte proliferation and interleukin-2 generation in a dose-dependent manner. When cows were treated repeatedly with dexamethasone and their leukocytes assayed, suppression of phytohemagglutinin-induced lymphocyte proliferation, interleukin-2 generation, natural cytotoxicity of mononuclear cells and polymorphonuclear neutrophilic granulocyte functions were observed. In contrast, concanavalin A induced lymphocyte proliferation was increased following treatment.

Bovine interferon: its biology and application in veterinary medicine.

Investigations of the production and potential use of bovine interferons against viral infections have occurred since the first descriptions of interferons in other systems. The recent advent of recombinant DNA-technology has facilitated such studies and furthered our knowledge about the bovine interferon system in general. This review gives an overview of the biology, antiviral and immunomodulatory activities of bovine interferons. Areas in which the interferons are now applied or have potential application in viral diseases in cattle are described. Finally, the value of studies of the bovine interferon system with respect to comparative interferon research is discussed.

Effect of bovine alpha 1 interferon on bovine herpesvirus type 1-induced respiratory disease.

Treatment of calves with bovine recombinant alpha 1 interferon prior to challenge with bovine herpesvirus type 1 increased the animals' ability to withstand a subsequent Pasteurella haemolytica challenge. The reduction in viral-bacterial synergy observed following interferon treatment did not appear to be due to a direct effect of the interferon on virus replication in the upper respiratory tract. Thus, even though interferon-treated animals shed slightly less virus from their nasal passages than did untreated animals, this reduction was not statistically significant. Furthermore, there was no difference in the level of intranasal interferon secreted by control or interferon-treated animals. These results suggest that interferon treatment does not affect the production of endogenous interferon. In contrast, a significant difference was observed between the number of days that control animals were sick, the levels of serum fibrinogen and the functional activity of polymorphonuclear neutrophilic granulocytes obtained from infected calves. These results suggest that bovine recombinant alpha 1 interferon may have a greater immunomodulatory effect than a direct antiviral effect in this model. This is further supported by the observation that bovine herpesvirus type 1 is relatively resistant to the direct antiviral effect of bovine recombinant alpha 1 interferon in vitro.

In vitro and systemic effects of recombinant bovine interferons on natural cell-mediated cytotoxicity in healthy and bovine herpesvirus-1-infected cattle.

There is now evidence to suggest that the beneficial effect of treatment of calves with recombinant bovine interferons (IFNs) on the outcome of an infection with bovine herpes-virus-1 (BHV-1) may in part be due to effects other than its direct antiviral activity. This evidence prompted us to study the effect of IFNs on natural cell-mediated cytotoxicity (NC) as a possible mechanism in curtailing clinical disease. In vitro treatment of blood leukocytes not only augmented cytotoxicity to NC-susceptible target cells, but also to cell types usually resistant to killing. In vivo treatment of healthy cattle with IFN-gamma caused a transient rise in NC activity, which was both dose and route dependent. Treatment of calves with IFN-alpha 1 or IFN-gamma prior to BHV-1 infection prevented or diminished the suppression of NC activity usually seen following virus infection. The effect could neither be correlated with serum IFN titers nor with conventional hematologic parameters. A possible mechanism for the IFN effect involving the ontogeny of bovine NC-effector cells and the biological consequences for the local antiviral defense in the lung are discussed.

Effect of recombinant DNA-produced bovine interferon alpha (BoIFN-alpha 1) on the interaction between bovine alveolar macrophages and bovine herpesvirus type 1.

Treatment of bovine alveolar macrophages (BAM) with bovine leukocyte interferon (BoIFN-alpha 1) resulted in reduced bovine herpesvirus type 1 replication and spread. This was demonstrated by reduced virus yields and number of cells infected. BoIFN-alpha 1 treatment of BAM also induced enhanced Fc receptor expression and/or avidity by the cells, increased their activity in antibody-dependent cellular cytotoxicity, and augmented their extrinsic antiviral activity as measured by a reduction in the development of plaques in a susceptible cell line. These results are discussed in the context of the possible role of interferon in activation of AM during the early phases of a virus infection.