Changes in the bovine herpesvirus 1 genome during acute infection, after reactivation from latency, and after superinfection in the host animal

ABSTRACT: A glycoprotein E-deleted Brazilian bovine herpesvirus 1 (BoHV-1gEΔ) was tested regarding to safety and immunogenicity. Intramuscular inoculation of young calves with a high virus dose did not result in clinical signs or virus shedding during acute infection or after dexamethasone administration. Calves vaccinated once IM (group I) or subcutaneously (group II) with live BoHV-1gEΔ or twice with inactivated virus plus aluminum hydroxide (group IV) or Montanide™ (group V) developed VN titers of 2 to 8 (GMT:2); 2 to 4 (GMT:1.65); 2 to 16 (GMT:2.45) and 2 to 128 (GMT:3.9), respectively. All BoHV-1gEΔ vaccinated calves remained negative in an anti-gE ELISA. Lastly, six young calves vaccinated with live BoHV-1gEΔ and subsequently challenged with a virulent BoHV-1 strain shed less virus and developed only mild and transient nasal signs comparing to unvaccinated calves. Thus, the recombinant BoHV-1gEΔ is safe and immunogenic for calves and allows for serological differentiation by a gE-ELISA test.

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Infection of Cattle with a Bovine Herpesvirus 1 Strain That Contains a Mutation in the Latency-Related Gene Leads to Increased Apoptosis in Trigeminal Ganglia during the Transition from Acute Infection to Latency

Journal of Virology ◽

10.1128/jvi.77.8.4848-4857.2003 ◽

2003 ◽

Vol 77 (8) ◽

pp. 4848-4857 ◽

Cited By ~ 54

Author(s):

Luciane Lovato ◽

Melissa Inman ◽

Gail Henderson ◽

Alan Doster ◽

Clinton Jones

Keyword(s):

Nasal Cavity ◽

Acute Infection ◽

Bovine Herpesvirus ◽

Pcr Analysis ◽

Viral Gene ◽

Trigeminal Ganglia ◽

Dexamethasone Treatment ◽

Bovine Herpesvirus 1 ◽

Latently Infected ◽

Herpesvirus 1

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.

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The Latency-Related Gene of Bovine Herpesvirus 1 Inhibits Programmed Cell Death

Journal of Virology ◽

10.1128/jvi.73.12.9734-9740.1999 ◽

1999 ◽

Vol 73 (12) ◽

pp. 9734-9740 ◽

Cited By ~ 59

Author(s):

Janice Ciacci-Zanella ◽

Melissa Stone ◽

Gail Henderson ◽

Clinton Jones

Keyword(s):

Gene Expression ◽

Cell Death ◽

Programmed Cell Death ◽

Acute Infection ◽

Bovine Herpesvirus ◽

Productive Infection ◽

Gene Products ◽

Bovine Herpesvirus 1 ◽

Transfected Cells ◽

Herpesvirus 1

ABSTRACT Although viral gene expression occurs in the peripheral nervous system during acute infection, bovine herpesvirus 1 (BHV-1) gene expression is extinguished, many neurons survive, and latency ensues. 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. Devireddy and C. Jones, J. Virol. 72:7294–7301, 1998). A subset of neurons express a protein encoded by the LR gene and the LR protein (LRP) is associated with cyclin-dependent kinase 2 (Cdk2)/cyclin complexes during productive infection (Y. Jiang, A. Hossain, M. T. Winkler, T. Holt, A. Doster, and C. Jones, J. Virol. 72:8133–8142, 1998). LR gene products inhibit cell cycle progression, perhaps as a result of LRP interacting with Cdk2/cyclin complexes. During acute infection, expression of cyclin A occurs in trigeminal ganglionic neurons (L. M. Schang, A. Hossain, and C. Jones, J. Virol. 70:3807–3814, 1996). Inappropriate expression of G1- and S-phase cyclins can initiate programmed cell death (PCD), apoptosis, in neurons, suggesting that LR gene products inhibit PCD. To test this hypothesis, we modified an assay to measure PCD frequency in transiently transfected cells. C6-ceramide, fumonisin B1(FB1), or etoposide was used to initiate PCD following transfection of cells with plasmids expressing LR gene products and the β-galactosidase gene. Transfected cells that survived were quantified by counting β-galactosidase-positive cells. Plasmids that expressed LR gene products promoted survival of monkey kidney (CV-1), human lung (IMR-90), or mouse neuroblastoma (neuro-2A) cells after induction of PCD. Plasmids with termination codons at the beginning of LR open reading frames or deletion of sequences that mediate splicing of LR RNA did not promote cell survival following PCD induction. We hypothesize that LR gene products play a role in promoting survival of postmitotic neurons during acute infection or reactivation.

