Wt Bovine Herpesvirus 1 Research Articles

Bovine herpesvirus-1 gE protein inhibits IFN-β production to enhance replication by promoting MAVS ubiquitination and interfering with the interaction between IRF3 and CBP/p300

Bovine herpesvirus-1 (BoHV-1) can infect all breeds of cattle and cause respiratory and genital tract diseases. In the process of viral infection, viruses can use their own proteins to suppress the innate immunity of the host and promote its replication; however, the mechanism by which BoHV-1 evades the innate immune response is not fully understood. In this study, we found that rabbits inoculated with the live gene deletion vaccine BoHV-1-△gI/gE/TK generated higher interferon-β (IFN-β) production in the serum, liver, lung and kidney than rabbits inoculated with wt BoHV-1, which led to milder lesions in the lung and kidney. We performed gene deletion and ectopic expression experiments on viral proteins and found that gE was the major protein that inhibited IFN-β expression. Further studies showed that MAVS and IRF3 were the targets of gE, and the specific mechanism was that gE inhibited IFN-β production by promoting MAVS ubiquitination and interfering with the interaction between IRF3 and CBP/p300. These results suggest a new way of BoHV-1 inhibition of IFN-β production to evade the host innate immunity.

Identification of a novel protein encoded by the latency-related gene of bovine herpesvirus 1

The latency-related (LR) RNA encoded by bovine herpesvirus 1 (BHV-1) is abundantly expressed and alternatively spliced in trigeminal ganglia. A mutant BHV-1 strain that contains three stop codons at the beginning of LR open reading frame (ORF)-2 (LR mutant virus) does not express ORF-2 or an adjacent reading frame that lacks an initiating ATG (RF-C). Calves latently infected with wild-type (wt) BHV-1, but not with the LR mutant virus, reactivate from latency, indicating that proteins encoded by the LR gene regulate the latency-reactivation cycle. The LR gene also contains another large ORF (ORF-1) that is approximately 200 bp downstream of stop codons inserted at the N-terminus of ORF-2. To test whether the LR mutant virus can expresses ORF-1, the authors developed antiserum directed against ORF-1. The ORF-1 antiserum recognizes specific proteins in bovine cells productively infected with wt BHV-1. ORF-1 protein expression is reduced, but not blocked, when bovine cells are infected with the LR mutant virus. Confocal microscopy demonstrated ORF-1 is present in the cytoplasm and nucleus of productively infected cells, whereas RF-C or a fusion protein containing RF-C localizes to the cytoplasm. Trigeminal ganglia from calves latently infected with wt BHV-1 contain neurons specifically stained with the ORF-1 antiserum. These studies suggest ORF-1 expression may be important for the BHV-1 latency-reactivation cycle.

Analysis of a bovine herpesvirus 1 recombinant virus that does not express the bICP0 protein

Bovine herpesvirus 1 (BHV-1) infected-cell protein 0 (bICP0) stimulates productive infection by activating viral gene expression. In this study, an attempt was made to construct a recombinant virus with point mutations in the C(3)HC(4) zinc RING finger of bICP0, as this domain is necessary for activating viral transcription and productive infection. A virus was identified in bovine cells that induced small clusters of infected cells resembling a small plaque. Instead of the expected mutations within the zinc RING finger, this virus contained a point mutation within the initiating ATG of bICP0, a point mutation two bases downstream from the ATG mutation and deletion of flanking plasmid sequences used for homologous recombination. The bICP0 mutant was rescued with wild-type (wt) bICP0 sequences and the bICP0-rescued virus produced wt plaques. The bICP0-rescued virus and wt BHV-1, but not the mutant, expressed the bICP0 protein during productive infection of bovine cells, suggesting that the mutant virus was a null mutant. Consequently, the mutant was designated the bICP0 null mutant. Infection of bovine cells with the bICP0 null mutant resulted in at least 100-fold lower virus titres, indicating that bICP0 protein expression is important, but not required, for virus production. When bovine cells infected with the bICP0 null mutant virus were subcultured, the cells continued to divide, but viral DNA could be detected after more than 35 passages, suggesting that the bICP0 null mutant induced a persistent-like infection in bovine cells and that it may be useful for generating additional bICP0 mutants.

Latency-Related Gene Encoded by Bovine Herpesvirus 1 Promotes Virus Growth and Reactivation from Latency in Tonsils of Infected Calves

Infection of calves with bovine herpesvirus 1 (BHV-1) results in transient immunosuppression that may lead to bacterium-induced pneumonia and, occasionally, death. Although sensory neurons in the trigeminal ganglia (TG) are the primary site of BHV-1 latency, viral genomes are detected in the tonsils of latently infected calves. Dexamethasone (DEX) consistently induces reactivation from latency, and viral gene expression is detected in TG and tonsils. In sensory neurons of latently infected calves, the latency-related (LR) gene is abundantly expressed and is required for reactivation from latency. In the present study, we compared the abilities of wild-type (wt) BHV-1 and a strain with a mutation in the LR gene (the LR mutant strain) to grow in the tonsils of infected calves and reactivate from latency. Lower levels of the LR mutant virus were detected in the tonsils of acutely infected calves. LR mutant viral DNA was consistently detected by PCR in the tonsils of latently infected calves, suggesting that the establishment of a latent or persistent infection occurred. Although the LR mutant did not reactivate from latency in vivo after DEX treatment, explantation of tonsil tissue from calves latently infected with the LR mutant yielded infectious virus. Relative to wt BHV-1, the LR mutant did not induce explant-induced reactivation as efficiently. These studies indicate that the LR gene promotes virus shedding from tonsil tissue during acute infection and reactivation from latency in tonsil tissue in vivo. We suggest that incorporation of the LR gene mutation into existing modified live vaccines would prevent reactivation from latency in neural and nonneural sites and would thus prevent transmission to other animals.

