Recombinant bovine IL17A acts as an adjuvant for bovine herpesvirus vaccine.

The Bovine herpes virus type 5 glycoprotein D (gD) is essential for viral penetration into host permissive cells. The Herpes virus gD glycoprotein has been used for bovine immunization, being efficient in reduction of viral replication, shedding and clinical signs, however sterilizing immunity is still not achieved. Recombinant subunit vaccines are, in general, poorly immunogenic requiring additional adjuvant components. Interleukin 17A (IL17A) is a pro-inflammatory cytokine produced by T helper 17 cells that mediate mucosal immunity. IL17 production during vaccine-induced immunity is a requirement for mucosal protection to several agents. In this study, we investigated the potential of a recombinant IL17A to act as an adjuvant for a recombinant BoHV-5 glycoprotein D vaccine in cattle. Three cattle groups were divided as: group 1) rgD5 + alumen + rIL-17A; 2) rgD5 + alumen; and 3) PBS + alumen. The cattle (3 per group) received two doses of their respective vaccines at an interval of 21 days. The group that received rIL17 in its vaccine formulation at the 7th day after the prime immunization had significant higher levels of specific rgD-IgG than the alumen group. Addition of rIL17 also led to a significant fold increase in specific anti-rgD IgG and neutralizing antibodies to the virus, respectively, when compared with the alumen group. Cells stimulated with rIL17A responded with IL17 transcription, as well IL2, IL4, IL10, IL15, Bcl6 and CXCR5. Our findings suggest that the rIL17A has adjuvant potential for use in vaccines against BoHV-5 as well as potentially other pathogens of cattle.

Molecular evidence of bovine herpesvirus 1 and 5 in cattle with suspected rabies in Rio Grande do Sul state, Brazil.

Rabies and herpetic encephalitis are the main viral infections in bovines with neurological symptoms. Bovine rabies has a high prevalence in Central and South America, while bovine encephalitis associated with herpesvirus is especially important in South America. Viral isolation is the classical way to confirm herpesvirus infection, but molecular evidence of the presence of the virus in affected animals is gaining importance in the diagnosis of the disease in the laboratory. This study investigated the presence of herpesvirus type 1 and 5 (BoHV-1 and BoHV-5) in 182 encephalon of rabies-suspected cattle in Rio Grande do Sul state (RS), Brazil using multiplex real-time polymerase chain reaction (mRT-PCR). The rabies virus was investigated by direct fluorescent antibody assay and intracerebral suckling mouse inoculation. The genomes of BoHV-1 and BoHV-5 were detected in 17% of samples. BoHV-5 and BoHV-1 were detected in 100% and 19% of BoHV positive samples, respectively, indicating the circulation of the pathogens in cattle herds in RS. The high Ct values and the absence of isolation suggest viral latency. Coinfection of herpesvirus and the rabies virus was detected in 28% of samples, although no significant association between pathogens was observed. Rabies was detected in 57.7% of suspected samples, confirming the importance of the disease in the state. Concerning the method by which samples were conserved, no significant difference was observed between the number of positive results in frozen and refrigerated samples.

Bovine alphaherpesvirus type 5 replicates more efficiently than bovine alphaherpesvirus type 1 in undifferentiated human neural cells.

Bovine herpesvirus (BoHV) types 1 and 5 are two closely related alpha-herpesviruses of cattle with neuroinvasive potential. BoHV-5 causes non-suppurative meningoencephalitis in calve whereas encephalitis caused by BoHV-1 has been occasionally reported. As an initial step to understand the biology of both BoHV types in neural cells, undifferentiated SH-SY5Y human neuroblastoma cells were infected with BoHV-1 strains Cooper and Los Angeles (LA), BoHV-5 strain 97/613 and A663, a BoHV-5/BoHV-1 natural recombinant. Cytopathic effect (CPE) in these cells was evident earlier for BoHV-5 strain 97/613 and CPE progression was slower for BoHV-1, particularly for Cooper strain. Virus antigen was detected as early as 8 h post-infection (hpi) for all strains, with the exception of BoHV-1 Cooper for which antigen expression was detectable by 24 hpi. All strains released detectable infectious virus in the extracellular medium by 8 hpi, confirming that undifferentiated SH-SY5Y cells are fully permissive to BoHV infection. Significantly different extracellular virus titers among the different strains were detected by 24 hpi, with BoHV-5 97/613 reaching the maximal virus production. The lowest extracellular titer was recorded for BoHV-1 Cooper at all the evaluated time-points. BoHV-1 Cooper, BoHV-1 LA and BoHV-5 97/613 had a steady increase in intracellular virus production. The evaluation of lysis plaques formation revealed that BoHV-5 A663 produced the largest plaques followed by BoHV-5 97/613. Both BoHV-1 strains produced smaller plaques when compared with BoHV-5. Despite a slower replicative cycle, strain A663 is more efficient in cell to cell dissemination. Thus, it is evident that BoHV-5 strains have growth advantages in undifferentiated neural cells compared with BoHV-1. This in vitro model might be useful to analyze the neuropathogenic potential of bovine alphaherpesviruses.

