VIRUS HOST INTERACTIONS THAT REGULATE THE LATENCY-REACTIVATION CYCLE OF BOVINE HERPESVIRUS 1 (BHV-1).

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 0216420
• Project Start Date: Sep 15, 2008
• Project End Date: Sep 14, 2013
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF NEBRASKA
(N/A)
LINCOLN,NE 68583

Performing Department
Veterinary and Biomedical Sciences

Non Technical Summary
Infection of cattle by bovine herpesvirus 1 (BHV-1) can lead to upper respiratory tract disorders, conjunctivitis, genital disorders, and immune-suppression. BHV-1 induced immune-suppression initiates "shipping fever", which is also referred to as bovine respiratory complex (BRDC). BRDC is an upper respiratory tract disorder that costs the US cattle industry more than $3 billion/year. BHV-1 induced immune-suppression leads to secondary bacterial infections that can cause pneumonia. Secondary infections correlates with depressed cell-mediated immunity after BHV-1 infection. BHV-1 infections, in large part due to vaccine breaks and reactivation from latency, are serious problems for feedlot cattle. Following acute infection, BHV-1 establishes latency in sensory neurons in trigeminal ganglia (TG). Stressful stimuli initiate reactivation from latency, which leads to productive infection and virus transmission. The BHV-1 latency-reactivation cycle is crucial for viral transmission, immune-suppression, and pathogenesis. The latency-related (LR) gene and ORF-E are abundantly expressed in sensory neurons of trigeminal ganglia (TG) of latently infected calves. ORF-E may promote efficient establishment of latency because it can induce neurite-like outgrowth in mouse neuroblastoma cells. We predict that ORF-E enhances restoration of mature neuronal functions following infection, which may promote maintenance of latency. A fraction of LR-RNA is polyadenylated and alternatively spliced in TG, suggesting more than one LR protein is expressed. LR gene products inhibit cell proliferation, bICP0 RNA expression, and apoptosis. LR protein expression is necessary for inhibiting apoptosis, but not cell growth or bICP0 expression. We have previously generated a LR mutant virus that has a small deletion at the 5' end of the gene and an insertion of three stop codons. This virus grows fine in cultured cells, but does not reactivate from latency suggesting LR gene products are required for the latency-reactivation cycle. Although modified live vaccines are available, they can be pathogenic in young calves or cause abortion in pregnant cows. The BHV-1 latency-reactivation cycle maintains the virus in nature, and complicates disease control. Understanding how the LR gene regulates latency and pathogenesis may lead to a superior modified-live vaccine that does not reactivate from latency, but protects vaccinated cattle from clinical symptoms following exposure to virulent field strains. It is also possible that understanding how ORF-E functions will help to design better vaccines.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3310	1101	34%
311	3410	1101	33%
311	4030	1101	33%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3310 - Beef cattle, live animal; 3410 - Dairy cattle, live animal; 4030 - Viruses;

Field Of Science
1101 - Virology;

Keywords

bovine herpesvirus 1

latency

sensory neurons

bovine respiratory disease

shipping fever

vaccines

apoptosis

gene expression

neurogenesis

Goals / Objectives
Infection of cattle by bovine herpesvirus 1 (BHV-1) can lead to upper respiratory tract disorders, conjunctivitis, genital disorders, and immune-suppression. BHV-1 induced immune-suppression initiates "shipping fever" (bovine respiratory complex), is a serious problem for the US cattle industry. The BHV-1 latency-reactivation cycle is crucial for viral transmission, immune-suppression, and pathogenesis. The latency-related (LR) gene is abundantly expressed in sensory neurons of trigeminal ganglia (TG) of latently infected calves. A LR mutant virus that contains 3 stop codons at the first LR open reading frame (ORF) does not reactivate from latency, and does not express three proteins encoded by the LR gene (ORF-1, ORF-2 and RF-C). Functional studies have demonstrated that LR proteins inhibit: 1) apoptosis in TG of acutely infected calves, and 2) reactivation from latency. Recent studies have demonstrated that: 1) ORF-2 inhibits apoptosis in transiently transfected cells, and 2) during reactivation from latency, the bICP0 early promoter is specifically activated. Our long-term goals are to identify virus host interactions that regulate the latency-reactivation cycle, understand the mechanism by which dexamethasone (DEX) induces reactivation from latency, and develop a superior modified live vaccine. We hypothesize that proteins encoded by the LR gene promote establishment of latency in a pool of neurons that support reactivation from latency. The major function of the LR gene that promotes establishment of latency is inhibiting apoptosis. We also hypothesize that DEX alters cellular gene expression, which subsequently stimulates reactivation from latency. To test these two predictions, we will conduct studies guided by the following three objectives: 1. Mutagenesis of ORF2 and functional analysis. 2. Construction and analysis of an LR mutant that disrupts ORF2 and ORF-E. 3. Analysis of DEX induced reactivation.

