ABSTRACT Like many Alphaherpesvirinae subfamily members, bovine herpesvirus 1 (BoHV-1) expresses an abundant transcript in latently infected sensory neurons, the latency-related (LR)-RNA. LR-RNA encodes a protein (ORF2) that inhibits apoptosis, interacts with Notch family members, interferes with Notch-mediated transcription, and stimulates neurite formation in cells expressing Notch. An LR mutant virus containing stop codons at the amino terminus of ORF2 does not reactivate from latency or replicate efficiently in certain tissues, indicating that LR gene products are important. In this study, β-catenin, a transcription factor activated by the canonical Wnt signaling pathway, was frequently detected in ORF2-positive trigeminal ganglionic neurons of latently infected, but not mock-infected, calves. Conversely, the lytic cycle regulatory protein (BoHV-1 infected cell protein 0, or bICP0) was not frequently detected in β-catenin-positive neurons in latently infected calves. During dexamethasone-induced reactivation from latency, mRNA expression levels of two Wnt antagonists, Dickkopf-1 (DKK-1) and secreted Frizzled-related protein 2 (SFRP2), were induced in bovine trigeminal ganglia (TG), which correlated with reduced β-catenin protein expression in TG neurons 6 h after dexamethasone treatment. ORF2 and a coactivator of β-catenin, mastermind-like protein 1 (MAML1), stabilized β-catenin protein levels and stimulated β-catenin-dependent transcription in mouse neuroblastoma cells more effectively than MAML1 or ORF2 alone. Neuroblastoma cells expressing ORF2, MAML1, and β-catenin were highly resistant to cell death following serum withdrawal, whereas most cells transfected with only one of these genes died. The Wnt signaling pathway interferes with neurodegeneration but promotes neuronal differentiation, suggesting that stabilization of β-catenin expression by ORF2 promotes neuronal survival and differentiation. IMPORTANCE Bovine herpesvirus 1 (BoHV-1) is an important pathogen of cattle, and like many Alphaherpesvirinae subfamily members establishes latency in sensory neurons. Lifelong latency and the ability to reactivate from latency are crucial for virus transmission. Maintaining the survival and normal functions of terminally differentiated neurons is also crucial for lifelong latency. Our studies revealed that BoHV-1 gene products expressed during latency stabilize expression of the transcription factor β-catenin and perhaps its cofactor, mastermind-like protein 1 (MAML1). In contrast to expression during latency, β-catenin expression in sensory neurons is not detectable following treatment of latently infected calves with the synthetic corticosteroid dexamethasone to initiate reactivation from latency. A viral protein (ORF2) expressed in a subset of latently infected neurons stabilized β-catenin and MAML1 in transfected cells. ORF2, β-catenin, and MAML1 also enhanced cell survival when growth factors were withdrawn, suggesting that these genes enhance survival of latently infected neurons.

Bovine herpesvirus 1 (BHV-1) establishes latency in sensory neurons. The synthetic corticosteroid dexamethasone consistently induces reactivation from latency. Within 90 min after latently infected calves are treated with dexamethasone, two BHV-1 regulatory proteins, BHV-1-infected cell protein 0 (bICP0) and viral protein 16 (VP16), are expressed in the same neuron. In this study, we demonstrate that VP16 and bICP0 can be detected at 22 and 33 min after dexamethasone (DEX) treatment of latently infected calves. However, we were unable to discern whether VP16 or bICP0 was expressed at early times after reactivation. VP16+ neurons consistently express the glucocorticoid receptor suggesting corticosteroid-mediated activation of its receptor rapidly stimulates reactivation from latency.

