How kelch domain-containing protein 3 distinguishes between the C-end degron of herpesviral protein UL49.5 and its mutants - Insights from molecular dynamics.

The C-terminal residues of proteins can function as degrons recognized by ubiquitin ligases for proteasomal degradation. Kelch domain-containing protein 3 (KLHDC3) is a substrate receptor for E3 ubiquitin ligase (Cullin2-RING ligase) that targets the C-terminal degrons. UL49.5 is 96 amino-acid type 1 transmembrane protein from bovine herpesvirus 1. Herpesviruses have evolved highly effective strategies to evade the antiviral immune response. One of these strategies is inhibition of the antigen processing and presentation pathway by MHC I, thereby reducing the presentation of the antigenic peptides on the surface of the infected cell. Recently, it has been demonstrated that UL49.5 triggers TAP degradation via recruiting the E3 ubiquitin ligase to TAP. Moreover, the mutagenesis revealed that the mutations within the UL49.5 C-degron sequence (93RGRG96) affect binding of UL49.5 to KLHDC3. In this work the molecular dynamics of KLHDC3 in complexes with the C-terminal decapeptide of the herpesviral protein UL4.95 and its three mutants has been employed to provide a framework for understanding molecular recognition of UL49.5 by KLHDC3. The findings of this study give insights into the interactions of the various degrons with KLHDC3. During the molecular dynamics, an active RGKG mutant adopts a conformation similar to that of the wild type decapeptide, whereas the conformations of two inactive mutants, KGRG and RGRD are significantly different. Both R93K and G96D mutations impair the interactions of the C-terminal glycine with KLHDC3. The findings of this study expand the existing knowledge about the mechanism of protein recognition by Cullin2-RING ligases thus contributing to the design of antiviral and anticancer drugs that can selectively promote or inhibit degradation of the proteins of interest.

A cell cycle regulator, E2F2, and glucocorticoid receptor cooperatively transactivate the bovine alphaherpesvirus 1 immediate early transcription unit 1 promoter.

Bovine alphaherpesvirus 1 (BoHV-1) infection causes respiratory tract disorders and immune suppression and may induce bacterial pneumonia. BoHV-1 establishes lifelong latency in sensory neurons after acute infection. Reactivation from latency consistently occurs following stress or intravenous injection of the synthetic corticosteroid dexamethasone (DEX), which mimics stress. The immediate early transcription unit 1 (IEtu1) promoter drives expression of infected cell protein 0 (bICP0) and bICP4, two viral transcriptional regulators necessary for productive infection and reactivation from latency. The IEtu1 promoter contains two glucocorticoid receptor (GR) responsive elements (GREs) that are transactivated by activated GR. GC-rich motifs, including consensus binding sites for specificity protein 1 (Sp1), are in the IEtu1 promoter sequences. E2F family members bind a consensus sequence (TTTCCCGC) and certain specificity protein 1 (Sp1) sites. Consequently, we hypothesized that certain E2F family members activate IEtu1 promoter activity. DEX treatment of latently infected calves increased the number of E2F2+ TG neurons. GR and E2F2, but not E2F1, E2F3a, or E2F3b, cooperatively transactivate a 436-bp cis-regulatory module in the IEtu1 promoter that contains both GREs. A luciferase reporter construct containing a 222-bp fragment downstream of the GREs was transactivated by E2F2 unless two adjacent Sp1 binding sites were mutated. Chromatin immunoprecipitation studies revealed that E2F2 occupied IEtu1 promoter sequences when the BoHV-1 genome was transfected into mouse neuroblastoma (Neuro-2A) or monkey kidney (CV-1) cells. In summary, these findings revealed that GR and E2F2 cooperatively transactivate IEtu1 promoter activity, which is predicted to influence the early stages of BoHV-1 reactivation from latency. IMPORTANCE: Bovine alpha-herpesvirus 1 (BoHV-1) acute infection in cattle leads to establishment of latency in sensory neurons in the trigeminal ganglia (TG). A synthetic corticosteroid dexamethasone consistently initiates BoHV-1 reactivation in latently infected calves. The BoHV-1 immediate early transcription unit 1 (IEtu1) promoter regulates expression of infected cell protein 0 (bICP0) and bICP4, two viral transcriptional regulators. Hence, the IEtu1 promoter must be activated for the reactivation to occur. The number of TG neurons expressing E2F2, a transcription factor and cell cycle regulator, increased during early stages of reactivation from latency. The glucocorticoid receptor (GR) and E2F2, but not E2F1, E2F3a, or E2F3b, cooperatively transactivated a 436-bp cis-regulatory module (CRM) in the IEtu1 promoter that contains two GR responsive elements. Chromatin immunoprecipitation studies revealed that E2F2 occupies IEtu1 promoter sequences in cultured cells. GR and E2F2 mediate cooperative transactivation of IEtu1 promoter activity, which is predicted to stimulate viral replication following stressful stimuli.