Project Methods
Objective 1. Functional analysis of ORF2. Objective 1A. Identification of domains within ORF2 that inhibit apoptosis. To identify ORF2 sequences necessary for inhibiting apoptosis, transposon insertion mutants will be prepared, and the mutant ORF2 constructs examined for their anti- apoptosis activity. Objective 1B. Identification of cellular proteins that interact with ORF2. We will identify cellular proteins that interact with ORF-2 by performing two-hybrid screen. Objective 2. Construction and analysis of an LR mutant that disrupts ORF2 and ORF-E. Objective 2A. Constructing viral strains with mutations in ORF2. We will use a bacterial artificial chromosome (BAC) that contains the Cooper strain of BHV-1 (pBHV-1BAC) to develop site directed mutations in ORF2. We will construct recombinant viruses containing an ATG→TTA and ATG-→TTG mutations at the 5' end of ORF2. Objective 2B. Constructing viral strains with mutations in ORF-E. We will use the virulent wt BHV-1 Bac clone to construct an ORF-E mutant virus that has its initiating ATG converted to TTA or TTG. Objective 2C. Analysis of growth properties of the ORF2 or ORF-E mutant. Bovine cells will be infected with wt virus, the ORF2 mutant viruses, the ORF-E mutant viruses or the respective rescued viruses using a moi of 1 or 10 pfu/cell. Virus titers will be measured at different times after infection. Objective 2C. Growth properties of ORF2 or ORF-E mutants in calves. Calves will be infected with the ORF2 or ORF-E mutants. Ocular and nasal swabs will be obtained from calves at 1, 2, 4, 6, 8, 10, 12, and 14 d after infection. The amount of virus present in swabs will be quantified. Objective 2D. Do the ORF2 or ORF-E mutants reactivate from latency Latently infected calves (60 d after infection; 5 calves/virus) will be treated with dexamethasone (DEX) to initiate reactivation. DEX treatment of rabbits or cattle latently infected with BHV-1 leads to efficient virus reactivation. Latently infected calves will be injected intravenously (jugular vein) with 100 mg DEX. Nasal and ocular swabs will be collected at 2, 4, 6, 8, 12, 16, and 24 days after DEX treatment. Infectious virus in swabs will be measured. Objective 3. Identification of novel cellular factors expressed in trigeminal ganglia (TG) during reactivation from latency. To begin to understand the effects of DEX on cellular transcription during reactivation from latency, calves latently infected with BHV-1 will be given an IV injection of DEX (100 mg/calf) and cellular gene expression measured at 4 or 8 hours later by Microarray analysis. Total RNA from trigeminal ganglia will be prepared. As a control, uninfected calves will be treated with DEX for 4 or 8 hours. A commercially available bovine genome array is available from Affymetrix. This Chip contains more than 23,000 transcripts that spans the bovine genome. Cellular genes that exhibit significant changes in steady state levels will be further analyzed by RT-PCR and Northern blotting.