Isolation and characterization of bovine herpes virus 5 (BoHV5) from cattle in India.

Bovine herpesvirus 1 (BoHV1) and 5 (BoHV5) are genetically and antigenically related alphaherpesviruses. Infection with one virus induces protective immunity against the other. However, disease associated with BoHV1 and BoHV5 varies significantly; whereas BoHV1 infection is usually associated with rhinotracheitis and abortion, BoHV5 causes encephalitis in cattle. BoHV5 outbreaks are sporadic and mainly restricted to the South American countries. We report BoHV5 infection for the first time from aborted cattle in India. Based on the characteristic cytopathic effects in MDBK cells, amplification of the viral genome by PCR, differential PCR for BoHV1/BoHV5, nucleotide sequencing and restriction endonuclease patterns, identity of the virus was confirmed as BoHV5 subtype A. Serum samples from the aborted cattle strongly neutralized both BoHV1 and BoHV5 suggesting an active viral infection in the herd. Upon UL27, UL44 and UL54 gene-based sequence and phylogenetic analysis, the isolated virus clustered with BoHV5 strains and showed highest similarity with the Brazilian BoHV5 strains.

Feedback inhibition of bovine herpesvirus 5 replication by dual-copy bhv5-miR-B10-3p.

Bovine herpesvirus 5 (BoHV-5) is a pathogen of cattle responsible for fatal meningoencephalitis. Like alpha herpesvirus subfamily members, BoHV-5 also encodes microRNA in lytic infections of epithelial cells. BoHV-5-miR-B10 was the most abundant miRNA detected in a high-throughput sequencing study. Here, we evaluated the kinetics of miR-B10 expression after BoHV-5 productive infection by stem-loop real-time quantitative PCR. miR-B10 candidate target sites in the virus were predicted, and BoHV-5 UL39 was confirmed as a target gene by dual-luciferase assay with the design of an miR-B10 tough decoy (TuD). The UL39 gene encoding ribonucleotide reductase (RR) large subunit plays an important role in the early stage of BoHV-5 lytic infection. As BoHV-5-miR-B10 is located in internal and terminal repeat regions, we generated a TuD gene-integrated BoHV-5 strain, which effectively down-regulated miR-B10-3p. Strikingly, the suppression of miR-B10-3p significantly improved BoHV-5 replication. Taking these findings together, our study established an efficient method to deliver and express TuD RNA for viral miRNA suppression, and demonstrated that virus-encoded miRNA suppresses viral-genome biogenesis with a feedback mode, which might serve as a brake for viral replication. Herpesviruses infect humans and a variety of animals. Almost all herpesviruses can encode miRNAs, but the functions of these miRNAs remain to be elucidated. Most herpesvirus-encoded miRNA harbours dual copies, which is difficult to be deleted by current genetic modulation. Here, we developed an efficient method to deliver and express TuD RNA to efficiently suppress viral miRNA with multiple copies. Using this method, we demonstrated for the first time that viral miRNA feedback regulates viral replication by suppressing the expression of RR.

Functional analysis of the latency-related gene of bovine herpesvirus types 1 and 5.

Bovine herpesvirus type 1 and type 5 (BoHV-1 and BoHV-5) are two alphaherpesviruses that affect cattle with two different syndromes. While BoHV-1 mainly produces respiratory symptoms, BoHV-5 is highly neuropathogenic and responsible for meningoencephalitis in young cattle. The latency-related (LR) gene, which is not conserved between these two herpesviruses, is the only viral gene abundantly expressed in latently infected neurons. The antiapoptotic action of this gene has been demonstrated during acute infection and reactivation from latency and seems to be mainly mediated by a LR protein (ORF-2) which is truncated in amino acid 51 in the case of BoHV-5. In this work, we show that the BoHV-5 LR gene is less efficient at cell survival and apoptosis inhibition in transient as well as in established neuronal cell lines compared to its BoHV-1 homolog. We hypothesize that the BoHV-5 LR gene may have novel functions that are lacking in the BoHV-1 LR gene and that these differences may contribute to its enhanced neuropathogenesis.