Bovine herpesvirus 1 (BHV-1) establishes a lifelong latent infection in sensory neurons following acute infection. Increased corticosteroid levels, due to stress, increases the incidence of reactivation from latency. Within minutes, corticosteroids activate the glucocorticoid receptor and transcription of promoters containing a glucocorticoid receptor element. A single intravenous injection of the synthetic corticosteroid dexamethasone consistently induces reactivation from latency in calves. Lytic cycle viral gene expression is detected within 6 h after dexamethasone treatment of calves latently infected with BHV-1. Cellular transcription factors are induced by dexamethasone in trigeminal ganglionic neurons within 1.5 h after dexamethasone treatment, suggesting they promote viral gene expression during the early phases of reactivation from latency, which we operationally defined as the escape from latency. In this study, immunohistochemistry was utilized to examine viral protein expression during the escape from latency. Within 1.5 h after dexamethasone treatment, bICP0 and a late protein (VP16) were consistently detected in a subset of trigeminal ganglionic neurons. Most neurons expressing bICP0 also expressed VP16. Additional studies revealed that neurons expressing the glucocorticoid receptor also expressed bICP0 or VP16 at 1.5 h after dexamethasone treatment. Two other late proteins, glycoprotein C and D, were not detected until 6 h after dexamethasone treatment and were detected in only a few neurons. These studies provide evidence that VP16 and the promiscuous viral trans-activator (bICP0) are expressed during the escape from latency, suggesting they promote the production of infectious virus in a small subset of latently infected neurons.

Bovine herpesvirus 1 (BHV-1), an alphaherpesvirinae subfamily member, establishes latency in sensory neurons. Elevated corticosteroid levels, due to stress, reproducibly triggers reactivation from latency in the field. A single intravenous injection of the synthetic corticosteroid dexamethasone (DEX) to latently infected calves consistently induces reactivation from latency. Lytic cycle viral gene expression is detected in sensory neurons within 6 h after DEX treatment of latently infected calves. These observations suggested that DEX stimulated expression of cellular genes leads to lytic cycle viral gene expression and productive infection. In this study, a commercially available assay-Bovine Gene Chip-was used to compare cellular gene expression in the trigeminal ganglia (TG) of calves latently infected with BHV-1 versus DEX-treated animals. Relative to TG prepared from latently infected calves, 11 cellular genes were induced more than 10-fold 3 h after DEX treatment. Pentraxin three, a regulator of innate immunity and neurodegeneration, was stimulated 35- to 63-fold after 3 or 6 h of DEX treatment. Two transcription factors, promyelocytic leukemia zinc finger (PLZF) and Slug were induced more than 15-fold 3 h after DEX treatment. PLZF or Slug stimulated productive infection 20- or 5-fold, respectively, and Slug stimulated the late glycoprotein C promoter more than 10-fold. Additional DEX-induced transcription factors also stimulated productive infection and certain viral promoters. These studies suggest that DEX-inducible cellular transcription factors and/or signaling pathways stimulate lytic cycle viral gene expression, which subsequently leads to successful reactivation from latency in a small subset of latently infected neurons.

In this study, we examined the functional role of bovine herpesvirus type 1 (BHV-1) Us9 acidic domain residues 83–90 in the anterograde axonal transport of the virus in calves (natural host), rabbits, and in cultured neurons. A mutant virus strain lacking Us9 residues 83–90 (BHV-1 Us9 Δ83–90) and the rescued virus (BHV-1 Us9 R83–90) replicated efficiently in the nasal and ocular epithelium during primary infection and established latency in the trigeminal ganglia (TG). However, upon reactivation from latency, only the BHV-1 Us9 R83–90 virus was detected in nasal and ocular swabs of animals. In compartmentalized, rabbit primary dorsal root ganglia (DRG) neuron cultures, the Us9-deleted BHV-1, BHV-1 Us9 Δ83–90 and BHV-1 Us9 R83–90 viruses were transported efficiently in the retrograde direction. However, only the BHV-1 Us9 R83–90 virus was transported in an anterograde direction. These studies suggested that the Us9 acidic domain residues located between 83 and 90 were required for axonal anterograde transport.

