Bovine herpesvirus 5 (BHV-5) Us9 is essential for BHV-5 neuropathogenesis.
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Bovine herpesvirus 5 (BHV-5) is a neurovirulent alphaherpesvirus that causes fatal encephalitis in calves. In a rabbit model, the virus invades the central nervous system (CNS) anterogradely from the olfactory mucosa following intranasal infection. In addition to glycoproteins E and I (gE and gI, respectively), Us9 and its homologue in alphaherpesviruses are necessary for the viral anterograde spread from the presynaptic to postsynaptic neurons. The BHV-5 Us9 gene sequence was determined, and the predicted amino acid sequence of BHV-5 Us9 was compared with the corresponding Us9 sequences of BHV-1.1. Alignment results showed that they share 77% identity and 83% similarity. BHV-5 Us9 peptide-specific antibody recognized a doublet of 17- and 19-kDa protein bands in BHV-5-infected cell lysates and in purified virions. To determine the role of the BHV-5 Us9 gene in BHV-5 neuropathogenesis, a BHV-5 Us9 deletion recombinant was generated and its neurovirulence and neuroinvasive properties were compared with those of a Us9 rescue mutant of BHV-5 in a rabbit model. Following intranasal infection, the Us9 rescue mutant of BHV-5 displayed a wild-type level of neurovirulence and neural spread in the olfactory pathway, but the Us9 deletion mutant of BHV-5 was virtually avirulent and failed to invade the CNS. In the olfactory mucosa containing the olfactory receptor neurons, the Us9 deletion mutant virus replicated with an efficiency similar to that of the Us9 rescue mutant of BHV-5. However, the Us9 deletion mutant virus was not transported to the bulb. Confocal microscopy of the olfactory epithelium detected similar amounts of virus-specific antigens in the cell bodies of olfactory receptor neuron for both the viruses, but only the Us9 rescue mutant viral proteins were detected in the processes of the olfactory receptor neurons. When injected directly into the bulb, both viruses were equally neurovirulent, and they were transported retrogradely to areas connected to the bulb. Taken together, these results indicate that Us9 is essential for the anterograde spread of the virus from the olfactory mucosa to the bulb. (+info)
Primary infection, latency, and reactivation of bovine herpesvirus type 5 in the bovine nervous system.
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Bovine herpesvirus type 5 (BHV-5) infection in calves causes meningoencephalitis, a fatal disease highly prevalent in South America. To study the pathogenesis of BHV-5 infection in cattle, 12 calves (group 1: acute infection) and 11 calves (group 2: latent infection) were intranasally inoculated with an Argentinean BHV-5 isolate at 10(8) and 10(4.7) tissue culture infective doses, respectively; six calves (control group) were mock infected. At 3 months postinoculation, all of the calves in group 2 and three calves in group 3 were given dexamethasone to reactivate the virus. The animals were euthanatized between days 6 and 17 postinoculation (group 1) and between days 6 and 16 postreactivation (group 2). Seventy-five percent and 91% of animals in groups 1 and 2, respectively, excreted BHV-5 in nasal and ocular discharges. Following dexamethasone administration, 45% of calves shed virus in both types of secretions. Spontaneous virus reactivation and shedding was observed in one calf. Neurologic signs consisting of circling, teeth grinding, ptyalism, jaw chomping, tongue protrusion, and apathy were observed in two animals in group 1 and, during the reactivation period, in four animals in group 2. Macroscopic findings consisted of softening of the cerebral tissue, meningeal hemorrhages and swelling, and edema and hemorrhages of prescapular, retropharyngeal and submandibular lymph nodes. Histologic lesions consisted of meningitis, mononuclear perivascular cuffing, neuronophagia, satellitosis, gliosis, hemorrhage, and necrosis and edema. Lesions in anterior cerebral cortex, medulla, and pons were consistently seen in all the animals of group 1. In the acutely infected animals, lesions in the diencephalon appeared at day 10 postinoculation, whereas in the latently infected calves these lesions were observed as early as at day 6 postreactivation. Latently infected animals developed lesions simultaneously in anterior cortex, medulla, pons, and diencephalon, showing a remarkable difference from the acutely infected group. Trigeminal ganglionitis appeared relatively early in animals of both groups (day 7 postinoculation in group 1 and day 8 postreactivation in group 2). (+info)
Glycoprotein G isoforms from some alphaherpesviruses function as broad-spectrum chemokine binding proteins.