Evidence of natural interspecific recombinant viruses between bovine alphaherpesviruses 1 and 5.

Closely related bovine alphaherpesviruses 1 (BoHV-1) and 5 (BoHV-5) co-circulate in certain countries, rendering cattle co-infection possible. This is a prerequisite for BoHV recombination. Here, we report the first identification of homologous recombination between field isolates of BoHV-1 and BoHV-5, two alphaherpesviruses belonging to two distinct species with an average genomic similarity of 82.3%. Three isolates of BoHV-5, previously classified as subtype "BoHV-5b", were phylogenetically studied and analyzed via eight PCR sequencing assays dispersed at regular intervals throughout the genome to discriminate between BoHV-1 and BoHV-5. In the phylogenetic analysis, differences of clustering were found in the UL27 gene which encodes the glycoprotein B (gB). We detected two recombination breakpoints in the open reading frame of the UL27 gene. We compared the amino acid sequences of the gB of BoHV-1.1 and 1.2, BoHV-5a and recombinant formerly named BoHV-5b (chimeric gB) and subsequently performed molecular modeling. All structures were alike and, simultaneously, similar to the chimeric gB. Neutralizing antibodies against BoHV-1, BoHV-5 and recombinant viruses were analyzed via serum virus neutralization test using polyclonal sera and a monoclonal antibody against gB to demonstrate an absence of viral escape for both assays. Our results show that homologous recombination between two related species of ruminant alphaherpesviruses can occur in natural field conditions. We found three recombinant field isolates, previously classified as BoHV-5b subtypes, between BoHV-1 and BoHV-5.

The latency related gene of bovine herpesvirus types 1 and 5 and its modulation of cellular processes.

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.

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.

Bovine herpesviruses induce different cell death forms in neuronal and glial-derived tumor cell cultures.

Oncolytic viruses have the ability to infect tumor cells and leave healthy cells intact. In this study, bovine herpesvirus 1 (BHV1; Los Angeles, Cooper, and SV56/90 strains) and bovine herpesvirus 5 (BHV5; SV507/99 and GU9457818 strains) were used to infect two neuronal tumor cell lineages: neuro2a (mouse neuroblastoma cells) and C6 (rat glial cells). BHV1 and BHV5 strains infected both cell lines and positively correlated with viral antigen detection (p < 0.005). When neuro2a cells were infected by Los Angeles, SV507/99, and GU9457818 strains, 40 % of infected cells were under early apoptosis and necroptosis pathways. Infected C6 cells were >40 % in necroptosis phase when infected by BHV5 (GU9457818 strain). Blocking caspase activation did not interfere with cell death. However, when necroptosis was blocked, 60-80 % of both infected cells with either virus switched to early apoptosis pathway with no interference with virus replication. Moreover, reactive oxygen species production and mitochondrial membrane dysfunction were detected at high levels in both infected cell lines. In spite of apoptosis and necroptosis blockage, tumor necrosis factor alpha (TNFA) and virus transcription were positively correlated for all viral strains studied. Thus, these results contribute to the characterization of BHV1 and BHV5 as potential oncolytic viruses for non-human cells. Nonetheless, the mechanisms underlying their oncolytic activity in human cells are still to be determined.

Comparison between DNA Detection in Trigeminal Nerve Ganglia and Serology to Detect Cattle Infected with Bovine Herpesviruses Types 1 and 5.