ABSTRACT Sensory neurons within trigeminal ganglia (TG) are the primary site for bovine herpesvirus 1 (BHV-1) latency. During latency, viral gene expression is restricted to the latency-related (LR) gene and the open reading frame ORF-E. We previously constructed an LR mutant virus that expresses LR RNA but not any of the known LR proteins. In contrast to calves latently infected with wild-type (wt) BHV-1 or the LR rescued virus, the LR mutant virus does not reactivate from latency following dexamethasone (DEX) treatment. In this study, we demonstrated that bICP0, but not bICP4, transcripts were consistently detected in TG of calves infected with the LR mutant or LR rescued virus following DEX treatment. Calves latently infected with the LR rescued virus but not the LR mutant virus expressed late transcripts, which correlated with shedding of infectious virus following DEX treatment. The bICP4 and bICP0 genes share a common immediate-early promoter, suggesting that this promoter was not consistently activated during DEX-induced reactivation from latency. The bICP0 gene also contains a novel early promoter that was activated by DEX in mouse neuroblastoma cells. Expression of a cellular transcription factor, C/EBP-alpha, was stimulated by DEX, and C/EBP-alpha expression was necessary for DEX induction of bICP0 early promoter activity. C/EBP-alpha directly interacted with bICP0 early promoter sequences that were necessary for trans activation by C/EBP-alpha. In summary, DEX treatment of latently infected calves induced cellular factors that stimulated bICP0 early promoter activity. Activation of bICP0 early promoter activity does not necessarily lead to late gene expression and virus shedding.

Bovine herpesvirus type 1 (BHV-1) is an important component of the bovine respiratory disease complex (BRDC) in cattle. The ability of BHV-1 to transport anterogradely from neuronal cell bodies in trigeminal ganglia (TG) to nerve ending in the noses and corneas of infected cattle following reactivation from latency plays a significant role in the pathogenesis of BRDC and maintenance of BHV-1 in the cattle population. We have constructed a BHV-1 bacterial artificial chromosome (BAC) clone by inserting an excisable BAC plasmid sequence in the long intergenic region between the glycoprotein B (gB) and UL26 genes. A BAC-excised, reconstituted BHV-1 containing only the 34-bp loxP sequence within the gB-UL26 intergenic region was highly infectious in calves, retained wild-type virulence properties, and reactivated from latency following treatment with dexamethasone. Using a two-step Red-mediated mutagenesis system in Escherichia coli, we constructed a gE cytoplasmic tail-truncated BHV-1 and a gE-rescued BHV-1. Following primary infection, the gE cytoplasmic tail-truncated virus was efficiently transported retrogradely from the nerve endings in the nose and eye to cell bodies in the TG of calves and rabbits. However, following dexamethasone-induced reactivation from latency, the gE mutant virus was not isolated from nasal and ocular sheddings. Reverse transcriptase PCR assays detected VP5 transcription in the TG of rabbits infected with gE-rescued and gE cytoplasmic tail-truncated viruses during primary infection and after dexamethasone treatment but not during latency. Therefore, the BHV-1gE cytoplasmic tail-truncated virus reactivated in the TG; however, it had defective anterograde transport from TG to nose and eye in calves and rabbits.

ABSTRACT Bovine herpesvirus 1 (BHV-1) infected cell protein 0 (bICP0) stimulates productive infection, in part by activating viral gene expression. The C 3 HC 4 zinc RING finger of bICP0 is crucial for activating viral transcription and productive infection. In this study, we used a bacterial artificial chromosome containing a wild-type (wt) virulent BHV-1 strain to generate a single amino acid mutation in the C 3 HC 4 zinc RING finger of bICP0. This virus (the 51g mutant) contains a cysteine-to-glycine mutation (51st amino acid) in the C 3 HC 4 zinc RING finger of bICP0. A plasmid expressing the 51g mutant protein did not transactivate viral promoter activity as efficiently as wt bICP0. The 51g mutant virus expressed higher levels of the bICP0 protein than did the 51g rescued virus (51gR) but yielded reduced virus titers following infection of permissive bovine cells. The 51g mutant virus, but not the 51gR virus, grew poorly in bovine cells pretreated with imiquimod to stimulate interferon production. During acute infection of calves, levels of infectious virus were 2 to 3 logs lower in ocular or nasal swabs with 51g than with 51gR. Calves latently infected with the 51g mutant did not reactivate from latency because virus shedding did not occur in ocular or nasal cavities. As expected, calves latently infected with 51gR reactivated from latency following dexamethasone treatment. These studies demonstrate that mutation of a single well-conserved cysteine residue in the C 3 HC 4 zinc RING finger of bICP0 has dramatic effects on the growth properties of BHV-1.

