Functional analysis of the latency-related gene of bovine herpesvirus types 1 and 5.

Bovine herpesvirus type 1 and type 5 (BoHV-1 and BoHV-5) are two alphaherpesviruses that affect cattle with two different syndromes. While BoHV-1 mainly produces respiratory symptoms, BoHV-5 is highly neuropathogenic and responsible for meningoencephalitis in young cattle. The latency-related (LR) gene, which is not conserved between these two herpesviruses, is the only viral gene abundantly expressed in latently infected neurons. The antiapoptotic action of this gene has been demonstrated during acute infection and reactivation from latency and seems to be mainly mediated by a LR protein (ORF-2) which is truncated in amino acid 51 in the case of BoHV-5. In this work, we show that the BoHV-5 LR gene is less efficient at cell survival and apoptosis inhibition in transient as well as in established neuronal cell lines compared to its BoHV-1 homolog. We hypothesize that the BoHV-5 LR gene may have novel functions that are lacking in the BoHV-1 LR gene and that these differences may contribute to its enhanced neuropathogenesis.

A DNA aptamer efficiently inhibits the infectivity of Bovine herpesvirus 1 by blocking viral entry.

Bovine herpesvirus 1 (BoHV-1) is an important pathogen of domestic and wild cattle responsible for major economic losses in dairy and beef industries throughout the world. Inhibition of viral entry plays a crucial role in the control of BoHV-1 infection and aptamers have been reported to inhibit viral replication. In this study, nine DNA aptamers that target BoHV-1 were generated using systemic evolution of ligands by exponential enrichment. Of the nine candidates, aptamer IBRV-A4 exhibited the highest affinity and specificity for BoHV-1, which bound to BoHV-1 with a Kd value of 3.519 nM and demonstrated the greatest virus binding as shown by fluorescence imaging. The neutralizing ability of aptamer IBRV-A4 was determined using neutralization assays and real time PCR in BoHV-1 infected Madin-darby bovine kidney cells. Virus titration, immunofluorescence and confocal laser scanning microscopy showed virus replication significantly decreased when aptamer IBRV-A4 was added to BoHV-1 infected MDBK cells at 0 and 0.5 hours post-infection, whereas no change was seen when IBRV-A4 was added 2 hours post-infection. This concludes that aptamer IBRV-A4 efficiently inhibits viral entry of BoHV-1 in MDBK cells and is therefore a novel tool for diagnosis and treatment of BoHV-1 infection in cattle.

BHV-1 induced oxidative stress contributes to mitochondrial dysfunction in MDBK cells.

The levels of cellular reactive oxygen species (ROS) and ATP as well as the mitochondrial membrane potential (MMP) in response to bovine herpesvirus 1 (BHV-1) infection of MDBK cells were measured, respectively. BHV-1 infection increased ROS production which depended on viral entry, and de novo protein expression and/or DNA replication. Vice versa, excessive ROS was required for efficient viral replication. Levels of both ATP and MMP were significantly decreased after BHV-1 infection. Interestingly, the loss of MMP was ameliorated by ROS depression. Collectively, ROS dependent mitochondrial damage and ultimately disruption of energy metabolism (ATP depletion) are a potential pathogenic mechanism for BHV-1 infection.

Evaluation of total oxidative stress and total antioxidant status in cows with natural bovine herpesvirus-1 infection.

Viruses, including herpes viruses, can alter oxidative balance by either increasing the formation of free radicals or inhibiting synthesis of enzymes involved in oxidative defense within host cells. This study examined the occurrence of oxidative and antioxidative balance in cows naturally infected with bovine herpesvirus type 1 (BHV-1) under field conditions. Clinical history indicated that cows had been sick and showed mild to severe respiratory signs, characterized by dullness, coughing, and lacrimation, and a high febrile response. All samples obtained from the infected animals during clinical examination were confirmed as positive for bovine herpesvirus type 1 by PCR. Control cows showed no clinical abnormalities and PCR results were negative. Total antioxidative status, total oxidant status, oxidative stress index, and some biochemical parameters were measured. The level of total antioxidative status was significantly lower in infected animals, compared with the healthy control group (P = 0.025). However, there was no statistically significant difference between the 2 groups for total oxidant status and oxidative stress index levels. Furthermore, there was a significant decrease in the infected groups, with respect to concentrations of alkaline phosphatase, alanine transferase, γ glutamyl transferase, monocyte, and erythrocyte (P < 0.05). On the other hand, aspartate aminotransferase and creatinine kinase concentrations significantly increased in the cows infected with BHV-1. In conclusion, the data obtained hereby explained that animals with infected BHV-1 seemed to have more oxidative stress and low antioxidant defense. Moreover, future research conductance is needed on antioxidative and oxidative balance to understand pathophysiology of BHV-1 infections.