Progress 09/15/08 to 09/14/13

Outputs
Target Audience: The target audience includes veterinarians, cattle producers, herpes virologists, and animal health companies. Changes/Problems: No major changes were made. What opportunities for training and professional development has the project provided? Three graduate students have worked on these projects during the last couple fo years. Two have graduated and are currently employed by CEVA, an animal health company. How have the results been disseminated to communities of interest? Published papers, seminars at various universities, seminars to veterinarians for continuing education about cattle diseases (sponsored by Novartus), and presentations at the International Herpesvirus Workshop. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1. We have identified functional domains in ORF2 that regulate apoptosis, DNA binding properties, and the ability to inhibit Notch-dependent signaling. 2. We have identified stress-induced cellular transcription factors that are induced in sensory neurons during reaction from latency. These cellular factors were shown to stimulate bovine herpesvirus 1 (BHV-1) productive infection. Certain cellular transcription factors also trans-activate viral promoters. Collectively, these studies demonstrated that the stress-induced cellular transcription facrtors promote BHV-1 reactivation from latency in sensory neurons. 3. The stress-induced cellular transcription factors were also found to trans-activate the herpes simplex virus type 1 (HSV-1) ICP0 promoter more than 400 fold. The ICP0 promoter dirves expression of a critical regulatory promoter suggesting that activation of this promoter during stress is crucial for HSV-1 reactivation from latency. These studies also suggest that common factors are needed for BHV-1 and HSV-1 reactivation from latency in sensory neurons. 4. We found that two BHV-1 regulatory proteins (VP16 and bICP0) are expressed in many neurons during the early stages of reactivation from latency. These studies indicate that VP16 and bICP0 are important for reactivation from latency.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: Sinani, D., L. Frizzo da Silva, and C. Jones. 2013. A bovine herpesvirus 1 protein expressed in latently infected neurons (ORF2) promotes neurite sprouting in the presence of activated Notch1 or Notch3. J of Virology, 87:1183-1192.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Pittayakhajonwut, D., D. Sinani, and C. Jones. 2013. A protein (ORF2) encoded by the latency related gene of bovine herpesvirus 1 interacts with DNA. J of Virology, 87: 5943-5501.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Frizzo da Silva, L. I. Kook, A. Doster, and C. Jones. Bovine herpesvirus 1 regulatory proteins, bICP0 and VP16, are readily detected in trigeminal ganglionic neurons expressing the glucocorticoid receptor during the early stages of reactivation from latency. J of Virology, 87: 11214-11222.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Devis Sinani, Ethan Cordes, Aspen Workman, Prasanth Thunuguntia, and Clinton Jones. 2013. Stress-induced cellular transcription factors expressed in trigeminal ganglionic neurons stimulate the herpes simplex virus type 1 (HSV-1) infected cell protein 0 (ICP0) promoter. J of Virology, 87:1183-1192.
• Type: Book Chapters Status: Published Year Published: 2013 Citation: Jones, C. 2013. Bovine herpesvirus 1 (BHV-1) and herpes simplex virus type 1 (HSV-1) promote survival of latently infected sensory neurons, in part by inhibiting apoptosis. J of Cell Death, 6:1-16.

Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: We identified ten cellular transcription factors that are induced during bovine herpesvirus 1 (BHV-1) reactivation from latency. These cellular transcription factors were shown to induce productive infection and to activate certain BHV-1 transcriptional promoters. We believe these cellular transcripts are important for inducing reactivation from latency. A viral protein (ORF2) that is expressed in latently infected sensory neurons was shown to interact with Notch1 and Notch3, which are two cellular transcription factors that regulate neuronal development and activate viral transcription. Recent studies demonstrated that ORF2 induces neurite sprouting in the presence of Notch1 or Notch3. This may be important for live long latency because Notch family members interfere with axonal repair, and this leads to neuronal degeneration. Neurite sprouting is a precursor for axaonal repair. Finally, two viral micro-RNAs that are expressed during latency were shown to interfere with cell death, which we believe helps to promote neuronal survival. Since BHV-1 is an important cofactor in the development of bovine respiratory disease complex, these studies shed light on the mechanism by which BHV-1 contributes to this poly-microbial disease. These studies were discussed at the International Herpesvirus meeting and at two seminars that I presented at MS State University and the Brazilian Virology meeting. PARTICIPANTS: Graduate Students involved in this work include: Devis Sinani, Aspen Workman, Leticia Frizzo da Silva, and Natasha Gadreault. Collaborations include Shafiqul Chowdhury (LSU Vet School), scientists at Boehringer Ingelheim, and Novartus. TARGET AUDIENCES: Cattle producers, veterinarians, Virologists, and herpes-virologists. I gave lectures at producer meetings (in person and via webinars) and the International Herpesvirus meeting to update these individuals about this topic. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
These studies have provide novel insight into how the BHV-1 latency related gene controls latency in sensory neurons. These studies have led to developing a novel modified live vaccine. we are also working with industry to pursue this idea and the role that BHV-1 plays in reproductive problems in the cattle industry.

