Distinctive features of bovine alphaherpesvirus types 1 and 5 and the virus-host interactions that might influence clinical outcomes.

Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) are two closely related alphaherpesviruses. BoHV-1 causes several syndromes in cattle, including respiratory disease and sporadic cases of encephalitis, whereas BoHV-5 is responsible for meningoencephalitis in calves. Although both viruses are neurotropic, they differ in their neuropathogenic potential. This review summarizes the findings on the specific mechanisms and pathways known to modulate the pathogenesis of BoHV-1 and BoHV-5, particularly in relation to respiratory and neurological syndromes, which characterize BoHV-1 and BoHV-5 infections, respectively.

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.

In vitro-generated interspecific recombinants between bovine herpesviruses 1 and 5 show attenuated replication characteristics and establish latency in the natural host.

BACKGROUND: Interspecific recombinant viruses R1ΔgC and R2ΔgI were isolated after in vitro co-infection with BoHV-1 and BoHV-5, two closely related alphaherpesviruses that infect cattle. The genetic characterization of R1ΔgC and R2ΔgI showed that they are composed of different sections of the parental genomes. The aim of this study was the characterization of the in vivo behavior of these recombinants in the natural host. RESULTS: Four groups of four 3-month-old calves of both genders were intranasally inoculated with either the recombinant or parental viruses. A control group of two animals was also included. Viral excretion and clinical signs were monitored after infection. Histopathological examination of the central nervous system (CNS) was performed and the establishment of latency in trigeminal ganglia was analyzed by PCR. The humoral response was also evaluated using ELISA tests. Three out of four animals from the BoHV-5 infected group excreted virus for 4-10 days. Two calves shed R1ΔgC virus for one day. In R2ΔgI and BoHV-1.2ΔgCΔgI groups, infectious virus was isolated only after two or three blind passages. None of the infected animals developed neurological signs, although those infected with BoHV-5 showed histopathological evidence of viral infection. Latent viral DNA was detected in at least one calf from each infected group. Serum and/or mucosal antibodies were detected in all groups. CONCLUSION: Both BoHV-1/-5 recombinants and the BoHV-1 parental strain are attenuated in calves, although they are able to replicate in animals at low rates and to establish latent infections.

Asymptomatic encephalitis in calves experimentally infected with bovine herpesvirus-5.

This study demonstrated that bovine herpesvirus 5 (BoHV)-5 infected calves can develop encephalitis and remain asymptomatic. Seven calves were infected intranasally and monitored for 30 days. Cerebrospinal fluid (CSF) analysis was performed from the onset of neurological signs. Multiple sections of brain and the trigeminal ganglion were submitted to histopathology. Virus detection (PCR and isolation) was performed on CSF and tissues. Four calves developed signs of neurologic disease and died. Three calves remained asymptomatic and were euthanized 30 days post-infection. Cerebrospinal fluid mononuclear pleocytosis occurred in symptomatic and asymptomatic calves. BoHV-5 was isolated and viral DNA was detected in multiple areas of the encephalon of all calves. The viral DNA was detected in the CSF of 2 calves showing neurological signs. Histologically, inflammation was noted in the brain of all calves and confirmed that the encephalitis caused by BoHV-5 may be mild and asymptomatic.

Bovine herpesvirus 5 induces an overproduction of nitric oxide in the brain of rabbits that correlates with virus dissemination and precedes the development of neurological signs.

We herein report an investigation of nitric oxide (NO) levels, a candidate molecule for neuronal toxicity and dysfunction, in the brain of rabbits during experimental neurological infection by bovine herpesvirus 5 (BoHV-5). Spectrophotometry for NO products (NO(2) and NO(3)) revealed that NO levels were significantly increased (F(4, 40) = 3.33; P <.02) in several regions of the brain of rabbits with neurological disease, correlating with moderate to high BoHV-5 titers. Immunohistochemistry of brain regions revealed a group of cells with neuronal and astrocyte morphology expressing the enzyme inducible NO synthase (iNOS) close to virus antigen-positive neurons. In addition, the investigation of nitric oxide levels between 2 and 6 days post infection (d.p.i.) revealed an initial increase in NO levels in the olfactory bulb and cortex (OB/OC) and anterior cortex (AC) at day 3 p.i., correlating with the initial detection of virus. As the infection proceeded, increased NO levels-and infectivity-were progressively being detected in the OB/CO and AC at day 4 p.i. (F(12, 128) = 2.82; P <.003); at day 5 p.i. in several brain regions (P <.003 in the OB/OC); and at day 6 p.i. in all regions (P <.003) but the thalamus. These results show that BoHV-5 replication in the brain of rabbits induces an overproduction of NO. The increase in NO levels in early infection correlated spatially and temporally with virus dissemination within the brain and preceded the development of neurological signs. Thus, the overproduction of NO in the brain of BoHV-5-infected rabbits may be a component of the pathogenesis of BoHV-5-induced neurological disease.