The zinc RING finger of bovine herpesvirus 1-encoded bICP0 protein is crucial for viral replication and virulence.

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.

Effect of dietary lipoic acid on metabolic hormones and acute-phase proteins during challenge with infectious bovine rhinotracheitis virus in cattle.

OBJECTIVE: To determine the effect of dietary supplemental lipoic acid (LA) on serum concentrations of metabolic hormones and acute-phase proteins of steers challenged with infectious bovine rhinotracheitis virus (IBRV). ANIMALS: 32 steers. PROCEDURES: Steers were randomly assigned to 4 treatments: negative control (NC; no LA, no IBRV challenge), control (CON; no LA, IBRV challenge), 16 mg of LA/kg of body weight (BW)/d plus IBRV challenge (LA16), and 32 mg of LA/kg of BW/d plus IBRV challenge (LA32). Following a 21-day adaptation period, CON, LA16, and LA32 steers received IBRV (2 mL/nostril [day 0]); NC steers received saline (0.9% NaCl) solution. Blood samples, nasal swab specimens, BW, and rectal temperatures were obtained 0, 1, 3, 5, 7, 14, and 21 days after challenge. Serum was analyzed for concentrations of haptoglobin, amyloid-A, leptin, and anti-IBRV antibodies. RESULTS: Steers fed LA32 began gaining BW by day 7, whereas BW of CON and LA16 steers declined. Serum haptoglobin concentration of LA32 steers was lower than that of CON and LA16 steers on day 7. Serum neutralization titers for 30 of 32 steers were negative for anti-IBRV antibodies before challenge; however, all steers (including NCs) had antibodies on day 21. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that LA supplementation augmented certain aspects of the immune response; LA32 steers had a more rapid recovery from IBRV viral challenge than did others.

Ectodysplasin-1 deficiency in a German Holstein bull associated with loss of respiratory mucous glands and chronic rhinotracheitis.

A 2-year-old German Holstein bull was identified as a carrier of a mutation within the X-chromosomal ED1 gene, which encodes a TNF-related signalling molecule mainly involved in ectodermal development. The clinicopathological appearance was associated with hypotrichosis, hypodontia, and a reduced number of eccrine glands, in addition to chronic rhinotracheitis and partial squamous metaplasia. Furthermore, for the first time in an ED1-deficient animal, a complete lack of respiratory mucous glands was observed. This suggests that the ED1 gene plays a role in the development of mucous glands, the absence of which resembles a feature of X-linked anhidrotic ectodermal dysplasia (ED1) in human patients.

Effects of copper deficiency and copper deficiency coupled with high dietary iron or molybdenum on phagocytic cell function and response of calves to a respiratory disease challenge.

A study was conducted to determine the effects of supplementing a diet marginally deficient in copper (Cu) with iron (Fe), molybdenum (Mo), or Cu on phagocytic cell function and disease resistance of calves. Thirty-one calves were born to heifers fed a corn silage-based diet containing 4.5 mg of Cu/kg. Treatments consisted of 1) control (CON; no supplemental Cu, Fe, or Mo), 2) 600 mg of Fe added/kg (FE), 3) 5 mg of Mo added/kg (MO), or 4) 10 mg of Cu added/kg of DM (CU). Activity of superoxide dismutase was lower (P < .06) in neutrophils from MO vs CON or CU calves at 170 d of age. bactericidal activity of neutrophils from MO calves tended (P = .15) to be lower compared with those from CU calves at 70 d of age. Calves were inoculated intranasally with live infectious bovine rhinotracheitis virus (IBRV) 2 d after weaning, followed by intratracheal administration of Pasteurella hemolytica 5 d later. Iron- and Cu-supplemented calves exhibited higher (P < .01) body temperatures and lower (P < .06) feed intakes following IBRV inoculation compared with CON and MO calves. Copper-supplemented calves had higher levels of plasma tumor necrosis factor (TNF) than MO calves at weaning (P < .05) and tended to have higher plasma TNF (P = .11) than FE and MO calves 5 d after IBRV inoculation. These data indicate that dietary levels of Mo and Cu can affect body temperature and feed intake responses to disease by affecting TNF and perhaps other cytokines.