Bovine herpesvirus type 1 (BHV-1) is an important pathogen that can initiate bovine respiratory disease complex. Like other members of the subfamily Alphaherpesvirinae, BHV-1 establishes latency in sensory neurons. The latency-related (LR) gene expresses a family of alternatively spliced transcripts in infected sensory neurons that have the potential to encode several LR proteins. An LR mutant virus that contains three stop codons near the 5' terminus of the first open reading frame in the LR gene does not express two LR proteins or reactivate from latency. In addition, the LR mutant virus induces higher levels of apoptosis in trigeminal ganglionic neurons and grows less efficiently in certain tissues of infected calves. In spite of the reduced pathogenesis, the LR mutant virus, wild-type BHV-1 and the LR rescued virus exhibit identical growth properties in cultured bovine cells. In this study, we demonstrated that during early phases of productive infection the LR mutant virus expressed higher levels of LR-RNA relative to the LR rescued virus or wt BHV-1. Bovine kidney cells infected with the LR mutant virus also induced higher levels of beta interferon RNA and interferon response genes. These results suggest that inappropriate expression of LR-RNA, in the absence of LR protein expression, may influence the latency-reactivation cycle and pathogenic potential of BHV-1.

In this study, the authors examined the role of bovine herpesvirus type 1 (BHV-1) Us9 in the anterograde transport of the virus from trigeminal ganglia (TG) to nose and eye upon reactivation from latency. During primary infection, both BHV-1 Us9-deleted and BHV-1 Us9-rescued viruses replicated efficiently in the nasal and ocular epithelium. However, upon reactivation from latency, only the BHV-1 Us9-rescued virus could be isolated in the nasal and ocular shedding. By real-time polymerase chain reaction, comparable DNA copy numbers were detected in the TGs during latency and reactivation for both the viruses. Therefore, Us9 is essential for reactivation of the virus in the TG and anterograde axonal transport from TG to nose and eye.

Bovine herpes virus 1 (BHV-1), like other α-herpesvirinae subfamily members, establishes latency in sensory neurons. Periodically BHV-1 reactivates from latency, resulting in virus shedding and spread to uninfected cattle. Although reactivation from latency does not usually lead to recurrent disease, the latency-reactivation cycle is crucial for virus transmission. The latency-related (LR) RNA is abundantly expressed during latency, and expression of a LR encoded protein is necessary for dexamethasone-induced reactivation from latency in cattle. Within LR promoter sequences, a small open reading frame (ORF) was identified (ORF-E) that is antisense to the LR-RNA, and downstream of the bICP0 gene. ORF-E transcription is consistently detected in trigeminal ganglia (TG) of latently infected calves, suggesting ORF-E expression plays a role in the latency-reactivation cycle. Polyclonal antiserum directed against an ORF-E peptide or the entire ORF-E protein specifically recognizes the nucleus of sensory neurons in TG of latently infected calves. The ORF-E peptide—specific antiserum also recognizes a protein when mouse neuroblastoma cells (neuro-2A) are transfected with an ORF-E expression construct. In contrast to the growth inhibiting properties of the LR gene, stably transfected ORF-E—expressing cells were obtained. Neuro-2A cells stably transfected with a plasmid expressing ORF-E induced morphological changes that resembled neurite-like projections. In contrast, neurite-like projections were not observed following transfection of neuro-2A cells with an empty vector. These studies suggest that a protein encoded by ORF-E has the potential to alter the physiology or metabolism of neuronal cell types, which may be important for long-term latency.

During latency, the bovine herpesvirus 1 (BHV-1) latency-related (LR) RNA is abundantly expressed in neurons within trigeminal ganglia (TG). A LR mutant virus that does not express two LR proteins is unable to reactivate from latency following dexamethasone treatment. Increased infiltration of inflammatory cells occurs in TG of calves acutely infected with the LR mutant virus. Throughout acute infection, wild-type BHV-1 DNA is detected in neurons surrounded by mononuclear infiltrates and in non-neuronal cells comprising the infiltrate. Conversely, LR mutant DNA is only detected in neurons near the end of acute infection, suggesting LR gene products promote virus spread in TG.