Integrated analysis of microRNA and messenger RNA expression profiles reveals functional microRNA in infectious bovine rhinotracheitis virus-induced mitochondrial damage in Madin-Darby bovine kidney cells.

BACKGROUND: Studies have confirmed that Infectious bovine rhinotracheitis virus (IBRV) infection induces mitochondrial damage. MicroRNAs (miRNAs) are a class of noncoding RNA molecules, which are involved in various biological processes and pathological changes associated with mitochondrial damage. It is currently unclear whether miRNAs participate in IBRV-induced mitochondrial damage in Madin-Darby bovine kidney (MDBK) cells. RESULTS: In the present study, we used high-throughput sequencing technology, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis to screen for mitochondria-related miRNAs and messenger RNAs (mRNAs). In total, 279 differentially expressed miRNAs and 832 differentially expressed mRNAs were identified in 6 hours (IBRV1) versus 24 hours (IBRV2) after IBRV infection in MDBK cells. GO and KEGG enrichment analysis revealed that 42 differentially expressed mRNAs and 348 target genes of differentially expressed miRNAs were correlated with mitochondrial damage, and the miRNA-mitochondria-related target genes regulatory network was constructed to elucidate their potential regulatory relationships. Among the 10 differentially expressed miRNAs, 8 showed expression patterns consistent with the high-throughput sequencing results. Functional validation results showed that overexpression of miR-10a and miR-182 aggravated mitochondrial damage, while inhibition of miR-10a and miR-182 alleviated mitochondrial damage. CONCLUSIONS: This study not only revealed the expression changes of miRNAs and mRNAs in IBRV-infected MDBK cells, but also revealed possible biological regulatory relationship between them. MiR-10a and miR-182 may have the potential to be developed as biomarkers for the diagnosis and treatment of IBRV. Together, Together, these data and analyses provide additional insights into the roles of miRNA and mRNA in IBRV-induced mitochondria damage.

Bovine papular stomatitis virus as a vaccine vector for cattle.

Virus vectored vaccines are not available commercially for cattle even though compelling potential applications exist. Bovine papular stomatitis virus (BPSV), a highly prevalent parapoxvirus, causes self-limited oral lesions in cattle. Ability of virus to accommodate large amounts of foreign DNA, induce low level of antiviral immunity, and circulate and likely persist in cattle populations, make BPSV an attractive candidate viral vector. Here, recombinant BPSV were constructed expressing either Bovine herpesvirus 1 (BoHV-1) glycoprotein gD (BPSVgD), or gD and gB (BPSVgD/gB). Immunization of BPSV serologically-positive calves with BPSVgD or BPSVgD/gB induced BoHV-1 neutralization antibodies and provided protection for three of four animals following a high dose BoHV-1 challenge at day 70 pi. Results indicate BPSV suitability as a candidate virus vector for cattle vaccines.

Evidence of a Protein-Coding Gene Antisense to the UL5 Gene in Bovine Herpesvirus I.

Bovine herpesvirus type 1 (BoHV-1) is an important agricultural pathogen that infects cattle and other ruminants worldwide. Though it was first sequenced and annotated over twenty years ago, the Cooper strain, used in this study, was sequenced as recently as 2012 and is currently said to encode 72 unique proteins. However, tandem mass spectrometry has identified several peptides produced during active infection that align with the BoHV-1 genome in unannotated regions. One of these abundant peptides, "ORF M", aligned antisense to the DNA helicase/primase protein UL5. This study characterizes the novel transcript and its protein product and provides evidence to support the existence of homolog protein-coding genes in other Herpesviruses.

Inhibition of Stress-Induced Viral Promoters by a Bovine Herpesvirus 1 Non-Coding RNA and the Cellular Transcription Factor, ß-Catenin.

