Bovine herpesvirus type 5 replication and induction of apoptosis in vitro and in the trigeminal ganglion of experimentally-infected cattle.

Bovine herpesvirus (BoHV) types 1 and 5 are neuroinvasive. Cases of BoHV-1-induced encephalitis are not as frequent as those caused by BoHV-5. In this study, the capability of BoHV-5 to induce apoptosis in cell cultures and in the trigeminal ganglion during acute infection of experimentally-infected cattle was analyzed. Apoptotic changes in cell cultures agree with the ability of the viral strains to replicate in each cell line. Marked differences were observed between the in vitro induction of apoptosis by BoHV-1Cooper and BoHV-5 97/613 strains. Apoptotic neurons were clearly evident in the trigeminal ganglion of BoHV-1-infected calves. For BoHV-5 a fewer number of positive neurons was observed. There is an association between the magnitude of bovine herpesviruses replication and the induction of apoptosis in trigeminal ganglion. These findings suggest that the induction of apoptosis and the innate immune response orchestrate the final outcome of alpha herpesviruses infection of the bovine nervous system.

Role of the suppressor of cytokine signaling 2 (SOCS2) during meningoencephalitis caused by Bovine herpesvirus 5 (BoHV-5).

The role of suppressors of cytokine signaling (SOCS) in meningoencephalitis caused by Bovine herpesvirus 5 (BoHV-5) was evaluated by intracranial infection in C57BL/6 wild-type mice (WT) and SOCS2 deficient mice (SOCS2(-/-)). Both infected groups presented weight loss, ruffled fur and hunched posture. Additionally, infected SOCS2(-/-) mice showed swollen chamfer and progressive depression. Infected WT animals developed mild meningitis, characterized by infiltration of mononuclear cells. Moreover, viral DNA was detected in liver and lung from infected WT group. This group also showed elevated brain levels of IFN-γ, IL-10, CXCL1 and CCL5, when compared with non-infected WT animals. Brain inflammation was exacerbated in infected SOCS2(-/-) mice with widespread distribution of the virus and increased brain levels of TNF-α, IFN-γ, IL-10, IL-12, CXCL1 and CCL5, when compared with WT infected mice. Moreover, infected SOCS2 deficient mice exhibited reduced brain mRNA expression of IFNα and IFNß and increased expression of mRNA of SOCS1, compared with infected WT mice. Taken together, our study provides an insight into the role of SOCS2 in modulating the immune response to BoHV-5 infection.

Sequence analysis of the 5' third of glycoprotein C gene of South American bovine herpesviruses 1 and 5.

Bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5) share high genetic and antigenic similarities, but exhibit marked differences in tissue tropism and neurovirulence. The amino-terminal region of glycoprotein C (gC), which is markedly different in each of the viruses, is involved in virus binding to cellular receptors and in interactions with the immune system. This study investigated the genetic and antigenic differences of the 5' region of the gC (5' gC) gene (amino-terminal) of South American BoHV-1 (n=19) and BoHV-5 (n=25) isolates. Sequence alignments of 374 nucleotides (104 amino acids) revealed mean similarity levels of 97.3 and 94.2% among BoHV-1 gC (gC1), respectively, 96.8 and 95.6% among BoHV-5 gC (gC5), and 62 and 53.3% between gC1 and gC5. Differences included the absence of 40 amino acid residues (27 encompassing predicted linear epitopes) scattered throughout 5' gC1 compared to 5' gC5. Virus neutralizing assays testing BoHV-1 and BoHV-5 antisera against each isolate revealed a high degree of cross-neutralization between the viruses, yet some isolates were neutralized at very low titers by heterologous sera, and a few BoHV-5 isolates reacted weakly with either sera. The virus neutralization differences observed within the same viral species, and more pronounced between BoHV-1 and BoHV-5, likely reflect sequence differences in neutralizing epitopes. These results demonstrate that the 5' gC region is well conserved within each viral species but is divergent between BoHV-1 and BoHV-5, likely contributing to their biological and antigenic differences.

Immunohistochemical approach to the pathogenesis of clinical cases of bovine Herpesvirus type 5 infections.