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.

Outbreak Control and Clinical, Pathological, and Epidemiological Aspects and Molecular Characterization of a Bovine Herpesvirus Type 5 on a Feedlot Farm in São Paulo State.

This paper describes the control, epidemiological, pathological, and molecular aspects of an outbreak of meningoencephalitis in calves due to bovine herpesvirus 5 at a feedlot with 540 animals in São Paulo State, Brazil. The introduction of new animals and contact between the resident animals and the introduced ones were most likely responsible for virus transmission. Bovine herpesvirus 1 vaccine was used, resulting in the efficacy of the outbreak control, although two bovine herpesvirus 1 positive animals, vaccinated and revaccinated, presented meningoencephalitis, thereby characterizing vaccinal failure.

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.

Partial protection induced by a BHV-1 recombinant vaccine against challenge with BHV-5.

Bovine herpesvirus type 5 (BHV-5) is the causative agent of bovine herpetic encephalitis, a major concern for cattle farming in Brazil and Argentina. We recently developed a differential, gE-negative vaccine (265 gE-), based on a Brazilian BHV-1 strain. The present study was carried out to examine whether such a vaccine would confer protection to BHV-5 infections. It was concluded that the recombinant BHV-1 vaccine tested here is not capable of conferring full protection to BHV-5 challenge.

Latent infection by bovine herpesvirus type-5 in experimentally infected rabbits: virus reactivation, shedding and recrudescence of neurological disease.

Latent infection with bovine herpesvirus type-5 (BHV-5) was established in rabbits inoculated with two South American isolates (EVI-88 and 613) by intranasal or conjunctival routes. Nine rabbits (613, 8/27; EVI-88, 1/34) developed neurological disease and died during acute infection and other three (613, n=2; EVI-88, n=1) developed a delayed neurological disease, at days 34, 41 and 56 post-inoculation (p.i.). Between days 56 and 62 p.i., the remaining rabbits were submitted to five daily administrations of dexamethasone (Dx) to reactivate the infection. Twenty-five out of 44 rabbits (56.8%) shed virus in nasal or ocular secretions after Dx treatment. Virus shedding was first detected at day two post-Dx and lasted from one to 11 days. The highest frequencies of virus reactivation were observed in rabbits inoculated conjunctivally (10/15 versus 15/29); and among rabbits infected with isolate 613 (12/16 versus 13/28). Virus reactivation upon Dx treatment was accompanied by neurological disease in nine rabbits (20.4%), resulting in six deaths (13.6%). Virus in moderate titers and mild to moderate non-suppurative inflammatory changes in the brain characterized the neurological infection. Three other rabbits showed severe neurological signs followed by death after 31 to 54 days of Dx treatment. Virus, viral nucleic acids and inflammatory changes were detected in their brains. The late-onset neurological disease, after acute infection or Dx treatment, was probably a consequence of spontaneous virus reactivation. These results demonstrate that BHV-5 does establish a latent infection in rabbits and that clinical recrudescence may occur upon reactivation.

[Retrospective analysis of cases with a diagnosis of cerebrocortical necrosis and its relation with type 5 bovine herpesvirus]. / Análisis retrospectivo de casos con diagnóstico de necrosis cerebrocortical y su relación con herpesvirus bovino tipo 5.

In order to demonstrate the association of bovine herpesvirus type 5 (BHV-5) and cerebrocortical necrosis (CCN), 89 such cases were examined in cattle from three regions of Buenos Aires Province, Argentina, registered between 1970-1999. Hematoxylin-eosin staining and BHV-5 in situ hybridization were performed on paraffin-embedded neural tissues. The severity of microscopic lesions was scored according to a 0-3 scale. Morbidity, mortality and lethality rates between groups depending on age and regions were determined. The highest prevalence of CCN was detected between 1979 and 1984, particularly during the spring. Differences in morbidity and mortality rates between groups of age and regions were not detected (P > 0.05). Amaurosis (48%), ptyalism (42%), circling (40%), ataxia (36%) and bruxism (37%) were frequently observed. Lesions were predominantly found in anterior and posterior cortex (90.6%) and diencephalon (36.5%). Meningitis and perivascular cuffing (94.4%) and focal (78%) or diffuse (73%) gliosis were predominant in cerebrum. Focal necrosis was observed in 66.6% of cases. BHV-5 was isolated from 9/19 cases since 1992 and BHV-5 DNA was detected by in situ hybridization in 3/9 cases. No virus was identified in brain tissues with severe lesions. These findings indicate the association of BHV-5 in neurological disease previously reported as CCN.

Toll-like receptor expression in the nervous system of bovine alpha-herpesvirus-infected calves.