The ability to establish, maintain, and reactivate from latency in sensory neurons within trigeminal ganglia (TG) is crucial for bovine herpesvirus 1 (BoHV-1) transmission. In contrast to lytic infection, the only viral gene abundantly expressed during latency is the latency-related (LR) gene. The synthetic corticosteroid dexamethasone consistently induces reactivation from latency, in part because the glucocorticoid receptor (GR) transactivates viral promoters that drive expression of key viral transcriptional regulator proteins (bICP0 and bICP4). Within hours after dexamethasone treatment of latently infected calves, LR gene products and ß-catenin are not readily detected in TG neurons. Hence, we hypothesized that LR gene products and/or ß-catenin restrict GR-mediated transcriptional activation. A plasmid expressing LR RNA sequences that span open reading frame 2 (ORF2-Stop) inhibited GR-mediated transactivation of the BoHV-1 immediate early transcription unit 1 (IEtu1) and mouse mammary tumor virus (MMTV) promoter activity in mouse neuroblastoma cells (Neuro-2A). ORF2-Stop also reduced productive infection and GR steady-state protein levels in transfected Neuro-2A cells. Additional studies revealed that the constitutively active ß-catenin mutant reduced the transactivation of the IEtu1 promoter by GR and dexamethasone. Collectively, these studies suggest ORF2 RNA sequences and Wnt/ß-catenin signaling pathway actively promote maintenance of latency, in part, by impairing GR-mediated gene expression.

A plasmid encoding the extracellular domain of CD40 ligand and Montanide™ GEL01 as adjuvants enhance the immunogenicity and the protection induced by a DNA vaccine against BoHV-1.

DNA vaccines are capable of inducing humoral and cellular immunity, and are important to control bovine herpesvirus 1 (BoHV-1), an agent of the bovine respiratory disease complex. In previous work, a DNA plasmid that encodes a secreted form of BoHV-1 glycoprotein D (pCIgD) together with commercial adjuvants provided partial protection against viral challenge of bovines. In this work, we evaluate new molecules that could potentiate the DNA vaccine. We show that a plasmid encoding a soluble CD40 ligand (CD40L) and the adjuvant Montanide™ GEL01 (GEL01) activate in vitro bovine afferent lymph dendritic cells (ALDCs). CD40L is a co-stimulating molecule, expressed transiently on activated CD4+ T cells and, to a lesser extent, on activated B cells and platelets. The interaction with its receptor, CD40, exerts effects on the presenting cells, triggering responses in the immune system. GEL01 was designed to improve transfection of DNA vaccines. We vaccinated cattle with: pCIgD; pCIgD-GEL01; pCIgD with GEL01 and CD40L plasmid (named pCIgD-CD40L-GEL01) or with pCIneo vaccines. The results show that CD40L plasmid with GEL01 improved the pCIgD DNA vaccine, increasing anti-BoHV-1 total IgGs, IgG1, IgG2 subclasses, and neutralizing antibodies in serum. After viral challenge, bovines vaccinated with pCIgD-GEL01-CD40L showed a significant decrease in viral excretion and clinical score. On the other hand, 80% of animals in group pCIgD-GEL01-CD40L presented specific anti-BoHV-1 IgG1 antibodies in nasal swabs. In addition, PBMCs from pCIgD-CD40L-GEL01 had the highest percentage of animals with a positive lymphoproliferative response against the virus and significant differences in the secretion of IFNγ and IL-4 by mononuclear cells, indicating the stimulation of the cellular immune response. Overall, the results demonstrate that a plasmid expressing CD40L associated with the adjuvant GEL01 improves the efficacy of a DNA vaccine against BoHV-1.

Cooperative activation of bovine herpesvirus 1 productive infection and viral regulatory promoters by androgen receptor and Krüppel-like transcription factors 4 and 15.

Bovine herpesvirus 1 (BoHV-1), a significant viral pathogen, establishes latency in sensory neurons. The viral genome contains more than 100 consensus glucocorticoid receptor (GR) regulatory elements (GREs): consequently, stress stimulates viral replication and reactivation from latency. The immediate early transcription unit 1 (IEtu1) and bICP0 early promoters are transactivated by GR and synthetic corticosteroid dexamethasone. The androgen receptor (AR), like GR, is a Type 1 nuclear hormone receptor that binds and stimulates certain promoters containing GREs. Consequently, we hypothesized AR and 5α-Dihydrotestosterone (DHT) stimulate productive infection and key viral promoters. New studies demonstrated AR, DHT, and Krüppel like transcription factor 4 (KLF4) cooperatively stimulated productive infection and bICP0 E promoter activity in mouse neuroblastoma cells (Neuro-2A). KLF15 also cooperated with AR and DHT to stimulate IEtu1 promoter activity. We suggest AR and testosterone increase the prevalence of virus in semen by stimulating viral gene expression and replication.

An Improved DNA Vaccine Against Bovine Herpesvirus-1 Using CD40L and a Chemical Adjuvant Induces Specific Cytotoxicity in Mice.

