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Bovine herpesvirus type 1 (BoHV-1) and bovine herpesvirus type 5 (BoHV-5) are important pathogens of cattle. The diseases they produce are quite different, with BoHV-5 being more neuropathogenic than BoHV-1 which mainly induces respiratory symptoms. The sequencing of the entire BoHV-5 genome has shown that most of the differences between these viruses are found in the immediate early and LR (latency related) genes. The LR gene is the only viral gene abundantly expressed in latently infected neurons, is essential for viral reactivation and seems to have an anti-apoptotic function which can be observed in vivo and in vitro. This gene spans two potential ORFs (1 and 2) which can also be found as a fused version, an ORF-E protein encoded within the promoter region and two miRNAs located within the 5' UTR segment. Most of the essential functions of the LR gene seem to be located within the ORF-2 which has been found to modulate components of cell signaling/cycle pathways. In this review we present a comparative sequence analysis of the LR gene of several BoHV-5 isolates, their differences with the BoHV-1 homologue and the potential impact this may have on its function. The LR gene was found to be highly conserved in all sequenced BoHV-5 strains. ORF-1 shares 60 % homology compared to BoHV-1 whereas the BoHV-5 homologue of ORF-2 is truncated at amino acid 51. Preliminary studies analyzing the emerging transcripts from the BoHV-5 LR gene in infected cells, as well as in stably transfected cells, indicates that their products are, in fact, missing crucial components of the anti-apoptotic function when compared to the BoHV-1 LR gene. In addition these transcripts maintain a region that, similar to what is found in BoHV-1, would produce a miRNA with the potential to recognize a region within the BoHV-5 immediate early gene. All together, these BoHV-5 characteristics suggest that this virus would not possess the same repertoire of latency maintaining functions as BoHV-1. Implications for BoHV-5 neuropathogenic potential are discussed.

Members of the subfamily Alphaherpesvirinae use the epithelium of the upper respiratory and/or genital tract as preferential sites for primary replication. However, bovine herpesvirus 5 (BoHV5) is neurotropic and neuroinvasive and responsible for meningoencephalitis in cattle and in animal models. A related virus, BoHV1 has also been occasionally implicated in natural cases of neurological infection and disease in cattle. The aim of the present study was to assess the in vitro effects of BoHV1 and BoHV5 replication in neuron-like cells. Overall, cytopathic effects, consisting of floating rounded cells, giant cells and monolayer lysis, induced by both viruses at 48 h postinfection (p.i.) resulted in a loss of cell viability and high virus titres (r = 0.978). The BoHV1 Cooper strain produced the lowest titres in neuron-like cells, although viral DNA was detected in infected cells during all experiments. Virus replication in infected cells was demonstrated by immunocytochemistry, flow cytometry and qPCR assays. BoHV antigens were better visualized at 48 h p.i. and flow cytometry analysis showed that SV56/90 and Los Angeles antigens were present at higher levels. In spite of the fact that BoHV titres dropped at 48 h p.i, viral DNA remained detectable until 120 h p.i. Sensitive TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) and annexin V assays were used to identify apoptosis. BoHV5 induced death in approximately 50% of cells within 24 h p.i., similar to what has been observed for BoHV1 Los Angeles. Infection with the BoHV1 Cooper strain resulted in 26.37% of cells being in the early stages of apoptosis; 63.69% of infected cells were considered viable. Modulation of mitochondrial function, as measured by mitochondrial membrane depolarization, was synchronous with the virus replication cycle, cell viability and virus titres at 48 h p.i. Our results indicate that apoptosis plays an important role in preventing neuronal death and provides a bovine-derived in vitro system to study herpesvirus-neuron interactions.

Several reports have previously shown that expression of the foot-and-mouth disease virus (FMDV) capsid precursor protein encoding region P1-2A together with the 3C protease (P1-2A/3C) results in correct processing of the capsid precursor into VP0, VP1 and VP3 and formation of FMDV capsid structures that are able to induce a protective immune response against FMDV challenge after immunization using naked DNA constructs or recombinant viruses. To elucidate whether bovine herpesvirus 1 (BHV-1) might also be suitable as a viral vector for empty capsid generation, we aimed to integrate a P1-2A/3C expression cassette into the BHV-1 genome, which, however, failed repeatedly. In contrast, BHV-1 recombinants that expressed an inactive 3C protease or the P1-2A polyprotein alone could be easily generated, although the recombinant that expressed P1-2A exhibited a defect in direct cell-cell spread and release of infectious particles. These results suggested that expression of the original, active FMDV 3C protease is not compatible with BHV-1 replication. This conclusion is supported by the isolation of recombinant BHV-1/3C*, which contained mutations within the 3C ORF (3C* ORF)--probably introduced spontaneously during generation of BHV-1/3C*--instead of the authentic 3C ORF contained in the transfer plasmids. Within the 3C* ORF, the codons for glycine 38 and phenylalanine 48 were both substituted by codons for serine. The resulting 3C* protease exhibits a highly reduced activity for proteolytic processing of the P1-2A polyprotein and thus might be a good candidate for the generation of live attenuated FMDV variants.