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Mimicry of host chemokines and chemokine receptors to modulate chemokine activity is a strategy encoded by beta- and gammaherpesviruses, but very limited information is available on the anti-chemokine strategies encoded by alphaherpesviruses. The secretion of chemokine binding proteins (vCKBPs) has hitherto been considered a unique strategy encoded by poxviruses and gammaherpesviruses. We describe a family of novel vCKBPs in equine herpesvirus 1, bovine herpesvirus 1 and 5, and related alphaherpesviruses with no sequence similarity to chemokine receptors or other vCKBPs. We show that glycoprotein G (gG) is secreted from infected cells, binds a broad range of chemokines with high affinity and blocks chemokine activity by preventing their interaction with specific receptors. Moreover, gG also blocks chemokine binding to glycosaminoglycans, an interaction required for the correct presentation and function of chemokines in vivo. In contrast to other vCKBPs, gG may also be membrane anchored and, consistently, we show chemokine binding activity at the surface of cells expressing full-length protein. These alphaherpesvirus vCKBPs represent a novel family of proteins that bind chemokines both at the membrane and in solution. (+info)
Genome of bovine herpesvirus 5.
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Here we present the complete genomic sequence of bovine herpesvirus 5 (BHV-5), an alphaherpesvirus responsible for fatal meningoencephalitis in cattle. The 138390-bp genome encodes 70 putative proteins and resembles the alpha2 subgroup of herpesviruses in genomic organization and gene content. BHV-5 is very similar to BHV-1, the etiological agent of infectious bovine rhinotracheitis, as reflected by the high level of amino acid identity in their protein repertoires (average, 82%). The highest similarity to BHV-1 products (>or=95% amino acid identity) is found in proteins involved in viral DNA replication and processing (UL5, UL15, UL29, and UL39) and in virion proteins (UL14, UL19, UL48, and US6). Among the least conserved (+info)
Distribution of bovine herpesvirus type 5 DNA in the central nervous systems of latently, experimentally infected calves.
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Bovine herpesvirus type 5 (BHV-5) is an alphaherpesvirus associated with meningoencephalitis, a disease highly prevalent in South America. In this study, we investigated the distribution of BHV-5 DNA in the brains of latently, experimentally infected calves by using a PCR for the glycoprotein B gene. Twelve calves inoculated intranasally with a Brazilian BHV-5 isolate were divided into two groups: group A calves (n = 4) were euthanized 55 days postinoculation (p.i.) for tissue collection; group B calves (n = 8) were submitted to dexamethasone administration at day 60 p.i. for reactivation of latent infection and were euthanized 50 days later. Latent infection was reactivated in all group B calves, as demonstrated by virus isolation from nasal secretions and/or seroconversion. Three calves developed neurological disease and died or were euthanized in extremis. For group A calves, viral DNA was consistently detected in the trigeminal ganglia (4/4), midbrain (4/4), thalamus (4/4), and olfactory cortex (4/4) and less frequently in the pons (3/4), cerebellum (3/4), anterior cerebral cortex (2/4), and olfactory bulb (2/4). For calves previously submitted to reactivation (group B), viral DNA was detected with roughly the same frequency in the same areas as for the group A calves. In addition, viral DNA was detected in the posterior (5/5) and dorso-lateral cortex (3/5). All DNA-positive tissues were negative for infectivity and viral antigens. These results demonstrated that latent BHV-5 DNA is present in several areas of the brain during latent infection and that virus reactivation may result in the establishment of latent infection in additional sites of the brain. (+info)
Production and characterization of monoclonal antibodies to a Brazilian bovine herpesvirus type 5.
