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.

The bovine herpesvirus 1 regulatory proteins, bICP4 and bICP22, are expressed during the escape from latency.

Following acute infection of mucosal surfaces by bovine herpesvirus 1 (BoHV-1), sensory neurons are a primary site for lifelong latency. Stress, as mimicked by the synthetic corticosteroid dexamethasone, consistently induces reactivation from latency. Two viral regulatory proteins (VP16 and bICP0) are expressed within 1 h after calves latently infected with BoHV-1 are treated with dexamethasone. Since the immediate early transcription unit 1 (IEtu1) promoter regulates both BoHV-1 infected cell protein 0 (bICP0) and bICP4 expressions, we hypothesized that the bICP4 protein is also expressed during early stages of reactivation from latency. In this study, we tested whether bICP4 and bICP22, the only other BoHV-1 protein known to be encoded by an immediate early gene, were expressed during reactivation from latency by generating peptide-specific antiserum to each protein. bICP4 and bICP22 protein expression were detected in trigeminal ganglionic (TG) neurons during early phases of dexamethasone-induced reactivation from latency, operationally defined as the escape from latency. Conversely, bICP4 and bICP22 were not readily detected in TG neurons of latently infected calves. In summary, it seems clear that all proteins encoded by known BoHV-1 IE genes (bICP4, bICP22, and bICP0) were expressed during early stages of dexamethasone-induced reactivation from latency.

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.

Comparative analysis of replication characteristics of BoHV-1 subtypes in bovine respiratory and genital mucosa explants: a phylogenetic enlightenment.

In general, members of the Alphaherpesvirinae use the epithelium of the upper respiratory and/or genital tract as a preferential site for primary replication. Bovine herpesvirus type 1 (BoHV-1) may replicate at both sites and cause two major clinical entities designated as infectious bovine rhinotracheitis (IBR) and infectious pustular vulvovaginitis/balanoposthitis (IPV/IPB) in cattle. It has been hypothesized that subtype 1.1 invades preferentially the upper respiratory mucosa whereas subtype 1.2 favors replication at the peripheral genital tract. However, some studies are in contrast with this hypothesis. A thorough study of primary replication at both mucosae could elucidate whether or not different BoHV-1 subtypes show differences in mucosa tropism. We established bovine respiratory and genital organ cultures with emphasis on maintenance of tissue morphology and viability during in vitro culture. In a next step, bovine respiratory and genital mucosa explants of the same animals were inoculated with several BoHV-1 subtypes. A quantitative analysis of viral invasion in the mucosa was performed at 0 h, 24 h, 48 h and 72 h post inoculation (pi) by measuring plaque latitude and penetration depth underneath the basement membrane. All BoHV-1 subtypes exhibited a more profound invasion capacity in respiratory tissue compared to that in genital tissue at 24 h pi. However, at 24 h pi plaque latitude was found to be larger in genital tissue compared to respiratory tissue and this for all subtypes. These similar findings among the different subtypes take the edge off the belief of the existence of specific mucosa tropisms of different BoHV-1 subtypes.

Evaluation of safety and efficacy of DNA vaccines against bovine herpesvirus-1 (BoHV-1) in calves.

Four DNA vaccines against BoHV-1 were evaluated for their efficacy in calves. Twelve animals were divided into four groups which were injected with four different DNA vaccines: pVAX-tgD (Vaccine A); pVAX-tgD co-immunised with pVAX-48CpG (Vaccine B); pVAX-UbiLacI-tgD-L (Vaccine C); pVAX-UbiLacI-tgD-L co-immunised with pVAX-48CpG (Vaccine D). Three additional calves were given the plasmid vector and served as controls. Ninety days after the first vaccination all calves were challenge infected with BoHV-1. All animals developed a severe form of infections bovine rhinotracheitis. Only the calves given the pVAX-tgD co-immunised with pVAX-48CpG (Vaccine B) developed humoral antibodies against BoHV-1 between 56 and 90 days after the first vaccination, whereas in calves of other groups and in the controls, antibodies appeared only after the infection. In the calves vaccinated with either pVAX-tgD (Vaccine A) or pVAX-tgD combined with pVAX-48CpG (Vaccine B), BoHV-1-specific IFN-γ secreting cells were detected in PBMCs 90 days after the first vaccination and their number increased after challenge exposure. In the other groups the IFN-γ secreting cells were detected after virus infection and at low values.