Recovery rate and plasma zinc and copper concentrations of steer calves fed organic and inorganic zinc and manganese sources with or without injectable copper and challenged with infectious bovine rhinotracheitis virus.

Two experiments were conducted using feeder calves in a randomized block design to determine the effects of organic and inorganic sources of Zn and Mn fed pre- and posttransit (Exp. 1 and 2) with or without injectable Cu (Exp. 2) on DMI, rectal temperature, BW changes, and plasma Zn and Cu concentrations of feedlot steers challenged with infectious bovine rhinotracheitis virus (IBRV). In Exp. 1, before weaning, all steers and their dams received the following free-choice mineral supplements: 1) control (no supplemental Zn or Mn), 2) ZnO+MnO (ZnMnO), and 3) Zn methionine+Mn methionine (ZnMnMet). In Exp. 2, 18 d before weaning and shipping, steers were allotted into two groups (22 steers/group) and fed 225 mg of Zn.steer-1 x d-1 in .9 kg of ground corn as ZnO or ZnMet. Half of the steers from each group were injected (s.c.) with 120 mg of Cu from Cu glycinate. Steers (Exp. 1 and 2) were weaned and shipped approximately 2,500 km to the feedlot, where they received the same supplements in the form of a complete diet for 34 d, during which time calves recovered from the stress due to shipment. All steers were initially sero-negative to IBRV. On d 34 (d 0 of IBRV), all steers were challenged with IBRV and DMI, rectal temperature, and BW change were monitored for 28 d.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunological and biochemical investigations of the infectious bovine rhinotracheitis. III. Alterations in pulmonary surfactant lipids.

The effect of experimental bovine herpes virus (BHV) type I rhinotracheitis on the surfactant system phospholipids in calves was examined. A stimulated exocytosis of pulmonary surfactant phospholipids in the acute phase of the disease was documented biochemically and ultrastructurally. The data presented were assumed as an evidence of the involvement of pulmonary surfactant in lung defense.

Changes in phospholipids of alveolar lining material in calves after aerosol exposure to bovine herpesvirus-1 or parainfluenza-3 virus.

Pulmonary lavage samples were collected from 90- to 130-day-old calves before and 6 days after aerosol inoculation with bovine herpesvirus-1 (BHV-1) or parainfluenza-3 (PI3) virus. Alveolar lining material was separated from lavage fluids by high-speed centrifugation and phospholipids were extracted from alveolar lining material and analyzed by high-performance liquid chromatography. Phosphatidylcholine and phosphatidylethanolamine were 74.2 +/- 6.5% and 13.3 +/- 2.8%, respectively, of the total phospholipid content in the surfactant obtained from calves before virus inoculation. Other phospholipids were represented by substantially lower percentages. Infection with either of the 2 viruses caused a significant (P less than or equal to 0.05) decrease in the percentage of phosphatidylcholine to 66.0 +/- 8.0% and 65.1 +/- 10.8% in the calves inoculated with BHV-1 and PI3 virus, respectively. A significant (P less than or equal to 0.05) increase in the percentage of phosphatidylethanolamine to 18.1 +/- 2.2% and 17.8 +/- 4.5% developed in calves inoculated with BHV-1 and PI3 virus, respectively. Infection with BHV-1 also induced an increase (not significant) in the percentage of phosphatidylinositol from 5.5 +/- 2.8% to 7.8 +/- 2.2%. A similar, but not significant, increase in the percentage of phosphatidylinositol was also seen in the calves inoculated with PI3 virus. Less substantial changes in the percentage of other phospholipids were detected after virus infection.

Nitrogen kinetics of infectious bovine rhinotracheitis-stressed calves.

Eight crossbred feeder steers were used in two consecutive N balance studies to investigate the effects of infectious bovine rhinotracheitis virus (IBRV) on N kinetics. Balance Study 1, which followed a 10-d acclimation phase, consisted of 7 d of sample collection referred to as the healthy phase (d -7 to d -1). Study 2, the IBRV-infected phase, began 2 d after a nasal IBRV challenge and continued for 6 d (d 2 to d 8). A stable isotope, [15N]-glycine, was used to determine N kinetics in both studies. Steers weighed 203 kg at the beginning of the study, 208 kg at IBRV infection and 194 kg at the end of study. Infection with IBRV increased (P less than .05) urinary N excretion from 17.9 to 31.5 g/d. Daily N balance was reduced (P less than .05) during infection from 21.2 to -3.3 g/d. Total serum proteins increased (P less than .05) during infection from 6.6 to 7.1 g/100 ml, the increase being predominantly in the alpha and gamma globulin fractions. Blood urea-N increased (P less than .05) during infection from 6.5 to 12.9 mg/100 ml. The urine excretion curve of the stable isotope and the N balance data indicated that IBRV infection increased N turnover and altered tissue utilization of N.