A mutation in the latency-related gene of bovine herpesvirus 1 disrupts the latency reactivation cycle in calves.

Bovine herpesvirus 1 (BHV-1) is an important pathogen of cattle, and infection is usually initiated via the ocular or nasal cavity. Following acute infection, the primary site for BHV-1 latency is the sensory neuron. Reactivation from latency occurs sporadically, resulting in virus shedding and transmission to uninfected cattle. The only abundant viral transcript expressed during latency is the latency-related (LR) RNA, suggesting that it mediates some aspect of latency. An LR mutant was constructed by inserting three stop codons near the beginning of the LR-RNA, suggesting that expression of LR proteins would be altered. The LR mutant grew with wild-type (wt) efficiency in bovine kidney cells (MDBK). When calves were infected with the LR mutant, a dramatic decrease (3 to 4 logs) in ocular, but not nasal, viral shedding occurred during acute infection relative to the wt or the LR-rescued virus (M. Inman, L. Lovato, A. Doster, and C. Jones, J. Virol. 75:8507-8515, 2001). In this study, we examined the latency reactivation cycle in calves infected with the LR mutant and compared these results to those from calves infected with wt BHV-1 or the LR-rescued virus. During acute infection, lower levels of infectious virus were detected in trigeminal ganglion homogenates from calves infected with the LR mutant. As judged by in situ hybridization, BHV-1-positive neurons were detected in trigeminal ganglia of calves infected with the wt but not the LR mutant. Although LR-RNA was detected by reverse transcription-PCR in calves latently infected with the LR mutant, a semiquantitative PCR analysis revealed that lower levels of viral DNA were present in trigeminal ganglia of calves infected with the LR mutant. Dexamethasone treatment of calves latently infected with wt BHV-1 or the LR-rescued virus, but not the LR mutant, consistently induced reactivation from latency, as judged by shedding of infectious virus from the nose or eyes and increases in BHV-1-specific antibodies. In summary, this study demonstrates that wt expression of LR gene products plays an important role in the latency reactivation cycle of BHV-1 in cattle.

Bovine Herpesvirus 1-Induced Apoptotic Cell Death: Role of Glycoprotein D

Bovine herpesvirus 1 (BHV-1) induces apoptotic cell death in peripheral blood mononuclear cells and in bovine B lymphoma (BL-3) cells. Attachment but not penetration of BHV-1 is necessary to induce apoptosis in target cells, suggesting that one or more BHV-1 envelope glycoproteins could be involved in the activation of the apoptotic process. In the present study, we demonstrate that, although BHV-1 virions devoid of glycoprotein D (BHV-1 gD−/−) still bind to BL-3 cells, they are no longer able to induce apoptosis. In contrast, virions that contain glycoprotein D (gD) in the viral envelope but do not genetically encode gD (BHV-1 gD−/+) induce a level of apoptosis comparable to that produced by wild-type (wt) BHV-1. In addition, monoclonal antibodies directed against gD, but not against gB or gC, strongly reduced the high levels of apoptosis induced by BHV-1. These observations demonstrate that the induction of apoptosis is directly due to BHV-1 viral particles harboring gD in the viral envelope. Interestingly, binding of affinity-purified gD to BL-3 cells did not induce apoptosis but inhibited the ability of wt BHV-1 to induce apoptosis. Altogether, these results provide evidence for the direct or indirect involvement of gD in the mechanism by which BHV-1 induces apoptosis.

A glycoprotein E deletion mutant of bovine herpesvirus 1 infects the same limited number of tissues in calves as wild-type virus, but for a shorter period.

To gain insight into the role of glycoprotein E of bovine herpesvirus 1 (BHV-1), we compared the distribution of wild-type (wt) BHV-1 with that of a gE deletion mutant (gE-) in calves after intranasal inoculation. The wt-infected calves had severe clinical signs, but the gE(-)-infected calves were virtually free of clinical signs. At 3, 4, 7, 8, 44, 45, 50 and 51 days post-infection (p.i.), one calf from each group was killed and tissues were collected for virus isolation and PCR analysis. At 3, 4, 7 and 8 days p.i., infectious virus could be isolated only from the nasopharyngeal mucosa, parotid gland and nearby lymphoid tissues for both the wt- and gE(-)-infected calves. At 3 and 4 days p.i., virus titres in these tissues were comparable in both the wt- and gE(-)-infected calves. However, the virus titres were significantly reduced at 7 and 8 days p.i. in the gE(-)-infected calves, but not in the wt-infected calves. Semi-quantitative PCR analysis revealed that for the entire infection period (3 to 51 days p.i.) significantly more BHV-1 DNA was detected in the trigeminal ganglia (TG) of the wt-infected calves than in those of the gE(-)-infected calves. We conclude that the gE- mutant infects the same limited number of tissues as wt BHV-1, but for a shorter period.