Bovine herpesvirus-1 (BoHV-1) uses many mechanisms to elude the immune system; one of them is spreading intracellularly, even in the presence of specific antiviral antibodies. Cytotoxic T lymphocytes (CTLs) are necessary to eliminate the virus. The main preventive strategy is vaccination based on inactivated virus. These vaccines are poor inducers of cellular immune responses, and complicate serological diagnosis and determination of the real prevalence of infection. DNA vaccines are a good option because of the capacity of Differentiating Infected from Vaccinated Animals-(DIVA vaccine)-and may be the best way to induce cytotoxic responses. Although this type of vaccines leads to only weak "in vivo" expression and poor immune responses, incorporation of molecular and/or chemical adjuvants can improve the latter, both in magnitude and in direction. In this study, we have investigated the specific immune responses elicited in mice by DNA vaccines based on the BoHV-1 glycoprotein D (pCIgD) with and without two different adjuvants: a plasmid encoding for murine CD40L (pCD40L) or Montanide™ 1113101PR (101). Mice vaccinated with pCIgD+CD40L, pCIgD+101, and pCIgD+CD40L+101 developed significantly higher specific antibody titers against BoHV-1 than the pCIgD group (p < 0.01). The animals vaccinated with pCgD+pCD40L+101 raised significantly higher levels of IgG2a and IgG2b (p < 0.01 and p < 0.001, respectively) than mice vaccinated with pCIgD alone. On the contrary, when the activity of CTL against cells infected with BoHV-1 was measured, the vaccine pCgD+pCD40L+101 induced significantly higher levels of cytotoxicity activity (p < 0.001) than pCIgD alone. A significant increase in the CD4+ populations in the group receiving pCIgD+CD40L+101 in comparison with the pCIgD group was observed and, also, interferon gamma, interleukin (IL)-6, and IL-17A levels were higher. Considering the results obtained from this study for humoral and cellular responses in mice, the inclusion of pCD40L and 101 as adjuvants in a BoHV-1 DNA vaccine for cattle is highly recommendable.

Time-course profiling of bovine alphaherpesvirus 1.1 transcriptome using multiplatform sequencing.

Long-read sequencing (LRS) has become a standard approach for transcriptome analysis in recent years. Bovine alphaherpesvirus 1 (BoHV-1) is an important pathogen of cattle worldwide. This study reports the profiling of the dynamic lytic transcriptome of BoHV-1 using two long-read sequencing (LRS) techniques, the Oxford Nanopore Technologies MinION, and the LoopSeq synthetic LRS methods, using multiple library preparation protocols. In this work, we annotated viral mRNAs and non-coding transcripts, and a large number of transcript isoforms, including transcription start and end sites, as well as splice variants of BoHV-1. Our analysis demonstrated an extremely complex pattern of transcriptional overlaps.

Two Separate Tyrosine-Based YXXL/Φ Motifs within the Glycoprotein E Cytoplasmic Tail of Bovine Herpesvirus 1 Contribute in Virus Anterograde Neuronal Transport.

Bovine herpesvirus 1 (BHV-1) causes respiratory infection and abortion in cattle. Following a primary infection, BHV-1 establishes lifelong latency in the trigeminal ganglia (TG). Periodic reactivation of the latent virus in TG neurons results in anterograde virus transport to nerve endings in the nasal mucosa and nasal virus shedding. The BHV-1 glycoprotein E cytoplasmic tail (gE-CT) is necessary for virus cell-to-cell spread in epithelial cells and neuronal anterograde transport. Recently, we identified two tyrosine residues, Y467 and Y563, within the tyrosine-based motifs 467YTSL470 and 563YTVV566, which, together, account for the gE CT-mediated efficient cell-to-cell spread of BHV-1 in epithelial cells. Here, we determined that in primary neuron cultures in vitro, the individual alanine exchange Y467A or Y563A mutants had significantly diminished anterograde axonal spread. Remarkably, the double-alanine-exchanged Y467A/Y563A mutant virus was not transported anterogradely. Following intranasal infection of rabbits, both wild-type (wt) and the Y467A/Y563A mutant viruses established latency in the TG. Upon dexamethasone-induced reactivation, both wt and the mutant viruses reactivated and replicated equally efficiently in the TG. However, upon reactivation, only the wt, not the mutant, was isolated from nasal swabs. Therefore, the gE-CT tyrosine residues Y467 and Y563 together are required for gE CT-mediated anterograde neuronal transport.

Specific Akt Family Members Impair Stress-Mediated Transactivation of Viral Promoters and Enhance Neuronal Differentiation: Important Functions for Maintaining Latency.