Bovine herpesvirus 1 (BoHV-1) UL3.5 is a tegument protein that interacts with BoHV-1 alpha-transinducing factor alphaBTIF). In this report, we show that a truncated UL3.5 lacking the last 40 amino acids (C40UL3.5) inhibited replication of BoHV-1. Stable expression of C40UL3.5 in MDBK cells inhibited replication of BoHV-1 300- to 500-fold in plaque assays. This inhibitory effect was specific for BoHV-1 as cells expressing C40UL3.5 supported replication of pseudorabies virus (species Suid herpesvirus 1, SuHV-1) and herpes simplex virus (species Human herpesvirus 1, HHV-1) to normal levels. In contrast, a truncated UL3.5 which lacked the first 20 amino acids and did not interact with alphaBTIF did not inhibit BoHV-1 replication. In the C40UL3.5-expressing cells infected with BoHV-1, expression of the viral immediate early gene BICP4 and the early protein gB were reduced and delayed. C40UL3.5, when either transiently or stably expressed, inhibited alphaBTIF-mediated transactivation of a BoHV-1 immediate-early promoter. C40UL3.5 may be useful for constructing transgenic cattle resistant to infection by BoHV-1.

In the aim to study the function of the bovine herpesvirus 1 (BoHV-1) UL28 protein during the replicative cycle, we characterized a UL28 deletion mutant of BoHV-1, BoHV-1 Delta UL28. Productive growth of BoHV-1 Delta UL28 was only observed in a specifically engineered complementing cell line expressing the native UL28 protein, demonstrating that UL28 is essential for virus replication. UL28 deficiency did not compromised viral protein synthesis of the late class as shown by the detection of the viral alpha gene trans-inducing factor protein encoded by UL48, a gene of the gamma2 class. Southern blotting analyses of total DNA extracted from BoHV-1 Delta UL28-infected normal cells revealed that viral DNA replication was not compromised but the process of cleavage of the newly synthesized DNA was defective. Transmission electron microscopy of non-complementing BoHV-1 Delta UL28-infected cells revealed an accumulation of capsids devoid of DNA, suggesting that the DNA packaging was impaired. We conclude that the BoHV-1 UL28 protein is essential for viral replication and is necessary for the formation of mature capsid.

Bovine Herpesvirus 1 (BHV-1) and Caprine Herpesvirus 1 (CapHV-1) are related members of the herpesvirus family. Since their natural hosts are often kept in close contact with each other, concern was raised that a reservoir might be established in the heterologous host in addition to the homologous host. To investigate this possibility, cross-infection experiments with BHV-1 in goats and CapHV-1 in calves were performed. BHV-1 infected goats developed mild disease signs during acute infection, whereas CapHV-1 infection in calves took a subclinical course. However, virus excretion and antibody production were indicative of successful cross-infection of both BHV-1 and CapHV-1. Reactivation of BHV-1 was achieved in 5 out of 8 goats as demonstrated by recurrent virus excretion and rising antibody titers. In constrast CapHV-1 in calves could not be reactivated experimentally. Nevertheless, PCR revealed that both viruses established latency in the trigeminal ganglia of the heterologous host. Based on these results we conclude that goats should indeed be regarded as a potential BHV-1 reservoir, which must be considered during IBR eradication programs.