In this study, the expression levels of viral Toll-like receptors (TLRs) in the nervous system of bovine herpesvirus type 5 (BoHV-5)-infected calves were investigated. A significant increase in the expression of TLRs 3 and 7-9 was found in the anterior cerebral cortex during acute infection and viral reactivation. In the trigeminal ganglia, only TLR9 expression was significantly affected. The magnitude of the increase was lower in BoHV-1-infected calves, suggesting that a restricted immune response might protect against exacerbated inflammatory responses in the brain. This work describes, for the first time, the involvement of TLRs 3 and 7-9 in the recognition of BoHV in the bovine nervous system, indicating that the expression of these receptors might be associated with the development of neurological disease. Modulation of the signalling pathways mediated by TLRs might provide an effective approach to control the neuro-immune response to BoHV-5, which may be responsible for neurological lesions.

Comparative study on the in vitro and in vivo properties of two bovine herpesvirus-5 reference strains.

BACKGROUND: Bovine herpesvirus 5 (BoHV-5) is an alphaherpesvirus responsible for meningoencephalitis in young cattle and it is antigenically and genetically related to bovine herpesvirus 1. BoHV-5 outbreaks are sporadic and restricted in their geographical distribution, being mostly detected in the Southern hemisphere. The N569 and A663 strains are prototypes of the "a" and "b" subtypes of BoHV-5, however, scarce information about their in vitro and in vivo properties is currently available. METHODS: For the in vitro comparison between BoHV-5 A663 and N569 strains, viral growth kinetics, lysis and infection plaque size assays were performed. Additionally, an experimental infection of cattle with BoHV-5 A663 and N569 strains was carried out. Viral excretion, development of neurological signs, presence of specific antibodies in serum and nasal swabs and presence of latent BoHV-5 DNA in trigeminal ganglion, were analyzed. Histopathological examination of samples belonging to inoculated animals was also performed. RESULTS: The lytic capacity and the cell-to-cell spread was lower for the A663 strain compared to the N569 strain, however, the production of total infectious viral particles was similar between both strains. Concerning the in vivo properties, the A663 and N569 strains are able to induce similar degrees of pathogenicity in cattle. CONCLUSIONS: Our results show that the A663 strain used in this study is less adapted to in vitro replication in MDBK cells than the N569 strain and, although slight differences were observed, both strains are able to induce a similar degree of virulence in the natural host.

Quillaja brasiliensis saponins are less toxic than Quil A and have similar properties when used as an adjuvant for a viral antigen preparation.

In this study, a preparation of saponins (QB-90U) extracted from leaves of Quillaja brasiliensis collected in Uruguay was evaluated as a vaccine adjuvant by comparison with alum and the well known saponin-based adjuvant, Quil A. The haemolytic activity and cellular toxicity of the saponin preparations were also evaluated. QB-90U was only slightly haemolytic and showed a low cytotoxicity when compared to Quil A. The adjuvant properties of QB-90U were assayed by sub-cutaneous immunization of mice with a preparation of inactivated bovine herpesvirus 5 (BoHV-5) either with no adjuvant or adjuvanted with QB-90U, Quil A or alum. Serum levels of anti-BoHV-5 IgG, IgG1, IgG2a, IgG2b and also IgG3 were significantly increased by QB-90U and were of the same order as those elicited by Quil A. Furthermore, high titres of neutralizing antibodies were found to be present in the serum of immunized animals from both groups. The cellular response induced by QB-90U did also reproduce the one elicited by Quil A. In fact, a robust DTH response was observed in mice immunized with both saponin preparations; as well as increased splenocytes levels of Th1-type cytokines, namely IFN-γ and IL-2. Taken together, the above results confirm and extend our previous observation regarding the similarity of the responses elicited by Quil A and the saponin preparation from Q. brasiliensis (Fleck et al., 2006) and indicate that QB-90U is worth of further studies as a safe and potent vaccine adjuvant.

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.

Biology of bovine herpesvirus 5.

Bovine herpesvirus 5 (BoHV-5) is an alphaherpesvirus responsible for meningoencephalitis in young cattle and is closely antigenically and genetically related to bovine herpesvirus 1 (BoHV-1). Both viruses have common aspects in their pathogenesis: (1) they infect epithelial cells at the portal of entry and (2) they establish a latent infection in the sensory nerve ganglia, i.e., the trigeminal ganglia. However, they have different neuroinvasion and neurovirulence capacities. Only in rare cases can BoHV-1 reach the brain of infected cattle. BoHV-5 infection induces different degrees of severity of neurological disease depending on both viral and host factors. Although a case of BoHV-5 associated disease in Europe and some outbreaks in USA and Australia have been reported, the current geographical distribution of BoHV-5 infection is mainly restricted to South America, especially Brazil and Argentina. This review focuses on the genomic characteristics, pathobiology and epidemiology of BoHV-5, in order to provide information on the possible basis of alphaherpesvirus neuropathogenesis.

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.

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.