ABSTRACT Herpes simplex virus type 1 (HSV-1) is the leading cause of virus-induced encephalitis; however, the viral genes that regulate encephalitis have not been well characterized. In this study, we tested whether the LAT (latency-associated transcript) locus regulates the frequency of encephalitis in male or female mice. Male BALB/c mice are more susceptible to HSV-1-induced encephalitis than age-matched female BALB/c mice. Deletion of LAT coding sequences reduced the frequency of encephalitis. A recombinant virus containing the first 1.5 kb of the LAT coding sequence induces levels of encephalitis in male BALB/c mice similar to those induced by wild-type HSV-1.

ABSTRACT 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.

ABSTRACT Bovine herpesvirus 1 (BHV-1) is an important pathogen of cattle and infection is usually initiated via the ocular or nasal cavity. After acute infection, the primary site for BHV-1 latency is sensory neurons in the trigeminal ganglia (TG). 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. An LR mutant was constructed by inserting three stop codons near the beginning of the LR RNA. This mutant grows to wild-type (wt) efficiency in bovine kidney cells and in the nasal cavity of acutely infected calves. However, shedding of infectious virus from the eye and TG was dramatically reduced in calves infected with the LR mutant. Calves latently infected with the LR mutant do not reactivate after dexamethasone treatment. In contrast, all calves latently infected with wt BHV-1 or the LR rescued mutant reactivate from latency after dexamethasone treatment. In the present study, we compared the frequency of apoptosis in calves infected with the LR mutant to calves infected with wt BHV-1 because LR gene products inhibit apoptosis in transiently transfected cells. A sensitive TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay and an antibody that detects cleaved caspase-3 were used to identify apoptotic cells in TG. Both assays demonstrated that calves infected with the LR mutant for 14 days had higher levels of apoptosis in TG compared to calves infected with wt BHV-1 or to mock-infected calves. Viral gene expression, except for the LR gene, is extinguished by 14 days after infection, and thus this time frame is operationally defined as the establishment of latency. Real-time PCR analysis indicated that lower levels of viral DNA were present in the TG of calves infected with the LR mutant throughout acute infection. Taken together, these results suggest that the antiapoptotic properties of the LR gene play an important role during the establishment of latency.

Following primary infection of the eye, oral cavity, and/or nasal cavity, bovine herpesvirus 1 (BHV-1) establishes latency in trigeminal ganglionic (TG) neurons. Virus reactivation and spread to other susceptible animals occur after natural or corticosteroid-induced stress. Infection of calves with BHV-1 leads to infiltration of lymphocytes in TG and expression of IFN-γ (interferon-gamma), even in latently infected calves. During latency, virus antigen and nucleic acid positive non-neural cells were occasionally detected in TG suggesting there is a low level of spontaneous reactivation. Since we could not detect virus in ocular or nasal swabs, these rare cells do not support high levels of productive infection and virus release or they do not support virus production at all. Dexamethasone (DEX) was used to initiate reactivation in latently infected calves. Foci of mononuclear or satellite cells undergoing apoptosis were detected 6h after DEX treatment, as judged by the appearance of TUNEL+ cells (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling). BHV-1 antigen expression was initially detected in lymphocytes and other non-neural cells in latently infected calves following DEX treatment. At 24h after DEX treatment, viral antigen expression and nucleic acid were readily detected in neurons. Our data suggest that persistent lymphocyte infiltration and cytokine expression occur during latency because a low number of cells in TG express BHV-1 proteins. Induction of apoptosis and changes in cytokine expression following DEX treatment correlates with reactivation from latency. We hypothesize that inflammatory infiltration of lymphoid cells in TG plays a role in regulating latency.

ABSTRACT 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.