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Antigens of a bovine herpesvirus type 5 (BHV-5), isolated from a cow with a neurological infection in Rio Grande do Sul State, Brazil, were used to immunize BALB/c mice to produce monoclonal antibodies (mAbs). Eleven hybridomas secreting mAbs directed at BHV-5 antigens were obtained after two fusions and screening of 356 hypoxanthine-aminopterin-thymidine-resistant clones. The mAbs reacted at dilutions up to 1:500 (hybridoma culture supernatant) and up to >1:10,000 (ascitic fluid) in an indirect fluorescent antibody assay (IFA) and in immunoperoxidase staining of BHV-5-infected cells. Four mAbs (1D12, 2E2, 2G10 and 4E4) showed virus-neutralizing activity against the parental BHV-5 isolate. Five mAbs (1F3, 2A6, 2F9, 2G10 and HB24L) reacted in Western immunoblotting with a protein of approximately 90 kDa. Three other mAbs (2E2, 3D6 and 4E4) reacted in IFA with antigens of a BHV-1 mutant glycoprotein C- negative strain, demonstrating that they are directed at a viral antigen other than glycoprotein C. The eleven mAbs tested reacted with 20 BHV-5 field isolates and nine mAbs reacted with 10 BHV-1 isolates. Two mAbs (1F3 and 2F9) failed to react with BHV-1 field isolates, although they displayed a weak and nonreproducible reaction with the BHV-1 reference strain Los Angeles. These mAbs may be very useful in distinguishing between BHV-1 and BHV-5 infections since most of the traditional reagents and techniques are unable to do so. One mAb (2F9) was shown to bind to viral antigens by immunohistochemistry of histological sections of the brain of a BHV-5-infected calf. These results demonstrate that the mAbs produced here are suitable for use in a variety of immunological techniques and therefore may be useful for diagnostic and research purposes. (+info)
Both viral and host factors contribute to neurovirulence of bovine herpesviruses 1 and 5 in interferon receptor-deficient mice.
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Herpes simplex virus (HSV) type 1 and bovine herpesviruses 1 and 5 (BHV-1 and BHV-5) can use the same cellular receptor for entry, but only HSV is known to cause disease in mice. We hypothesized that components of either the innate or the adaptive immune system, or a combination of both, were responsible for curbing replication of BHVs in mice. Therefore, wild-type mice as well as mice with various combined genetic deficiencies in the alpha/beta interferon receptor or gamma interferon receptor and in the ability to produce mature B and T lymphocytes (RAG-2 deletion) were infected with BHV-1 and BHV-5 and monitored clinically, serologically, histopathologically, and virologically. A functional immune system protected the mice from disease and death due to BHV infection, and the immune response was Th1 like. BHV-5 was transported to the central nervous system by the axonal pathway, whereas viremia was required for this outcome with BHV-1. The alpha/beta interferon system was able to obstruct quantitative spread of the viruses in the infected organism. The gamma interferon system had a protective effect against BHV-1, even in mice with the RAG-2 deletion. In contrast, the same mice succumbed to neurological disease and death upon infection with BHV-5. Productively infected neurons were detected only in BHV-5-infected mice with an intact gamma interferon system. We conclude that the alpha/beta interferon system had a protective effect, while an intact gamma interferon system was required for efficient replication of BHV-5 in mouse neurons and for the development of neurological disease. (+info)
A glycine-rich bovine herpesvirus 5 (BHV-5) gE-specific epitope within the ectodomain is important for BHV-5 neurovirulence.
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The bovine herpesvirus 5 (BHV-5) gE ectodomain contains a glycine-rich epitope coding region (gE5 epitope), residues 204 to 218, that is significantly different from the corresponding gE region of BHV-1. Deletion of the gE epitope significantly reduced the neurovirulence of BHV-5 in rabbits. Pulse-chase analyses revealed that the epitope-deleted and wild-type gE were synthesized as N-glycosylated endoglycosidase H-sensitive precursors with approximate molecular masses of 85 kDa and 86 kDa, respectively. Like the wild-type gE, epitope-deleted gE complexed with gI and was readily transported from the endoplasmic reticulum. Concomitantly, the epitope-deleted and wild-type gE acquired posttranslational modifications in the Golgi leading to an increased apparent molecular mass of 93-kDa (epitope-deleted gE) and 94-kDa (wild-type gE). The kinetics of mutant and wild-type gE processing were similar, and both mature proteins were resistant to endoglycosidase H but sensitive to glycopeptidase F. The gE epitope-deleted BHV-5 formed wild-type-sized plaques in MDBK cells, and the epitope-deleted gE was expressed on the cell surface. However, rabbits infected intranasally with gE epitope-deleted BHV-5 did not develop seizures, and only 20% of the infected rabbits showed mild neurological signs. The epitope-deleted virus replicated efficiently in the olfactory epithelium. However, within the brains of these rabbits there was a 10- to 20-fold reduction in infected neurons compared with the number of infected neurons within the brains of rabbits infected with the gE5 epitope-reverted and wild-type BHV-5. In comparison, 70 to 80% of the rabbits exhibited severe neurological signs when infected with the gE5 epitope-reverted and wild-type BHV-5. These results indicated that anterograde transport of the gE epitope-deleted virus from the olfactory receptor neurons to the olfactory bulb is defective and that, within the central nervous system, the gE5 epitope-coding region was required for expression of the full virulence potential of BHV-5. (+info)