Bovine herpesviruses (BoHVs) types 1 (BoHV-1) and 5 (BoHV-5) are alphaherpesviruses of major importance to the bovine production chain. Such viruses are capable of establishing latent infections in neuronal tissues. Infected animals tend to develop a serological response to infection; however, such response-usually investigated by antibody assays in serum-may eventually not be detected in laboratory assays. Nevertheless, serological tests such as virus neutralization (VN) and various enzyme-linked immunosorbent assays (ELISAs) are widely employed to check individual or herd status of BoHV infections. The correlation between detection of antibodies and the presence of viral nucleic acids as indicatives of infection in infected cattle has not been deeply examined. In order to investigate such correlation, 248 bovine serum samples were tested by VN to BoHV-1 and BoHV-5, as well as in a widely employed (though not type-differential) gB ELISA (IDEXX IBR gB X2 Ab Test) in search for antibodies to BoHVs. Immediately after blood withdrawal, cattle were slaughtered and trigeminal ganglia (TG) excised for DNA extraction and viral nucleic acid detection (NAD) by nested PCR. Neutralizing antibodies to BoHV-1 and/or BoHV-5 were detected in 44.8% (111/248) of sera, whereas the gB ELISA detected antibodies in 51.2% (127/248) of the samples. However, genomes of either BoHV-1, BoHV-5, or both, were detected in TGs of 85.9% (213/248) of the animals. These findings reveal that the assays designed to detect antibodies to BoHV-1 and/or BoHV-5 employed here may fail to detect a significant number of latently infected animals (in this study, 35.7%). From such data, it is clear that antibody assays are poorly correlated with detection of viral genomes in BoHV-1 and BoHV-5-infected animals.

Distribution of bovine alpha-herpesviruses and expression of toll-like receptors in the respiratory system of experimentally infected calves.

This study provides an initial analysis of the toll-like receptors (TLRs) that might be implicated in alpha-herpesvirus infection of the bovine respiratory system. A significant variation in the expression of TLR3 and TLRs 7-9 during bovine herpesvirus type 1 (BoHV-1) and 5 (BoHV-5) acute infections and particularly an up-regulation during viral reactivation in respiratory tissues has been demonstrated. Furthermore, viral distribution in the respiratory tract of BoHV-1- and BoHV-5-infected calves at different stages of the infectious cycle was analysed. The wide distribution of BoHV DNA in the respiratory tract during acute infection was restricted during latent infection and the subsequent reactivation of BoHV-1 and BoHV-5. Overall, the findings presented here contribute to the knowledge on the replication and dissemination of bovine alpha-herpesviruses. Furthermore, some of the immune factors triggered in the host that determine the different outcomes of infection by two closely related pathogens of cattle have been elucidated.

A new method for simultaneous detection and discrimination of Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) using real time PCR with high resolution melting (HRM) analysis.

Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) are antigenically and genetically similar. The aim of this study was to develop a simple and reliable one-step real time PCR assay with high resolution melting (HRM) analysis for the simultaneous detection and differentiation of BoHV-1 and BoHV-5. Optimization of assay conditions was performed with DNA from reference strains. Then, DNA from field isolates, clinical samples and tissue samples of experimentally infected animals were studied by real time PCR-HRM. An efficient amplification of real time PCR products was obtained, and a clear melting curve and appropriate melting peaks for both viruses were achieved in the HRM curve analysis for BoHV type identification. BoHV was identified in all of the isolates and clinical samples, and BoHV types were properly differentiated. Furthermore, viral DNA was detected in 12/18 and 7/18 samples from BoHV-1- and BoHV-5-infected calves, respectively. Real time PCR-HRM achieved a higher sensitivity compared with virus isolation or conventional PCR. In this study, HRM was used as a novel procedure. This method provides rapid, sensitive, specific and simultaneous detection of bovine alpha-herpesviruses DNA. Thus, this technique is an excellent tool for diagnosis, research and epidemiological studies of these viruses in cattle.

Comparative analysis of the replication of bovine herpesvirus 1 (BHV1) and BHV5 in bovine-derived neuron-like cells.

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.

Epidemiology and genetic characterization of BVDV, BHV-1, BHV-4, BHV-5 and Brucella spp. infections in cattle in Turkey.

The aim of the study was to determine the epidemiological data of bovine viral diarrhea virus (BVDV), bovine herpesvirus-1 (BHV-1), bovine herpesvirus-4 (BHV-4), bovine herpesvirus-5 (BHV-5) and Brucella-associated cattle that were previously reported to have abortion and infertility problems in Ankara, Corum, Kirikkale and Yozgat provinces, Turkey. Whole blood and sera samples were obtained from 656 cattle, and antibodies against Brucella spp. were detected in 45 (6.86%) and 41 (6.25%) animals by Rose Bengal plate and serum tube agglutination tests, respectively. The seropositivity rates against BVDV, BHV-1 and BHV-4 were 70.89%, 41.3% and 28.78%, respectively. RT-PCR and PCR were performed to detect RNA and DNA viruses in blood samples, respectively. The BVDV 5'-untranslated region and BHV-1 gB gene detected in this study were phylogenetically analyzed. The BVDV strains analyzed in this study were closely related to those previously reported from Turkey. The nucleotide sequence from the BHV-1 strain detected in this study is the first nucleotide sequence of BHV-1 circulating in this area of Turkey deposited in the GenBank. The presence of Brucella spp. and prevalence of BHV-1, BHV-4 and BVDV in cattle should be further investigated throughout these regions.