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Bovine Herpesvirus 1 Can Infect CD4+ T Lymphocytes and Induce Programmed Cell Death during Acute Infection of Cattle

Journal of Virology ◽

10.1128/jvi.73.10.8657-8668.1999 ◽

1999 ◽

Vol 73 (10) ◽

pp. 8657-8668 ◽

Cited By ~ 82

Author(s):

M. T. C. Winkler ◽

A. Doster ◽

C. Jones

Keyword(s):

T Cells ◽

T Lymphocytes ◽

Acute Infection ◽

Bovine Herpesvirus ◽

Cell Mediated Immunity ◽

Apoptotic Cells ◽

Bovine Herpesvirus 1 ◽

Herpesvirus 1 ◽

Pharyngeal Tonsil

ABSTRACT Acute infection of cattle with bovine herpesvirus 1 (BHV-1) represses cell-mediated immunity, which can lead to secondary bacterial infections. Since BHV-1 can induce apoptosis of cultured lymphocytes, we hypothesized that these virus-host interactions occur in cattle. To test this hypothesis, we analyzed lymph nodes and peripheral blood mononuclear cells (PBMC) after calves were infected with BHV-1. In situ terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) staining of lymphoid tissues (pharyngeal tonsil, cervical, retropharyngeal, and inguinal) was used to detect apoptotic cells. Calves infected with BHV-1 for 7 days revealed increased apoptotic cells near the corticomedullary junction in lymphoid follicles and in the subcapsular region. Increased frequency of apoptotic cells was also observed in the mucosa-associated lymphoid tissue lining the trachea and turbinate. Immunohistochemistry of consecutive sections from pharyngeal tonsil revealed that CD2+ T lymphocytes were positive for the BHV-1 envelope glycoprotein gD. The location of these CD2+ T lymphocytes in the germinal center suggested that they were CD4+ T cells. Electron microscopy and TUNEL also revealed apoptotic and herpesvirus-infected lymphocytes from this area. Fluorescence-activated cell sorting analyses demonstrated that CD4+ and CD8+ T cells decreased in lymph nodes and PBMC after infection. The decrease in CD4+ T cells correlated with an increase in apoptosis. CD4+ but not CD8+ lymphocytes were infected by BHV-1 as judged by in situ hybridization and PCR, respectively. Immediate-early (bovine ICP0) and early (ribonucleotide reductase) transcripts were detected in PBMC and CD4+ lymphocytes prepared from infected calves. In contrast, a late transcript (glycoprotein C) was not consistently detected suggesting productive infection was not efficient. Taken together, these results indicate that BHV-1 can infect CD4+T cells in cattle, leading to apoptosis and suppression of cell-mediated immunity.

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A Mutation in the Latency-Related Gene of Bovine Herpesvirus 1 Leads to Impaired Ocular Shedding in Acutely Infected Calves

Journal of Virology ◽

10.1128/jvi.75.18.8507-8515.2001 ◽

2001 ◽

Vol 75 (18) ◽

pp. 8507-8515 ◽

Cited By ~ 56

Author(s):

Melissa Inman ◽

Luciane Lovato ◽

Alan Doster ◽

Clinton Jones

Keyword(s):

Nasal Cavity ◽

Cell Cycle Progression ◽

Clinical Symptoms ◽

Acute Infection ◽

Bovine Herpesvirus ◽

Neutralizing Antibody ◽

Gene Products ◽

Viral Transcript ◽

Bovine Herpesvirus 1 ◽

Herpesvirus 1

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.

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A Mutation in the Latency-Related Gene of Bovine Herpesvirus 1 Disrupts the Latency Reactivation Cycle in Calves

Journal of Virology ◽

10.1128/jvi.76.13.6771-6779.2002 ◽

2002 ◽

Vol 76 (13) ◽

pp. 6771-6779 ◽

Cited By ~ 58

Author(s):

Melissa Inman ◽

Luciane Lovato ◽

Alan Doster ◽

Clinton Jones

Keyword(s):

Infectious Virus ◽

Acute Infection ◽

Bovine Herpesvirus ◽

Pcr Analysis ◽

Trigeminal Ganglia ◽

Viral Transcript ◽

Bovine Herpesvirus 1 ◽

Virus Shedding ◽

Latently Infected ◽

Herpesvirus 1

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.

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