Herpesvirus infection in woodland caribou in Alberta, Canada

Sera and genital swabs collected from 121 adult woodland caribou (Rangifer tarandus caribou) in five subpopulations in northern Alberta, Canada, between December 1997 and October 1999, were examined for evidence of infection with herpesviruses or pestiviruses. No virus was isolated from sera or swabs, and no antibodies against bovine viral diarrhea virus were detected. However, 63 (52%) of the 121 animals had neutralizing antibody titers against bovine herpesvirus 1. There was sufficient serum from 37 of the 121 caribou to allow parallel testing for antibodies against a new alphaherpesvirus isolated from an elk (Cervus elaphus nelsoni), and 20 animals had antibodies against this virus. Paired sera collected 11 mo apart from 14 caribou showed seroconversion in seven animals, indicating that an active herpesvirus infection was present. Virus neutralization data suggest that these caribou are infected with a distinct alphaherpesvirus.     

Serological survey of herpesvirus infections in wild ruminants of France and Belgium

The presence of antibodies against bovine herpesvirus 1 (BHV-1), bovid herpesvirus 6 (BHV-6), herpesvirus of Cervidae type 1 (HVC-1), reindeer herpesvirus, bovine herpesvirus 2 (BHV-2) and bovid herpesvirus 4 (BHV-4) was investigated in wild ruminants of France and Belgium between 1981 and 1986. There were no animals serologically positive for BHV-4. Antibodies against BHV-2 were demonstrated in roe deer (Cervus capreolus) (less than 1%) and chamois (Rupicapra rupicapra) (1%) in France. Animals seropositive to the four related viruses (BHV-1, BHV-6, HVC-1, reindeer herpesvirus) were detected in red deer (Cervus elaphus) in France and Belgium (1% and 11%, respectively), in roe deer (less than 1%) from France, in chamois (4%) in France and in ibex (Capra ibex) (4%) from France. The presence of antibodies against HVC-1, especially in red deer from Belgium, may suggest that wild ruminants in continental Europe are now infected with this virus, which previously has been isolated only in Scotland.     

Serologic survey of white-tailed deer on Anticosti Island, Quebec for bovine herpesvirus 1, bovine viral diarrhea, and parainfluenza 3.

In 1985 unusual mortality was observed among the 3- to 4-yr-old white-tailed deer (Odocoileus virginianus) on Anticosti Island, Québec (Canada). A viral pathogen was suspected to be the cause of the deaths. Thus, a serologic survey for bovine herpesvirus 1 (BHV-1), bovine viral diarrhea (BVD) virus and parainfluenza-3 (PI-3) virus was conducted. We examined 396 deer sera from 1985. Results indicated that the high mortality mainly afflicted 3- to 4-yr-old deer. In 1985, 57% of deer sampled were seropositive for viral neutralizing antibodies against BHV-1. Prevalences decreased over the next 2 yr of the survey. Prevalence of antibodies against PI-3 virus, determined by hemagglutination inhibition test, remained high (82% to 84%) for the 3 yr period. No deer were seropositive for neutralizing antibodies against BVD virus during the survey period. Analysis of antibodies against BHV-1 and PI-3 viruses according to sex, age and antibody titers revealed that an epizootic BHV-1 infection occurred in 1985; PI-3 infection appears to be enzootic in Anticosti deer.     

Characterization of bovine mononuclear cell populations with natural cytolytic activity against bovine herpesvirus 1-infected cells

Freshly isolated or overnight cultured peripheral blood mononuclear cells from immune or nonimmune animals had natural cytolytic activity against bovine herpesvirus 1 (BHV-1)-infected tumor target cells. No lysis was demonstrated against tumor target cells alone. This natural cytolytic activity was present in mononuclear cells from the spleen, lymph node, and peripheral blood but little or no cytolytic activity was detected in bone marrow or thymus cells. When monoclonal antibodies and complement to deplete bovine mononuclear cell subpopulations from the nonadherent cells were used, results indicated the effector cell was not a T cell, B cell, or activated monocyte. From nonadherent populations separated on density gradients, it was determined that the effector cells were large, low density mononuclear cells. These results indicate the nonadherent effector cells mediating lysis of BHV-1-infected xenogeneic adherent target cells were large null lymphocytes and/or immature monocytes.     