Neurotropic Alphaherpesvirinae subfamily members such as bovine herpesvirus 1 (BoHV-1) and herpes simplex virus 1 (HSV-1) establish and maintain lifelong latent infections in neurons. Following infection of ocular, oral, or nasal cavities, sensory neurons within trigeminal ganglia (TG) are an important site for latency. Certain external stressors can trigger reactivation from latency, in part because activation of the glucocorticoid receptor (GR) stimulates productive infection and promoters that drive expression of key viral transcriptional regulators. The Akt serine/threonine protein kinase family is linked to maintaining latency. For example, Akt3 is detected in more TG neurons during BoHV-1 latency than in reactivation and uninfected calves. Furthermore, Akt signaling correlates with maintaining HSV-1 latency in certain neuronal models of latency. Finally, an active Akt protein kinase is crucial for the ability of the HSV-1 latency-associated transcript (LAT) to inhibit apoptosis in neuronal cell lines. Consequently, we hypothesized that viral and/or cellular factors impair stress-induced transcription and reduce the incidence of reactivation triggered by low levels of stress. New studies demonstrate that Akt1 and Akt2, but not Akt3, significantly reduced GR-mediated transactivation of the BoHV-1 immediate early transcription unit 1 (IEtu1) promoter, the HSV-1 infected cell protein 0 (ICP0) promoter, and the mouse mammary tumor virus long terminal repeat (MMTV-LTR). Akt3, but not Akt1 or Akt2, significantly enhanced neurite formation in mouse neuroblastoma cells, which correlates with repairing damaged neurons. These studies suggest that unique biological properties of the three Akt family members promote the maintenance of latency in differentiated neurons.IMPORTANCE External stressful stimuli are known to increase the incidence of reactivation of Alphaherpesvirinae subfamily members. Activation of the glucocorticoid receptor (GR) by the synthetic corticosteroid dexamethasone (DEX) stimulates bovine herpesvirus 1 (BoHV-1) and herpes simplex virus 1 (HSV-1) reactivation. Furthermore, GR and dexamethasone stimulate productive infection and promoters that drive expression of viral transcriptional regulators. These observations lead us to predict that stress-induced transcription is impaired by factors abundantly expressed during latency. Interestingly, activation of the Akt family of serine/threonine protein kinases is linked to maintenance of latency. New studies reveal that Akt1 and Ak2, but not Akt3, impaired GR- and dexamethasone-mediated transactivation of the BoHV-1 immediate early transcription unit 1 and HSV-1 ICP0 promoters. Strikingly, Akt3, but not Akt1 or Akt2, stimulated neurite formation in mouse neuroblastoma cells, a requirement for neurogenesis. These studies provide insight into how Akt family members may promote the maintenance of lifelong latency.

Phylogenetic analysis and characterization of bovine herpesvirus-1 in cattle of China, 2016-2019.

Bovine herpesvirus type 1 (BoHV-1) is one of the most critical pathogens in cattle and is prevalent in China. BoHV-1 is divided into two gene types, BoHV-1.1 and 1.2, which are further differentiated into two subtypes, BoHV-1.2a and 1.2b. However, the phylogenetic analysis of BoHV-1 isolates has not been reported in China. To perform a molecular epidemiological survey based on isolates from cattle in China, 102 lung tissue samples of calves under ten months of age with respiratory disease (BRD) that died from 2016 to 2019 in China were used to isolate BoHV-1 with Madin-Darby bovine kidney (MDBK) cells. Part of the BoHV-1 isolates were applied to the phylogenetic analysis based on the region of the glycoprotein C (gC) gene of BoHV-1. Thirty BoHV-1 isolates were obtained, and the gC gene of 13 isolates was amplified by polymerase chain reaction (PCR) methods and sequenced. The result of the phylogenetic analysis according to the 451-nucleotide portion of the gC gene found that all of 13 isolates belonged to the BoHV-1.2b gene subtype, but these isolates had located two different phylogenetic tree branches. The gC gene sequence homology of isolates in group1 was higher with a reference strain of BoHV-1.2b EVI14 up to 98.0-100%, while in group 2, this was higher with reference strain BoHV-1.2b B589 up to 97.8-99.8%. The deduced amino acid sequence of gC from isolates in group 2 had two amino acid mutations with interference strain BoHV-1.2b K22 or BoHV-1.1 COOPER. The cytopathic effects (CPEs) of BoHV-1 isolates in group 2 were ulcered on the centration like a volcano on MDBK cell, and different from traditional CPEs of BoHV-1. Overall, BoHV-1.2b seems to be the primary strain of BoHV-1 in cattle in China and is also a critical cause of BRD. These BoHV-1.2b isolates had significant genetic variations.

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.