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.

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.

Bovine herpesvirus 5 encodes a unique pattern of microRNAs compared with bovine herpesvirus 1.

Bovine herpesvirus type 5 (BoHV-5) and bovine herpesvirus 1 (BoHV-1) are two closely related viruses. However, BoHV-5 is responsible for fatal meningitis in calves, while BoHV-1 is associated with infectious rhinotracheitis in cattle, and the mechanism by which the two viruses cause different symptoms is not well understood. In this study, we identified 11 microRNA (miRNA) genes, encoded by the BoHV-5 genome, that were processed into 16 detectable mature miRNAs in productive infection as determined by deep sequencing. We found that 6 out of 16 miRNA genes were present as two copies in the internal repeat and terminal repeat regions, resulting in a total of 17 miRNA-encoding loci distributed in both DNA strands. Surprisingly, BoHV-5 shared only one conservative miRNA with BoHV-1, which was located upstream of the origin of replication. Furthermore, in contrast to BoHV-1, no miRNAs were detected in the unique short region and locus within or near the bovine infected-cell protein 0 and latency-related genes. Variations in both the 5' and 3' ends of the reference sequence were observed, resulting in more than one isoform for each miRNA. All of the 16 miRNAs were detectable by stem-loop reverse transcriptase-PCR. The miRNAs with high read numbers were subjected to Northern blot analysis, and all pre-miRNAs and one mature miRNA were detected. Collectively, the data suggest that BoHV-5 encodes a different pattern of miRNAs, which may regulate the life cycle of BoHV-5 and might account for the different pathogenesis of this virus compared with BoHV-1.

Validation of a reference control for an SYBR-Green fluorescence assay-based real-time PCR for detection of bovine herpesvirus 5 in experimentally exposed bovine embryos.

The objective of this study was to optimize an internal control to improve SYBR-Green-based qPCR to amplify/detect the BoHV-5 US9 gene in bovine embryos produced in vitro and experimentally exposed to the virus. We designed an SYBR-Green-based binding assay that is quick to perform, reliable, easily optimized and compares well with the published assay. Herein we demonstrated its general applicability to detect BoHV-5 US9 gene in bovine embryos produced in vitro experimentally exposed to BoHV-5. In order to validate the assay, three different reference genes were tested; and the histone 2a gene was shown to be the most adequate for normalizing the qPCR reaction, by considering melting and standard curves (p < 0.05). On the other hand, no differences were found in the development of bovine embryos in vitro whether they were exposed to BoHV-5 reference and field strains comparing to unexposed embryos. The developed qPCR assay may have important field applications as it provides an accurate BoHV-5 US9 gene detection using a proven reference gene and is considerably less expensive than the TaqMan qPCR currently employed in sanitary programs.

Multiplex PCR followed by restriction length polymorphism analysis for the subtyping of bovine herpesvirus 5 isolates.

BACKGROUND: Several types and subtypes of bovine herpesviruses 1 and 5 (BoHV-1 and BoHV-5) have been associated to different clinical conditions of cattle, making type/subtype differentiation essential to understand the pathogenesis and epidemiology of BoHV infections. BoHV-5 subtyping is currently carried out by BstEII restriction enzyme analysis (REA) of the complete virus genome. This method allowed the description of three subtypes, one of which is the most widespread while the remaining two have so far only been found in South America. The present work describes a multiplex PCR followed by REA for BoHV-5 subtyping. RESULTS: The method consists in the simultaneous amplification of glycoprotein B and UL54 gene fragments of 534 and 669 base pairs (bp), respectively, BstEII digestion of amplicons, separation of products in 1% agarose gels, and analysis of fragment length polymorphims. The multiplex PCR detected up to 227 BoHV-5 genome copies and 9.2 × 105 BoHV-5 genome copies when DNA was extracted from purified virus or infected tissue homogenates, respectively. The applicability of multiplex PCR-REA was demonstrated on 3 BoHV-5 reference strains. In addition, subtyping of two new isolates and seventeen previously reported ones (17 BHV-5a and 2 BHV-5b) by this method gave coincident results with those obtained with the classic BstEII REA assay. CONCLUSIONS: Multiplex PCR-REA provides a new tool for the fast and simple diagnosis and subtyping of BoHV-5.