Neutralizing antibodies to bovine herpesvirus-1 (BHV-1) and bovine parainfluenza-3 (PI-3) viruses in cattle in Tunisia

A serological survey was conducted in an attempt to detect antibodies to bovine respiratory viruses in cattle from several localities around Tunis. Blood was collected from approximately 10% of the animals in each of the 44 farms visited and tested for specific antibodies against bovine herpesvirus-1 (BHV-1) and bovine parainfluenza-3 (PI-3) viruses, by ELISA and serum neutralization (SN). Antibodies to PI-3 and BHV-1 viruses were demonstrated in 55.3% and 25.9% animals, respectively. An overall 21.2% of the 170 animals tested had antibodies to both viruses. The incidence of antibody presence varied at different location. A correlation of the presence of BHV-1 antibody with breed and age of the animals was observed; however, no such relationship for PI-3 antibodies appeared to exist.     

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.     

Antibodies to ruminant alpha-herpesviruses and pestiviruses in Norwegian cervids

A serologic survey revealed that Norwegian populations of free-ranging reindeer (Rangifer tarandus tarandus), roe deer (Capreolus capreolus), red deer (Cervus elaphus), and moose (Alces alces) have been exposed to alpha-herpesviruses and pestiviruses. A total of 3,796 serum samples collected during the period 1993-2000 were tested in a neutralization test for antibodies against bovine herpesvirus 1 (BHV-1) or cervid herpesvirus 2 (CerHV-2), and 3,897 samples were tested by a neutralization test and/or enzyme-linked immunosorbent assay for antibodies against bovine viral diarrhea virus (BVDV). Antibodies against alpha-herpesvirus were found in 28.5% of reindeer, 3.0% of roe deer, and 0.5% of red deer, while all moose samples were negative. In reindeer, the prevalence of seropositive animals increased with age and was higher in males than females. Antibodies against BVDV were detected in 12.3% of roe deer, 4.2% of reindeer, 2.0% of moose and 1.1% of red deer. The results indicate that both alpha-herpesvirus and pestivirus are endemic in reindeer and pestivirus is endemic in roe deer in Norway. The viruses may be specific cervid strains. Seropositive red deer and moose may have become exposed as a result of contact with other ruminant species.     

Inactivated bovine herpesvirus 1 induces apoptotic cell death of mitogen-stimulated bovine peripheral blood mononuclear cells.

Bovine herpesvirus 1 (BHV-1) is able to inhibit the proliferation of bovine peripheral blood mononuclear cells. Here, we have demonstrated that live BHV-1 and, interestingly, inactivated BHV-1 can induce apoptosis of mitogen-stimulated bovine peripheral blood mononuclear cells in vitro.     

Synthesis, cellular location, and immunogenicity of bovine herpesvirus 1 glycoproteins gI and gIII expressed by recombinant vaccinia virus.

Two of the major glycoproteins of bovine herpesvirus 1 (BHV-1) are gI, a polypeptide complex with apparent molecular weights of 130,000, 74,000, and 55,000, and gIII (a 91,000-molecular-weight [91K] glycoprotein), which also exists as a 180K dimer. Vaccinia virus (VAC) recombinants were constructed which carry full-length gI (VAC-I) or gIII (VAC-III) genes. The genes for gI and gIII were each placed under the control of the early VAC 7.5K gene promoter and inserted within the VAC gene for thymidine kinase. The recombinant viruses VAC-I and VAC-III retained infectivity and expressed both precursor and mature forms of glycoproteins gI and gIII. The polypeptide backbones, partially glycosylated precursors, and mature gI and gIII glycoproteins were indistinguishable from those produced in BHV-1-infected cells. Consequently, they were apparently cleaved, glycosylated, and transported in a manner similar to that seen during authentic BHV-1 infection, although the processing efficiencies of both gI and gIII were generally higher in recombinant-infected cells than in BHV-1-infected cells. Immunofluorescence studies further demonstrated that the mature gI and gIII glycoproteins were transported to and expressed on the surface of cells infected with the respective recombinants. Immunization of cattle with recombinant viruses VAC-I and VAC-III resulted in the induction of neutralizing antibodies to BHV-1, which were reactive with authentic gI and gIII. These data demonstrate the immunogenicity of VAC-expressed gI and gIII and indicate the potential of these recombinant glycoproteins as a vaccine against BHV-1.     

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.     

Prevalence of Toxoplasma gondii antibodies in barren-ground caribou (Rangifer tarandus groenlandicus) from the Canadian Arctic

Prevalence of antibodies to Toxoplasma gondii was determined in 147 barren-ground caribou (Rangifer tarandus groenlandicus) from 5 herds in the Northwest Territories and Nunavut, northern Canada, by the modified agglutination test (MAT). In the mainland herds (Bluenose, Bathurst, and Beverly), antibodies were found in 43 (37%) of 117 caribou, and MAT titers were 1:25 in 10, 1:50 in 24, and 1:500 in 9. In the island herds, only 1 (4.3%) of 23 animals sampled from the North Baffin Island herd was positive (titer = 1:25) and no antibodies were detected in 7 caribou from the Dolphin and Union herd. The high prevalence of antibodies to T. gondii in the mainland caribou herds indicates that caribou meat may contain viable T. gondii.     