Molecular and antigenic characterization of bovine herpesvirus type 1 (BoHV-1) strains from cattle with diverse clinical cases in Turkey.

The present study reports the molecular and antigenic characterization of 13 bovine herpesvirus type 1 (BoHV-1) field viruses obtained from cattle with different clinical cases in Turkey between 1992 and 2017. We selected glycoprotein C (gC) of BoHV-1 as a target to detect and/or verify presence of the virus in suspect materials followed by virus isolation (VI) in MDBK cells. In seven out of 13 BoHV-1 positive samples, cytophatic effects (CPEs) were observed in MDBK cell cultures, although only four virus samples reached a sufficient titer to use in phylogenetic assay, restriction endonuclease analysis (REA), and virus neutralization test (VNT). According to the results of sequence analysis of the 13 BoHV-1 positive samples, nine BoHV-1 field viruses were determined as BoHV-1.1 and four as BoHV-1.2. Using REA, we demonstrated that two of our isolated viruses could be categorized as BoHV-1.1 while the other two isolates were BoHV-1.2 subtypes. Differences between the BoHV-1.1 and BoHV-1.2 isolates were also detected in the VNT results by assaying 125 suspected serum samples after testing with isolated (KY748023, KY748022, KY748020, and KY748021) and reference viruses (BoHV-1 Cooper and BoHV-5 Texas 89). These results are indicating the need to correctly identify BoHV-1 field isolates to better understand the epidemiology and pathogenesis of infection. In addition, it would be useful to identify the subtypes circulating in the specific geographical area while determining vaccination preferences.

Infecção latente pelo herpesvírus bovino tipo 1 em búfalos (Bubalus bubalis) no Rio Grande do Sul / Bovine herpesvirus type 1 latent infection in buffalo (Bubalus bubalis) in Rio Grande do Sul

Apesar dos bovinos serem considerados os hospedeiros naturais do BoHV-1, estudos sorológicos têm sugerido que búfalos podem ser suscetíveis ao BoHV-1 e a outros alfa-herpesvírus geneticamente relacionados. O objetivo deste estudo foi detectar a presença de DNA viral de BoHV-1 em 202 amostras de gânglios trigêmeos de búfalos, pela técnica de semi-nested PCR, para detecção de um segmento do gene codificante da glicoproteína D (gD) do BoHV-1. Além disso, 242 amostras de soro foram analisadas pela técnica de soroneutralização (SN) para a detecção de anticorpos neutralizantes contra BoHV-1, BoHV-5 e BuHV. Todas as amostras clínicas foram coletadas em um matadouro na cidade de Pelotas, RS, Brasil. O DNA de BoHV-1 foi detectado em 61 (30,1%) gânglios, e os resultados da SN demonstraram que 27,6% dos animais apresentaram anticorpos contra, pelo menos, um dos vírus testados. O sequenciamento genômico e a análise de 14 amplicons confirmaram a presença do DNA do BoHV-1 nos tecidos analisados. Em resumo, os resultados indicam que o BoHV-1 está distribuído em rebanhos bubalinos provenientes da região Sul do Brasil. Entretanto, são necessárias investigações adicionais, no sentido de elucidar o papel exato dos búfalos na epidemiologia das infecções pelo BoHV-1.(AU)

Although bovines are natural hosts for BoHV-1, serologic studies in several countries have suggested that buffaloes (Bubalus bubalis) may be susceptible to BoHV-1 and other genetically related alphaherpesvirus. This study aimed to investigate the presence of BoHV-1 DNA in trigeminal ganglia from 202 buffaloes by a semi-nested PCR to amplify partially the glycoprotein D (gD) gene of BoHV-1. Additionally, 242 serum samples were tested by serum neutralization (SN) for the detection of antibodies against BoHV-1, BoHV-5 and BuHV. All clinical samples were collected in a slaughterhouse located in Pelotas, RS, Brazil. BoHV-1 DNA was detected in 61 (30.1%) of the samples and SN revealed 27.6% of the animals with neutralizing antibodies against at least one of the tested viruses. Nucleotide sequencing of 15 amplicons followed by BLAST analysis confirmed the presence of BoHV-1 DNA in the analyzed tissues. Taken together, these data indicate that BoHV-1 infection is distributed in buffaloes in southern Brazil. However, the role of buffaloes in the BoHV-1 epidemiology needs further investigation.(AU)

Mapping a highly conserved linear neutralizing epitope on gD glycoprotein of bovine herpesvirus type I using a monoclonal antibody.