ABSTRACT Bovine herpesvirus 1 (BHV-1) is an important pathogen of cattle, and infection is usually initiated in the ocular or nasal cavity. Like other alphaherpesviruses, BHV-1 establishes latency in sensory neurons but has the potential of reactivating from latency and spreading. The only abundant viral transcript expressed during latency is the latency-related (LR) RNA, which is alternatively spliced in trigeminal ganglia during acute infection (L. R. Devireddy and C. Jones, J. Virol. 72:7294–7301, 1998). LR gene products inhibit cell cycle progression (Y. Jiang, A. Hossain, M. T. Winkler, T. Holt, A. Doster, and C. Jones, J. Virol. 72:8133–8142, 1998) and chemically induced apoptosis (J. Ciacci-Zannela, M. Stone, G. Henderson, and C. Jones. J. Virol. 73:9734–9740, 1999). Although these studies suggest that LR gene products play an important role in the latency/pathogenesis of BHV-1, construction of a mutant is necessary to test this hypothesis. Because the bICP0 gene overlaps and is antisense to the LR gene, it was necessary to mutate the LR gene without altering bICP0 expression. This was accomplished by inserting three stop codons near the beginning of the LR RNA, thus interfering with expression of proteins expressed by the LR RNA. The LR mutant virus grew with wild-type (WT) efficiency in bovine kidney (MDBK) cells and expressed bICP0 at least as efficiently as WT BHV-1 or the LR rescued virus. When calves were infected with the LR mutant, we observed a dramatic decrease (3 to 4 log units) in ocular shedding during acute infection relative to WT or the LR rescued virus. In contrast, shedding of the LR mutant from the nasal cavity was not significantly different from that of the WT or the LR rescued virus. Calves infected with the LR mutant exhibited mild clinical symptoms, but they seroconverted. Neutralizing antibody titers were lower in calves infected with the LR mutant, confirming reduced growth. In summary, this study suggests that an LR protein promotes ocular shedding during acute infection of calves.

Following acute infection of cattle with bovine herpesvirus-1 (BHV-1), cyclin expression was examined in trigeminal ganglia (TG). Cyclin A was primarily detected in the nucleus of TG neurons. In contrast, cyclin D1 and cyclin E were detected in the nucleus and cytoplasm of TG neurons. Uninfected or latently infected calves did not express detectable levels of these cyclins in TG neurons. Following dexamethasone-induced reactivation, cyclins D1, E and A were also detected in TG neurons. In situ hybridization of consecutive sections demonstrated that many neurons expressing cyclins contained viral nucleic acid, demonstrating that they were infected. Based on these observations, we hypothesize that BHV-1 infection activates neuronal cyclin expression to enhance productive infection. It is also possible that the stress of neuronal infection or reactivation leads to cyclin expression.

ABSTRACT Bovine herpesvirus 1 (BHV-1), like other members of the Alphaherpesvirinae subfamily, establishes latent infection in sensory neurons. Reactivation from latency can occur after natural or corticosteroid-induced stress culminating in recurrent disease and/or virus transmission to uninfected animals. Our previous results concluded that CD4 + T cells in the tonsil and other adjacent lymph nodes are infected and undergo apoptosis during acute infection (M. T. Winkler, A. Doster, and C. Jones, J. Virol. 73:8657–8668, 1999). To test whether BHV-1 persisted in lymphoreticular tissue, we analyzed tonsils of latently infected calves for the presence of viral DNA and gene expression. BHV-1 DNA was consistently detected in the tonsils of latently infected calves. Detection of the latency-related transcript (LRT) in tonsils of latently infected calves required nested reverse transcription-PCR (RT-PCR) suggesting that only a few cells contained viral DNA or that LRT is not an abundant transcript. bICP0 (immediate-early and early transcripts), ribonucleotide reductase (early transcript), and glycoprotein C (late transcript) were not detected by RT-PCR in latently infected calves. When reactivation was initiated by dexamethasone, bICP0 and ribonucleotide reductase transcripts were detected. Following dexamethasone treatment, viral nucleic acid was detected simultaneously in trigeminal ganglionic neurons and lymphoid follicles of tonsil. LRT was detected at 6 and 24 h after dexamethasone treatment but not at 48 h. Dexamethasone-induced reactivation led to apoptosis that was localized to tonsillar lymphoid follicles. Taken together, these findings suggest that the tonsil is a site for persistence or latency from which virus can be reactivated by dexamethasone. We further hypothesize that the shedding of virus from the tonsil during reactivation plays a role in virus transmission.