[Evaluation of synthetic activity of lymphocyte nucleoli and phagocytic activity in peripheral blood leukocytes in heifers after infection with infectious bovine rhinotracheitis virus]. / Hodnocení syntetické aktivity jadérka lymfocytú a fagocytární aktivity leukocytú periferní krve u skotu po infekci virem IBR.

The biosynthetic activity of the nucleoli of peripheral blood lymphocytes in calves, and the proportion of lymphocytes with the micronucleoli, with compact and ring-shaped nucleoli, were evaluated after experimental intranasal and intratracheal infections with the infectious bovine rhinotracheitis (IBR) virus. IBR virus belongs to the group of herpes viruses, the control of which is dominated by cellular immunity. The results are compared with the values for phagocytizing neutrophils, phagocytic activity and phagocytic index of the leucocytes of peripheral blood and with some basic haematological data.

Nitrogen metabolism of calves inoculated with bovine adenovirus-3 or with infectious bovine rhinotracheitis virus.

Beef calves were inoculated with bovine adenovirus-3 or infectious bovine rhinotracheitis virus. After inoculation, plasma fibrinogen increased, serum phosphorus decreased, and nitrogen and phosphorus digestibility decreased compared with preinoculation values. Urinary N excretion increased when calves developed rectal temperatures greater than 39.7 C. Results indicated that clinical infection of calves with infectious bovine rhinotracheitis virus increases urinary N excretion and reduces N and phosphorus balance, and that clinical and subclinical infections with either virus reduce dietary N digestibility.

Interferon production and replication of infectious bovine rhinotracheitis virus in fasted calves.

Three groups of calves were inoculated intranasally with infectious bovine rhinotracheitis (IBR) virus, to determine any effects of a 3-day fast on virus replication and interferon (IFN) production measured in nasal secretions (NS). One group ("fasted") was inoculated 24 h after onset of the fast and another ("refed") at the end of fasting, immediately before refeeding. A third ("control") group was inoculated but not fasted. In fasted calves, overall mean virus excretion (during the first 5 postinoculation days) did not differ from that in control calves, though average virus excretion was higher on days 3 and 4, 24 and 48 h after refeeding. In refed calves, overall mean virus excretion was lower (p less than 0.05), yet on day 5 these calves secreted two times more IFN than nonfasted calves. Analysis of the overall data (all 5 postinoculation sampling days) showed that fasted calves produced more IFN (p less than 0.05), with IFN titers sometimes exceeding 1000, than either control nonfasted calves or refed calves. We conclude that fasting enhanced the ability of calves to produce IFN, and this did not result from increased IBR virus replication.

[Infectious bovine rhinotracheitis virus (bovine herpesvirus l): biochemical aspects]. / Le virus de la rhinotrachéite infectieuse bovine (bovine herpesvirus 1): aspects biochimiques.

The biochemical characteristics of infectious bovine rhinotracheitis virus (Bovine herpes-virus 1, BHV 1) are reviewed: description of the virion particle and virus purification, analysis of structural and non structural proteins, nucleic acid properties, restriction endonuclease analysis of the viral DNA. The authors emphasize the biochemical methods able to insure a better understanding of the molecular epidemiology of BHV 1 and of its latency; also to develop biochemical weapons against the infection.

DNA of bovine herpesvirus type 1 in the trigeminal ganglia of latently infected calves.

Twelve calves infected with bovine herpesvirus type 1 (BHV-1) were killed when in a latent state of infection. Latency was verified 30 days after virus inoculation of the calves by seroconversion, absence of virus shedding, and in 2 calves, by recrudescence of the infection after they were treated with dexamethasone. By in situ hybridization techniques and autoradiography, DNA of BHV-1 was detected in 13 of 23 trigeminal ganglia of latently infected calves. Viral DNA was restricted to the nucleus of nerve cells. Single neurons harboring BHV-1 DNA were observed in 4.9% of the sections (n = 325) of the trigeminal ganglia. The results obtained correspond to those known from herpes simplex virus infections in mice. The implications for the virus-host relationship are discussed.