We investigated whether the bovine herpesvirus 1.1 (BHV1) ORF, a homologue of the herpes simplex virus 1 (HSV1) UL25 gene, represented a functional gene. The BHV1 UL25 ORF, which is located at positions 60602<--62398 of the viral genome, generated a 4.5 kb transcript accumulating at low abundance as soon as 3 hours p.i. after which the levels increased up to 12h p.i. and remained constant up to 24 hours p.i. In addition, UL25 transcription initiated at 303 bases upstream from the translation initiation codon, corresponding to 26 and 354 b downstream from putative TATA and CAAT boxes, respectively, thus providing evidence that these elements function as the UL25 promoter. Western blotting of BHV1-infected cell lysates, using a BHV1-UL25 specific antiserum generated against a T7-Tag/UL25 fusion recombinant protein expressed in E. coli, detected a 63 kDa protein of the expected size (63,083 Da) whose expression profile followed that of its transcript. The synthesis of the 63 kDa protein was abrogated by a DNA synthesis inhibitor, unambiguously demonstrating that the viral specific protein expressed from the BHV1 UL25 ORF belongs to the gamma2 class.

Bovine herpesvirus 1 (BHV-1) is a major pathogen of cattle, causing significant disease including immunosuppression in infected animals. In vitro, the surface expression of major histocompatibility complex (MHC) class I molecules, crucial for an appropriate anti-viral immune response of the host, is down-regulated by BHV-1 infection. Northern blot analyses revealed that the mRNAs for MHC class I and class II molecules were significantly down-regulated in BHV-1 infected cells, starting as early as 2 h after infection. Furthermore, mRNA expression of the two house keeping genes actin and glyceraldehyde-6-phosphate dehydrogenase (GAPDH) was also repressed after infection. This BHV-1 induced effect on cellular metabolism resembled the virion host shutoff (vhs) activity of herpes simplex virus (HSV). Similar to the HSV vhs activity, the putative BHV-1 vhs activity was not abrogated in cells infected in the presence of actinomycin D (ActD) which suggested that no viral gene expression is required for the vhs function and the putative vhs protein is associated with the virion. Sequence comparison indicated a BHV-1 open reading frame having a 60% similarity to the HSV vhs sequence. This putative BHV-1 open reading frame contained the four conserved regions of the alphaherpesvirus vhs protein. Since an HSV vhs-mutant exhibited less virulence and good immunogenicity, we suggest that a BHV-1 vhs- mutant may hold promising potential as a candidate vaccine.

Glycoproteins gE and gI of bovine herpesvirus 1 (BHV-1) are type I transmembrane proteins that can form a complex that is involved in cell-to-cell spread mechanisms. The extracellular domains of both proteins have cysteine-rich regions that are also found in the homologous proteins of other alpha-herpesviruses. The extracellular domain of gE has two conserved cysteine-rich regions: C1 and C2. The other conserved regions in gE are located between C2 and transmembrane region and in the cytoplasmic domain of gE. We studied the complex formation between gE and gI using a series of truncated gE proteins and a full length form and a secreted form of gI. All proteins were expressed in recombinant baculoviruses. To analyse the complex formation between these polypeptides we used monoclonal antibodies (MAbs 67 and 75) that specifically react with the gE/gI complex and not with separately expressed glycoproteins gE and gI alone. This analysis showed that the BHV-1 gE/gI complex can be formed in insect cells after a co-infection with baculoviruses expressing gE and gI in their full length form. When secreted forms of gE and gI were expressed after co-infection, the gE/gI complex was still formed and could also be detected in the tissue culture medium. This gE/gI complex was also formed after mixing the tissue culture media of insect cells expressing the secreted form or gE or gI separately. The smallest part of gE that still formed a complex is encoded by the first 246 residues of gE. This extracellular domain contains only the C1 region, showing that the C2 region is not essential for gE/gI complex formation. Shorter forms of gE encoding the C1 region did not form a detectable complex. We also found that the formation of gE/gI complex is not sufficient for normal cell-to-cell spread of BHV-1. A recombinant BHV-1 gE TM-virus, expressing a truncated glycoprotein E from which the transmembrane and cytoplasmic domain were removed, forms plaques as small as a gE null mutant.

The 8.2 kb HindIII K-fragment of bovine herpesvirus 1 (BHV1) lies entirely within the short unique region of the genome and contains all or parts of 7 coding regions. We have probed this fragment for the presence of nonessential genes by a simple, rapid method of insertional mutagenesis. Our analysis indicates that all the genes present in the K-fragment, except the glycoprotein D gene, are nonessential for replication of BHV1 in tissue culture. After inserting a copy of the glycoprotein D gene into the thymidine kinase locus of BHV1 it was possible to delete the entire HindIII K-fragment and the contiguous 0.36 kb O-fragment in one step. The deletion mutant, which contains 7 kb less BHV1 DNA than wt virus and lacks ORF1, US2, the genes for protein kinase, glycoprotein G, glycoprotein I, and most of glycoprotein E was still replication-competent.