Meningoencephalitis by Herpesvirus type 5 (BoHV-5) in cattle has some features that are similar to those of herpetic encephalitis in humans and other animal species. Human Herpesvirus 3 (commonly known as Varicella-zoster virus 1), herpes simplex viruses (HSV), and equid Herpesvirus 1 (EHV-1) induce an intense inflammatory, vascular and cellular response. In spite of the many reports describing the histological lesions associated with natural and experimental infections, the immunopathological mechanisms for the development of neurological disorder have not been established. A total of twenty calf brains were selected from the Veterinary School, University of São Paulo State, Araçatuba, Brazil, after confirmation of BoHV-5 infection by virus isolation as well as by a molecular approach. The first part of the study characterized the microscopic lesions associated with the brain areas in the central nervous system (CNS) that tested positive in a viral US9 gene hybridization assay. The frontal cortex (Fc), parietal cortex (Pc), thalamus (T) and mesencephalon (M) were studied. Secondly, distinct pathogenesis mechanisms that take place in acute cases were investigated by an immunohistochemistry assay. This study found the frontal cortex to be the main region where intense oxidative stress phenomena (AOP-1) and synaptic protein expression (SNAP-25) were closely related to inflammatory cuffs, satellitosis and gliosis, which represent the most frequently observed neurological lesions. Moreover, MMP-9 expression was shown to be localized in the leptomeninges, in the parenchyma and around mononuclear infiltrates (p < 0.0001). These data open a new perspective in understanding the role of the AOP-1, MMP-9 and SNAP-25 proteins in mediating BoHV-5 pathogenesis and the strategies of host-virus interaction in order to invade the CNS.

Glycoprotein D of bovine herpesvirus 5 (BoHV-5) confers an extended host range to BoHV-1 but does not contribute to invasion of the brain.

Bovine herpesvirus 1 (BoHV-1) and BoHV-5 are closely related pathogens of cattle, but only BoHV-5 is considered a neuropathogen. We engineered intertypic gD exchange mutants with BoHV-1 and BoHV-5 backbones in order to address their in vitro and in vivo host ranges, with particular interest in invasion of the brain. The new viruses replicated in cell culture with similar dynamics and to titers comparable to those of their wild-type parents. However, gD of BoHV-5 (gD5) was able to interact with a surprisingly broad range of nectins. In vivo, gD5 provided a virulent phenotype to BoHV-1 in AR129 mice, featuring a high incidence of neurological symptoms and early onset of disease. However, only virus with the BoHV-5 backbone, independent of the gD type, was detected in the brain by immunohistology. Thus, gD of BoHV-5 confers an extended cellular host range to BoHV-1 and may be considered a virulence factor but does not contribute to the invasion of the brain.

A bovine herpesvirus 5 recombinant defective in the thymidine kinase (TK) gene and a double mutant lacking TK and the glycoprotein E gene are fully attenuated for rabbits.

Bovine herpesvirus 5 (BoHV-5), the agent of herpetic meningoencephalitis in cattle, is an important pathogen of cattle in South America and several efforts have been made to produce safer and more effective vaccines. In the present study, we investigated in rabbits the virulence of three recombinant viruses constructed from a neurovirulent Brazilian BoHV-5 strain (SV507/99). The recombinants are defective in glycoprotein E (BoHV-5gEDelta), thymidine kinase (BoHV-5TKDelta) and both proteins (BoHV-5gEDeltaTKDelta). Rabbits inoculated with the parental virus (N = 8) developed neurological disease and died or were euthanized in extremis between days 7 and 13 post-infection (pi). Infectivity was detected in several areas of their brains. Three of 8 rabbits inoculated with the recombinant BoHV-5gEDelta developed neurological signs between days 10 and 15 pi and were also euthanized. A more restricted virus distribution was detected in the brain of these animals. Rabbits inoculated with the recombinants BoHV-5TKDelta (N = 8) or BoHV-5gEDeltaTKDelta (N = 8) remained healthy throughout the experiment in spite of variable levels of virus replication in the nose. Dexamethasone (Dx) administration to rabbits inoculated with the three recombinants at day 42 pi did not result in viral reactivation, as demonstrated by absence of virus shedding and/or increase in virus neutralizing titers. Nevertheless, viral DNA was detected in the trigeminal ganglia or olfactory bulbs of all animals at day 28 post-Dx, demonstrating they were latently infected. These results show that recombinants BoHV-5TKDelta and BoHV-5gEDeltaTKDelta are attenuated for rabbits and constitute potential vaccine candidates upon the confirmation of this phenotype in cattle.

Construction and growth properties of bovine herpesvirus type 5 recombinants defective in the glycoprotein E or thymidine kinase gene or both.