Publications

• 1. Frizzo da Silva, L. and C. Jones. 2011. Infection of cultured bovine cells with bovine herpesvirus 1 (BHV-1) or Sendai virus induces different beta interferon subtypes. Virus Research, 157: 54-60. 2. Workman, A. and C. Jones. 2011. Analysis of the cell cycle regulatory protein (E2F1) after infection of cultured cells with bovine herpesvirus 1 (BHV-1) or herpes simplex virus type 1 (HSV-1). Virus Research 160: 66-73. 3. Frizzo da Silva, L.F., N. Gaudreault, and C. Jones. 2011. Cytoplasmic localized infected cell protein 0 (bICP0) encoded by bovine herpesvirus 1 inhibits beta interferon promoter activity and reduces IRF3 (interferon response factor 3) protein levels. Virus Research 169:143-149. 4. Chowdhury, S.I., M.C.S. Brum, C. Coats, A. Doster, H. Wei, C. Jones. 2011. Bovine Herpesvirus Type 1 envelope protein Us9 acidic domain is crucial for anterograde axonal transport. Vet Micro 28:270-279. 5. Sinani, D. and C. Jones. 2011. Localization of sequences in a protein encoded by the latency related gene of bovine herpesvirus 1 (ORF2) that inhibits apoptosis and interferes with Notch1 mediated trans-activation of the bICP0 promoter. J Virol 85: 12124-12133. 6. Da Silva, L.F. and C. Jones. 2012. Two micro-RNAs encoded within the BHV-1 latency related (LR) gene promote cell survival by interacting with RIG-I and stimulating nuclear factor-kappa B (NF-kB) dependent transcription and beta-interferon signaling pathways. J Virol, 86:1670-1682. 7. Workman, A., J. Eudy, L. Smith, L. Frizzo da Silva, D. Sinani, H. Bricker, E. Cook, A. Doster, and C. Jones. 2012. Cellular transcription factors induced in trigeminal ganglia during dexamethasone-induced reactivation from latency stimulate bovine herpesvirus 1 productive infection and certain viral promoters. J Virol 86: 2459-2473. 8. Frizzo da Silva, L., D. Sinani, and C. Jones. 2012. The ICP27 protein encoded by bovine herpesvirus type 1 (bICP27) interferes with promoter activity of the bovine genes encoding beta interferon 1 (IFN-β1) and IFN-β3. Virus Research, 169:162-168.

Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: Latency of alpha-herpesvirinae subfamily members is the focus of my research program. We are studying two alpha-herpesvirinae subfamily members, Bovine Herpes Virus Type 1 (BHV-1) and Herpes Simplex Virus Type 1 (HSV-1), to identify common signaling pathways that regulate the latency-reactivation cycle. BHV-1 is a significant viral pathogen of cattle because it causes respiratory disease, abortion, occasionally encephalitis, and is a causative agent of Bovine Respiratory Complex, which costs the cattle industry more than $1 billion/year in losses/ year. In contrast to the 70-80 viral genes expressed in epithelial cells, viral gene expression is restricted in latently infected neurons. The BHV-1 latency related (LR) gene and ORF-E are the known viral genes that are abundantly expressed in latently infected neurons. During the last year, we found that a protein encoded by the LR gene (ORF2) interacts with two cellular transcription factors (ORF2) that stimulate viral gene expression and productive infection. We also mapped functional domains in ORF2 that are necessary for inhibiting Notch functions and inhibiting apoptosis. Two micro-RNAs that are encoded by the LR gene were found to enhance cell survival by interacting with a RNA sensor (RIG-I). The interaction between RIG-I and the micro-RNAs also led to increased interferon, which we believe is important for maintaining latency. Studies were also performed to identify cellular transcription factors that induce reactivation from latency following dexamethasone treatment. We have identified 8 cellular transcription factors that are induced during dexamethasone induced reactivation from latency, which stimulate productive infection and viral gene expression. PARTICIPANTS: Graduate students who worked on this project are as follows: Aspen Workman, Devis Sinnani, Leticia Frizzo da Silva, and Natasha Gadreault. We have also collaborated with Dr. Jim Eudy who is at UNMC on some of these studies. TARGET AUDIENCES: Cattle producers, veterinarians, scientists, and virologists. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Understanding how the LR gene regulated latency in sensory neurons is important because the latency-reactivation cycle complicates development of BHV-1 vaccines. Since all of the commercially available modified live vaccines reactivate from latency, it is imperative to understand how reactivation from latency occurs. We have made a novel discovery that cellular factors initiate lytic cycle viral gene expression during the early phases of reactivation from latency. We believe this new knowledge will guide us to develop novel strategies for generating a superior modified live vaccine.