Degenerate PCR method for identification of an antiapoptotic gene in BHV-1

To investigate on the hypothetical presence of an antiapoptotic gene, we utilized the CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primers) strategy amplifying unknown sequences from a background of genomic (bovine herpesvirus type-1) BHV-1 DNA. An alignment of carboxyl-terminal domains belonging to three proteins encoded by gamma34.5, MyD116 and GADD34 genes, was carried out to design degenerate PCR primers in highly conserved regions. This allowed the amplification of a 110 bp fragment. This fragment was subjected to automatic sequencing and DNA sequence analysis revealed that its position resided between the nt 14363 and the nt 14438 in bovine herpesvirus type-1 (BHV-1) Cooper strain sharing an identity of 86% (UL14). Transient transfections showed that UL14 protein is efficient in protecting MDBK and K562 cells from sorbitol induced apoptosis. The protein's anti-apoptotic function may derive from its heat shock protein-like properties.     

Bovine cells expressing bovine herpesvirus 1 (BHV-1) glycoprotein IV resist infection by BHV-1, herpes simplex virus, and pseudorabies virus.

We expressed the bovine herpesvirus 1 (BHV-1) glycoprotein IV (gIV) in bovine cells. The protein expressed was identical in molecular mass and antigenic reactivity to the native gIV protein but was localized in the cytoplasm. Expressing cells were partially resistant to BHV-1, herpes simplex virus, and pseudorabies virus, as shown by a 10- to 1,000-fold-lower number of plaques forming on these cells than on control cells. The level of resistance depended on the level of gIV expression and the type and amount of challenge virus. These data are consistent with previous reports by others that cellular expression of the BHV-1 gIV homologs, herpes simplex virus glycoprotein D, and pseudorabies virus glycoprotein gp50 provide partial resistance against infection with these viruses. We have extended these findings by showing that once BHV-1 enters gIV-expressing cells, it replicates and spreads normally, as shown by the normal size of BHV-1 plaques and the delayed but vigorous synthesis of viral proteins. Our data are consistent with the binding of BHV-1 gIV to a cellular receptor required for initial penetration by all three herpesviruses and interference with the function of that receptor molecule.     

Characterization of envelope proteins of infectious bovine rhinotracheitis virus (bovine herpesvirus 1) by biochemical and immunological methods.

Ten glycoproteins of molecular weights of 180,000, 150,000, 130,000, 115,000, 97,000, 77,000, 74,000, 64,000, 55,000, and 45,000 (designated as 180K, 150K, etc.) and a single nonglycosylated 107,000-molecular-weight (107K) protein were quantitatively removed from purified bovine herpesvirus 1 (BHV-1) virions by detergent treatment. Immunoprecipitations with monospecific and monoclonal antibodies showed that three sets of coprecipitating glycoproteins, 180K/97K, 150K/77K, and 130K/74K/55K, were the major components of the BHV-1 envelope. These glycoproteins were present in the envelope of the virion and on the surface of BHV-1-infected cells and reacted with neutralizing monoclonal and monospecific antibodies. Antibodies to 150K/77K protein had the largest proportion of virus-neutralizing antibodies, followed by antibodies to 180K/97K protein. Monoclonal antibodies to 130K/74K/55K protein were neutralizing but only in the presence of complement; however, monospecific antisera produced with 55K protein did not have neutralizing activity. Analysis under nonreducing conditions showed that the 74K and 55K proteins interact through disulfide bonds to form the 130K molecule. Partial proteolysis studies showed that the 180K protein was a dimeric form of the 97K protein and that the 150K protein was a dimer of the 77K protein, but these dimers were not linked by disulfide bonds. The 107K protein was not glycosylated and induced antibodies that did not neutralize BHV-1. The 64K protein was not precipitated by anti-BHV-1 convalescent antisera, and monospecific antisera to this protein precipitated several polypeptides from uninfected cell lysates, suggesting that 64K is a protein of cellular origin associated with the BHV-1 virion envelope.     

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.     

Inhibition of T-lymphocyte mitogenic responses and effects on cell functions by bovine herpesvirus 1.