Bovine herpesvirus type 5 (BoHV-5) is an important pathogen of cattle in South America. We describe here the construction and characterization of deletion mutants defective in the glycoprotein E (gE) or thymidine kinase (TK) gene or both (gE/TK) from a highly neurovirulent and well-characterized Brazilian BoHV-5 strain (SV507/99). A gE-deleted recombinant virus (BoHV-5 gE) was first generated in which the entire gE open reading frame was replaced with a chimeric green fluorescent protein gene. A TK-deleted recombinant virus (BoHV-5 TK) was then generated in which most of the TK open reading frame sequences were deleted and replaced with a chimeric beta-galactosidase gene. Subsequently, using the BoHV-5 gE virus as backbone, a double gene-deleted (TK plus gE) BoHV-5 recombinant (BoHV-5 gE/TK) was generated. The deletion of the gE and TK genes was confirmed by immunoblotting and PCR, respectively. In Madin Darby bovine kidney (MDBK) cells, the mutants lacking gE (BoHV-5 gE) and TK + gE (BoHV-5 gE/TK) produced small plaques while the TK-deleted BoHV-5 produced wild-type-sized plaques. The growth kinetics and virus yields in MDBK cells for all three recombinants (BoHV-5 gE, BoHV-5 TK and BoHV-5 gE/TK) were similar to those of the parental virus. It is our belief that the dual gene-deleted recombinant (BoHV-5 gE/TK) produced on the background of a highly neurovirulent Brazilian BoHV-5 strain may have potential application in a vaccine against BoHV-5.

Glycoprotein E (gE) specified by bovine herpesvirus type 5 (BHV-5) enables trans-neuronal virus spread and neurovirulence without being a structural component of enveloped virions.

Bovine herpesvirus 5 (BHV-5) is a neurovirulent alpha-herpesvirus that causes fatal encephalitis in calves. We previously demonstrated that deletion of a glycine-rich epitope in the gE ectodomain dramatically reduced BHV-5 neurovirulence. To investigate the role of gE cytoplasmic tail sequences in the neuropathogenesis of BHV-5 in rabbits, we constructed a BHV-5gE recombinant virus with a short residual cytoplasmic domain lacking the YXXL motifs and the acidic (BHV-5gEAm480). In vitro, BHV-5gEAm480 produced on the average smaller plaques, compared with wild-type BHV-5, but it produced on the average substantially larger plaques than the gE ORF-deleted BHV-5. The truncated gE was not phosphorylated, and was not endocytosed from the cell surface. Importantly, the truncated gE was not incorporated into enveloped infectious virions, but its glycosylation and interaction with gI were not affected. In a rabbit model of infection, the BHV-5gEAm480 remained highly virulent, while the gE-null virus was avirulent. The gEAm480 mutant virus invaded most of the central nervous system (CNS) structures that are invaded by the wild-type BHV-5. The number of neurons infected by BHV-5gEAm480 was very similar to the number infected by BHV-5 wild-type and gEAm480-rescued viruses. Collectively, the results suggest that gE functions in transsynaptic transmission of BHV-5 and neurovirulence without being a structural component of the virion particle.

In the absence of glycoprotein I (gI), gE determines bovine herpesvirus type 5 neuroinvasiveness and neurovirulence.

Bovine herpesvirus type 5 (BHV-5) is an alphaherpesvirus that causes fatal encephalitis in calves. Envelope glycoproteins E (gE) and gI of alphaherpesviruses are important for the pathogenesis in vivo. Previously the authors determined that BHV-5 gE is important for BHV-5 neurovirulence. To determine the role of gI in BHV-5 neurovirulence, the authors have constructed gI-deleted and gI-revertant BHV-5 and analyzed their neuropathogenic properties in a rabbit seizure model. Following intranasal infection, 40% of the rabbits infected with the gI-deleted virus showed severe neurological signs. gI-deleted BHV-5 invaded all the central nervous system (CNS) structures invaded by the gI-revertant BHV-5; however, the number of neurons infected by the gI-deleted virus was similar or slightly reduced (two to four fold). Thus, the gI-deleted virus retained significant neurovirulence and/or neuroinvasive properties when compared with the gE-deleted BHV-5. Pulse-chase analysis revealed that the gE of gI-deleted virus was processed to a larger and a diffused 94- to 100-kDa protein (instead of 94 kDa). The 94- to 100-kDa protein was processed in the Golgi with delayed kinetics but it was endoglycosidase H (EndoH) resistant. In cells infected with gI-deleted virus, there was a reduction in cell-surface gE expression compared to wild-type, which correlated to reduced amount of gE processed in the Golgi. The authors believe that in the absence of gI, BHV-5 gE is sufficient for BHV-5 neurovirulence.

A glycine-rich bovine herpesvirus 5 (BHV-5) gE-specific epitope within the ectodomain is important for BHV-5 neurovirulence.

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.