Publications

• Workman, A., D. Sinani, D. Pittayakhajonwut, and C. Jones. 2011. A Protein (ORF2) Encoded by the Latency Related Gene of Bovine Herpesvirus 1 Interacts with Notch1 and Notch3. J Virology, 85: 2536-2546. Sinani, D. and C. Jones. 2011. Localization of sequences in a protein encoded by the latency related gene of bovine herpesvirus 1 (ORF2) that inhibits apoptosis and interferes with Notch1 mediated trans-activation of the bICP0 promoter. J Virol 85: 12124-12133. Jones, C., L.F. da Silva, and D. Sinani. 2011. Regulation of the latency-reactivation cycle by products encoded by the bovine herpesvirus 1 (BHV-1) latency-related gene. J Neurovirology, DOI 10.1007/s13365-011-0060-3 Da Silva, L.F. and C. Jones. Two micro-RNAs encoded within the BHV-1 latency related (LR) gene promote cell survival by interacting with RIG-I and stimulating nuclear factor-kappa B (NF-kB) dependent transcription and beta-interferon signaling pathways. J Virol, IN PRESS 121. Workman, A., J. Eudy, L. Smith, L. Frizzo da Silva, D. Sinani, H. Bricker, E. Cook, A. Doster, and C. Jones. Cellular transcription factors induced in trigeminal ganglia during dexamethasone-induced reactivation from latency stimulate bovine herpesvirus 1 productive infection and certain viral promoters. IN PRESS, J Virol.

Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: Like other members of this subfamily, a latent infection is established in sensory neurons following acute infection. However, the virus can reactivate and spread to other cattle. Reactivation from latency is the mechanism by which the virus survives in nature and is thus an important property of pathogenesis. During a latent infection, the viral latency related RNA (LR-RNA) is abundantly expressed. Expression of LR proteins is necessary for reactivation from latency and protecting infected neurons from apoptosis. We have demonstrated that a LR encoded protein (ORF-2) or ORF-2 fusion proteins encoded by alternatively spliced LR transcripts inhibit apoptosis in mouse neuroblastoma cells (neuro-2A). Frame shift mutants of ORF-2 do not inhibit apoptosis suggesting that protein expression, not LR-RNA expression, mediates the anti-apoptosis activity of the LR gene in transfected neuro-2A cells. Our results focused on the latency project are summarized below. PARTICIPANTS: Aspen Workman, graduate student. Devis Sinnani, graduate student. Tareq Jaber, graduate student. TARGET AUDIENCES: Herpesvirus scientists, cattle producers, stress biologists, and biologists. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Yeast two-hybrid analysis has revealed that ORF-2 sequences specifically interact with two cellular proteins that regulate cell survival and differentiation (Notch 1 and Notch 3). We have confirmed that ORF2 interacts with Notch 1 and 3 by confocal microscopy and pull-down assays. Interestingly, ORF2 inhibits the ability of Notch 1 to trans-activate promoters. A manuscript describing these studies is in press. We have also identified two viral encoded micro-RNA that inhibit bICP0 protein expression. A manuscript describing these studies was published this year. Dexamethasone consistently initiates reactivation from latency. Wild type BHV-1, but not the LR mutant virus, express all viral genes during reactivation from latency. A manuscript describing these studies was published recently. C/EBP-alpha is induced during reactivation from latency. C/EBP-alpha trans-activates viral promoters and is induced by dexamethasone. In a collaborative study, we demonstrated that BHV-1 glycoprotein (g) was necessary for neuronal transport and reactivation from latency. A study describing this study was published in 2009. A collaborative study was also performed with Pfizer to analyze BHV-1 strains identified during vaccine breaks. These studies provided evidence that these novel strains were not identical to vaccine strains, but they had enhanced virulence. A manuscript describing these studies was recently published.

Publications

• Jones, C. 2009. Regulation of Innate Immune Responses by Bovine Herpesvirus 1 and Infected Cell Protein 0 (bICP0). Viruses 1:255-275.
• Jaber, T., A. Workman, and C. Jones. 2010. Small non-coding RNAs encoded within the bovine herpesvirus 1 latency related gene can reduce steady state levels of infected cell protein 0 (bICP0). J Virology, 84: 6297-6307.
• Workman, A. and C. Jones. 2010. Bovine herpesvirus 1 (BoHV1) productive infection and bICP0 early promoter activity are stimulated by E2F1. J Virology, 84: 6308-6317.
• Workman, A., D. Sinani, D. Pittayakhajonwut, and C. Jones. 2010. A Protein (ORF2) Encoded by the Latency Related Gene of Bovine Herpesvirus 1 Interacts with Notch1 and Notch3. IN PRESS, J Virol.
• Chowdhury, S. and C. Jones. 2010. Bovine herpesvirus type 1 (BHV-1) is an important cofactor in the bovine respiratory disease complex. Veterinary Clinics of North America, Food Animal Practice, Bovine Respiratory Disease, eds V.L. Cooper and B. Broderson, 303-321.

Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: BHV-1 is a significant viral pathogen of cattle that can induce respiratory disease, abortion, or occasionally encephalitis. BHV-1 is also frequently found in buffalo, which is a growing food animal source in the US. BHV-1 is a important causative agent of "Shipping Fever" or Bovine Respiratory Disease Complex. As a consequence of the pathogenic potential of BHV-1, the cattle industry suffers more than $1 billion/year in losses. BHV-1 typically initiates infection in mucosal epithelial surfaces located in the eyes, nose, mouth, upper respiratory tract, or genital tract. Extensive viral gene expression occurs, virus is shed, and clinical symptoms are apparent. Virus then enters the peripheral nervous system, where it establishes a latent infection in sensory neurons. Viral DNA can persist in a latent state for the lifetime of the infected host or it can periodically reactivate. In contrast to the 70-80 viral genes expressed in epithelial cells, only one small region of the viral genome is transcriptionally active in latently infected neurons. This region is designated the latency related (LR) gene. Expression of LR proteins is necessary for reactivation from latency and protecting infected neurons from apoptosis. We have demonstrated that a LR encoded protein (ORF-2) or ORF-2 fusion proteins encoded by alternatively spliced LR transcripts inhibit cold shock or Fas ligand induced apoptosis in mouse neuroblastoma cells (neuro-2A). Frame shift mutants of ORF-2 do not inhibit apoptosis suggesting that protein expression, not LR-RNA expression, mediates the anti-apoptosis activity of the LR gene in transfected neuro-2A cells. Yeast two-hybrid analysis has revealed that ORF-2 sequences specifically interact with two cellular proteins that regulate cell survival and differentiation (Notch 1 and Notch 3). We have also identified sequences in the LR gene that inhibit bICP0 expression. It appears that a viral encoded micro-RNA inhibits bICP0 expression. PARTICIPANTS: Several graduate students worked on this project as well as a postdoctoral fellow. We have collaborated with a faculty member in the Diagnostic Center at UNL, and a faculty member at LSU Vet School. TARGET AUDIENCES: Pharmaceutical companies, other herpes virologists, ranchers, veterinarians, and neuro-biologists. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The latency-reactivation cycle is crucial for virus transmission in the field. Understanding the mechanism by which the LR gene regulates the latency-reactivation cycle will provide novel strategies for developing a superior modified live vaccine.

Publications

• Perez, S., F. Meyer, K. Saira, A. Doster, and C. Jones. 2008. Premature expression of the latency-related (LR) RNA correlates with higher levels of beta-interferon RNA expression in productively infected cells. J. Gen. Virology 89: 1338 - 1345.
• Geiser, V., S. Rose, and C. Jones. 2008. Bovine herpesvirus 1 induces cell death by a cell type dependent fashion. Microbial Pathogenesis 44:459-466.
• Shen, W. and C. Jones. 2008. Open reading frame 2 encoded by the latency related gene of bovine herpesvirus 1 has anti-apoptosis activity in transiently transfected neuroblastoma cells. J. Virology, 82:10940-10945.
• Meyer, F. and C. Jones. 2009. C/EBP-alpha cooperates with bTIF to activate the bovine herpesvirus 1 immediate early transcription unit 1 promoter. J. Neurovirology 15:123-130.
• Workman, A., S. Perez, A. Doster, and C. Jones. 2009. Dexamethasone treatment of calves latently infected with bovine herpesvirus 1 (BHV-1) leads to activation of the bICP0 early promoter, in part by the cellular transcription factor C/EBP-alpha. J. Virology, 83:8800-8809.
• Ellis, J., S. Gow, N. Goji, C. Jones, A. Workman, G. Henderson, G. Alaniz, and T. Meinert. 2009. Efficacy of a combination viral vaccine in protecting cattle from experimental infection with bovine herpesviruses-1 isolated from recent vaccine breaks. J. of American Veterinary Medical Association, 235:563-572.
• Jones, C. and S. Chowdhury. 2008. A review of the biology of bovine herpesvirus type 1 (BHV-1), its role as a cofactor in the bovine respiratory disease complex, and development of improved vaccines. Adv in Animal Health, 8:187-205.