The mitogenic response of bovine peripheral blood mononuclear cells stimulated by concanavalin A (ConA) was suppressed by infectious bovine herpesvirus 1 (BHV-1). Proliferation in response to interleukin-2 (IL-2) by IL-2-dependent lymphocyte cultures was also inhibited by BHV-1. Although inhibition of mitogenesis approached 100%, less than 1 cell in 1,000 was productively infected by BHV-1 in ConA-stimulated cultures. Neither conditioned medium from mitogen-stimulated peripheral blood mononuclear cell cultures nor human recombinant IL-2 reversed suppression by the virus. Infection by BHV-1 did not influence the expression of IL-2 or IL-2 receptor mRNA in ConA-stimulated cultures, nor did it affect the cytolytic capabilities of lymphocytes. The data suggest that the inhibition of T-lymphocyte proliferation is the result of a nonproductive BHV-1 infection.     

Expression of bovine herpesvirus 1 glycoproteins gI and gIII in transfected murine cells.

Genes encoding two of the major glycoproteins of bovine herpesvirus 1 (BHV-1), gI and gIII, were cloned into the eucaryotic expression vectors pRSVcat and pSV2neo and transfected into murine LMTK- cells, and cloned cell lines were established. The relative amounts of gI or gIII expressed from the two vectors were similar. Expression of gI was cell associated and localized predominantly in the perinuclear region, but nuclear and plasma membrane staining was also observed. Expression of gI was additionally associated with cell fusion and the formation of polykaryons and giant cells. Expression of gIII was localized predominantly in the nuclear and plasma membranes. Radioimmunoprecipitation in the presence or absence of tunicamycin revealed that the recombinant glycoproteins were proteolytically processed and glycosylated and had molecular weights similar to those of the forms of gI and gIII expressed in BHV-1-infected bovine cells. However, both recombinant glycoproteins were glycosylated to a lesser extent than were the forms found in BHV-1-infected bovine cells. For gI, a deficiency in N-linked glycosylation of the amino-terminal half of the protein was identified; for gIII, a deficiency in O-linked glycosylation was implicated. The reactivity pattern of a panel of gI- and gIII-specific monoclonal antibodies, including six which recognize conformation-dependent epitopes, was found to be unaffected by the glycosylation differences and was identical for transfected or BHV-1-infected murine cells. Use of the transfected cells as targets in immune-mediated cytotoxicity assays demonstrated the functional recognition of recombinant gI and gIII by murine antibody and cytotoxic T lymphocytes. Immunization of mice with the transfected cells elicited BHV-1-specific virus-neutralizing antibody, thus verifying the antigenic authenticity of the recombinant glycoproteins and the important role of gI and gIII as targets of the immune response to BHV-1 in this murine model system.     

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.     

Impacts of spruce budworm defoliation on the habitat of woodland caribou,moose, and their main predators

Forest logging has contributed to the decline of several woodland caribou populations by causing the fragmentation of mature coniferous stands. Such habitat alterations could be worsened by spruce budworm (SBW) outbreaks. Using 6201 vegetation plots from provincial inventories conducted after the last SBW outbreak (1968–1992) in boreal forests of Québec (Canada), we investigated the influence of SBW‐caused tree defoliation and mortality on understory vegetation layers relevant to woodland caribou and its main predators. We found a positive association between severe outbreaks and the cover of most groups of understory plant species, especially in stands that were dominated by balsam fir before the outbreak, where a high canopy openness particularly benefited relatively fast‐growing deciduous plants. Such increases in early successional vegetation could provide high‐quality forage for moose, which is likely to promote higher wolf densities and increase predation pressure on caribou. SBW outbreaks may thus negatively affect woodland caribou by increasing predation risk, the main factor limiting caribou populations in managed forests. For the near future, we recommend updating the criteria used to define critical caribou habitat to consider the potential impacts of spruce budworm defoliation.     

Serological survey for potential disease agents of free-ranging cervids in six selected national parks from Germany

A total of 164 blood samples, collected from free-ranging red deer (Cervus elaphus), roe deer (Capreolus capreolus) and fallow deer (Dama dama) in six German national parks (NP) between 2000 and 2002, were assayed for antibodies against nine viral disease agents. Antibodies were only detected against the alpha-herpesviruses; specifically, bovine herpesvirus-1 (BHV-1) (22 of 157, 14%), cervid herpesvirus-1 (17 of 157, 10.8%), and caprine herpesvirus-1 (11 of 159, 6.9%). Titers ranged from 4 to 102. Most of the seropositive sera, and those with the highest antibody titers, were from red and roe deer in the Harz and Hochharz NP, which are connected and allow migration between the two. The distribution and specificity of antibodies detected in individual deer suggests that the three alpha-herpesviruses are circulating in these deer populations. No antibodies were detected against bovine viral diarrhea virus, epizootic hemorrhagic disease virus, bovine leukemia virus, bluetongue virus, foot-and-mouth disease virus, or sheep and goat poxvirus.