Bovine herpesvirus type 1 (BoHV-1), a member of the Alphaherpesvirinae, causes a variety of diseases, which result in significant economic losses worldwide. Envelope glycoprotein D (gD) of BoHV-1 plays an important role in viral entry into the permissive cells, and protective immune response. The fine mapping epitope on the gD will contribute to the understanding of viral pathogenesis and development of alternative vaccines against various diseases associated with BoHV-1. We previously reported the preparation of a monoclonal antibody (MAb) 2B6, which was raised by a truncated recombinant gD protein, demonstrating a neutralizing activity against BoHV-1 infection in Madin-Darby bovine kidney cells. This study described the identification of a linear B-cell epitope on gD using MAb 2B6. A series of partially overlapping gD proteins with glutathione S-transferase tag were generated to define the epitope recognized by MAb 2B6. The amino acid (aa) sequence 323GEPKPGPSPDADRPE337 was recognized by MAb 2B6 using Western blot with the variedly truncated recombinant proteins. Importantly, this epitope was highly conserved among the typical members of BoHV-1, indicating that the epitope may be utilized in diagnosis of diseases due to BoHV-1 infection. Furthermore, the minimal linear epitope sequence 323GEPKPGP329 on gD recognized by MAb 2B6 was confirmed using single-aa residue deletion mutation in carboxyl terminal. This finding not only contributes to our understanding of gD of BoHV-1 virion but also shows a potential for the development of vaccine candidates and diagnostic techniques.

Bovine alphaherpesvirus 1 and 5 in semen from bulls presenting genital lesions under field conditions in Brazil / Alfaherpesvírus bovino 1 e 5 em sêmen de touros com lesões genitais, sob condições a campo no Brasil

Bovine alphaherpesviruses 1 and 5 (BoHV-1/5) are main pathogens of respiratory, reproductive and neurological diseases in cattle. The aim of this study was to investigate the frequency of neutralizing antibodies against BoHV-1/5 in serum samples and to detect viral DNA in semen of bulls from beef cattle farms located in RS. A total of 372 serum and semen sample from bulls were collected in eighteen farms. Serum samples were submitted to virus neutralization (VN) assay, while semen samples were used to detect BoHV-1 and BoHV-5 DNA by PCR. VN results showed that BoHV-1/5 antibodies were detected in bulls of 66.7% (12/18) of the farms, 295 (79.5%) BoHV positive bulls, 287 for BoHV-1 and 234 for BoHV-; at 43 vaccinated bulls 72.1% (31/43) showing serology negative. BoHV-1/5 DNA was detected in the semen of three bulls; one of the them presenting BoHV-1, one out three presenting BoHV-5 and one BoHV-1/5.co-infection All BoHV DNA positive samples came from animals presenting posthitis and other genital lesions at sampling. Results showed a high seroprevalence of BoHV-1/5 antibodies in bulls as well as strong evidence that these viruses are actively circulating in the cattle farms. A remarkable finding is that in the presence of clinically evident lesions in the genital tract, both BoHV-1 and 5 may found in semen.(AU)

Os alfa-herpesvírus bovinos 1 e 5 (BoHV-1/5) são importantes patógenos de doença respiratória, reprodutiva e neurológica em bovinos. O objetivo deste estudo foi investigar a frequência de detecção de anticorpos neutralizantes contra BoHV-1/5 em amostra de soro e detectar DNA viral em sêmen de touros do rebanho bovino localizado nas fazendas de gado de corte do RS. Um total de 371 amostras de soro e sêmen foi coletado de touros em 18 fazendas, 325 das quais são provenientes de touros não vacinados e 43 de vacinados. Amostras de soro foram submetidas à técnica de vírus-neutralização (VN), enquanto as amostras de sêmen foram submetidas à extração de DNA e posterior PCR (polymerase chain reaction) para detecção de BoHV-1 e 5. Os resultados da VN demostraram que anticorpos contra BoHV-1/5 foram detectados nos touros não vacinados em 66,7% (12/18) das fazendas, 295 (79,5%) touros mostraram-se positivos para BoHV, 287 para BoHV-1 e 234 para BoHV-5; e para 43 touros vacinados, observou-se que 72,1% (31/43) foram negativos na sorologia DNA de BoHV-1/5, detectado no sêmen de três touros: um deles apresentava BoHV-1, outro BoHV-5 e em um foi detectada coinfecção por BoHV-1/5. Todas as amostras positivas para o DNA viral eram provenientes de animais que apresentavam lesões de postite e outras lesões genitais. Esses resultados demonstram que há uma alta soroprevalência de BoHV-1/5 em touros, bem como uma forte evidência de que esses vírus estão circulando ativamente no rebanho bovino dessas fazendas. Um achado interessante foi a detecção de BoHV-1 e 5 em touros com lesões na região do trato genital.(AU)

Transmembrane regions of bovine herpesvirus 1-encoded UL49.5 and glycoprotein M regulate complex maturation and ER-Golgi trafficking.

Bovine herpesvirus 1 (BoHV-1)-encoded UL49.5 (a homologue of herpesvirus glycoprotein N) can combine different functions, regulated by complex formation with viral glycoprotein M (gM). We aimed to identify the mechanisms governing the immunomodulatory activity of BoHV-1 UL49.5. In this study, we addressed the impact of gM/UL49.5-specific regions on heterodimer formation, folding and trafficking from the endoplasmic reticulum (ER) to the trans-Golgi network (TGN) - events previously found to be responsible for abrogation of the UL49.5-mediated inhibition of the transporter associated with antigen processing (TAP). We first established, using viral mutants, that no other viral protein could efficiently compensate for the chaperone function of UL49.5 within the complex. The cytoplasmic tail of gM, containing putative trafficking signals, was dispensable either for ER retention of gM or for the release of the complex. We constructed cell lines with stable co-expression of BoHV-1 gM with chimeric UL49.5 variants, composed of the BoHV-1 N-terminal domain fused to the transmembrane region (TM) from UL49.5 of varicella-zoster virus or TM and the cytoplasmic tail of influenza virus haemagglutinin. Those membrane-anchored N-terminal domains of UL49.5 were sufficient to form a complex, yet gM/UL49.5 folding and ER-TGN trafficking could be affected by the UL49.5 TM sequence. Finally, we found that leucine substitutions in putative glycine zipper motifs within TM helices of gM resulted in strong reduction of complex formation and decreased ability of gM to interfere with UL49.5-mediated major histocompatibility class I downregulation. These findings highlight the importance of gM/UL49.5 transmembrane domains for the biology of this conserved herpesvirus protein complex.

Synergistic Activation of Bovine Herpesvirus 1 Productive Infection and Viral Regulatory Promoters by the Progesterone Receptor and Krüppel-Like Transcription Factor 15.

Bovine herpesvirus 1 (BoHV-1), including modified live vaccines, readily infects the fetus and ovaries, which can lead to reproductive failure. The BoHV-1 latency reactivation cycle in sensory neurons may further complicate reproductive failure in pregnant cows. The immediate early transcription unit 1 (IEtu1) promoter drives expression of important viral transcriptional regulators (bICP0 and bICP4). This promoter contains two functional glucocorticoid receptor (GR) response elements (GREs) that have the potential to stimulate productive infection following stressful stimuli. Since progesterone and the progesterone receptor (PR) can activate many GREs, we hypothesized that the PR and/or progesterone regulates productive infection and viral transcription. New studies demonstrated that progesterone stimulated productive infection. Additional studies revealed the PR and Krüppel-like transcription factor 15 (KLF15) cooperated to stimulate productive infection and IEtu1 promoter activity. IEtu1 promoter activation required both GREs, which correlated with the ability of the PR to interact with wild-type (wt) GREs but not mutant GREs. KLF15 also cooperated with the PR to transactivate the bICP0 early promoter, a promoter that maintains bICP0 protein expression during productive infection. Intergenic viral DNA fragments (less than 400 bp) containing two GREs and putative KLF binding sites present within genes encoding unique long 52 (UL-52; component of DNA primase/helicase complex), Circ, bICP4, and IEtu2 were stimulated by KLF15 and the PR more than 10-fold, suggesting that additional viral promoters are activated by these transcription factors. Collectively, these studies suggest progesterone and the PR promote BoHV-1 spread to reproductive tissues, thus increasing the incidence of reproductive failure.IMPORTANCE Bovine herpesvirus 1 (BoHV-1) is the most frequently diagnosed cause of abortions in pregnant cows and can cause "abortion storms" in susceptible herds. Virulent field strains and even commercially available modified live vaccines can induce abortion, in part because BoHV-1 replicates efficiently in the ovary and corpus luteum. We now demonstrate that progesterone and the progesterone receptor (PR) stimulate productive infection. The BoHV-1 genome contains approximately 100 glucocorticoid receptor (GR) response elements (GREs). Interestingly, the PR can bind and activate many promoters that contain GREs. The PR and Krüppel-like transcription factor 15 (KLF15), which regulate key steps during embryo implantation, cooperate to stimulate productive infection and two viral promoters that drive expression of key viral transcriptional regulators. These studies suggest that the ability of progesterone and the PR to stimulate productive infection has the potential to promote virus spread in reproductive tissue and induce reproductive failure.