Bovine herpesvirus type 1 infection of bovine bronchial epithelial cells increases neutrophil adhesion and activation.

Respiratory infection of cattle with bovine herpesvirus type 1 (BHV-1) predisposes cattle to secondary pneumonia with Mannheimia haemolytica as part of the bovine respiratory disease complex (BRD). One cell type that has received limited investigation for its role in the inflammation that accompanies BRD is the respiratory epithelial cell. In the present study we investigated mechanisms by which BHV-1 infection of respiratory epithelial cells contributes to the recruitment and activation of bovine polymorphonuclear neutrophils (PMNs) in vitro. Primary cultures of bovine bronchial epithelial (BBE) cells were infected with BHV-1 and assessed for cytokine expression by real-time PCR. We found that BHV-1 infection elicits a rapid IL-1, IL-8 and TNF-alpha mRNA response by BBE cells. Bovine PMNs exhibited greater adherence to BHV-1 infected BBE cells than uninfected cells. The increased adherence was significantly reduced by the addition of an anti-IL-1beta antibody or human soluble TNF-alpha receptor (sTNF-alphaR). Pre-incubation of bovine PMNs with conditioned media from BHV-1 infected BBE cells increased PMN migration, which was inhibited by addition of an anti-IL-1beta antibody, sTNF-alphaR, or an IL-8 peptide inhibitor. Conditioned media from BHV-1 infected BBE cells activated bovine PMNs in vitro as demonstrated by PMN shape change, production of reactive oxygen species and degranulation. PMNs also exhibited increased LFA-1 expression and susceptibility to M. haemolytica LKT following incubation with BHV-1 infected BBE cell conditioned media. Our results suggest that BHV-1 infection of BBE cells triggers cytokine expression that contributes to the recruitment and activation of neutrophils, and amplifies the detrimental effects of M. haemolytica LKT.

[Pathogenesis of mixed experimental infection caused by diarrheal viruses--bovine mucosal disease and bovine infectious rhinotracheitis in calves].

The pathogenesis of mixed experimental infection caused by intranasal inoculation of seronegative calves aged 4-6 months with bovine viral diarrhea-mucosal disease (BVDMD) (cytopathogenic) and infectious bovine rhinotracheitis (BRT) viruses, was studied. Consecutive injections of viruses resulted in acute respiratory disease that was severer and accompanied by necrotic rhinotracheitis and acute catarrhal bronchopneumonia than individual injections. BVDMD virus was reisolated from the samples taken from the respiratory tract, intestine, and lymphoid system. The longer excretion of BRT virus with nasal swabs and its high concentration in the respiratory organs suggests its more potent pathogenic properties during reproduction of BVDMD virus.

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.

Modification of active immunization with live bovine herpesvirus 1 vaccine by passive viral antibody.

The study in BHV-1 naïve calves investigated the effect of intramuscularly (IM) administered BHV-1 neutralising bovine immunoglobulin on the efficacy of a live intranasally (IN) administered BHV-1 vaccine. Overall on daily basis there was between 40- and 5000-fold less vaccine virus shed by the passively immune calves compared with that shed by the naïve counterparts. The latter seroconverted to the vaccine whereas the virus neutralising (VN) antibody titres in the passively immune calves decreased after vaccination. Compared with unvaccinated naïve or passively immune calves, both groups of vaccinated calves shed significantly less challenge BHV-1 but the daily amount shed was significantly lower in vaccinated naïve calves. The latter were also significantly better protected against pyrexia following the IN BHV-1 challenge compared with vaccinated passively immune calves. Unlike vaccinated calves, clinical reaction to challenge in both unvaccinated groups also involved nasal discharge but the duration of both the nasal discharge and the severe pyrexia was significantly shorter in unvaccinated passively immune calves. Conclusions from the study are: (1) the circulating VN antibody is significantly protective against virus shedding and to alesser extent also against the febrile respiratory disease; (2) the passively immune calves are unlikely to seroconvert after an active infection and (3) the passive antibody does negatively affect vaccine efficacy.

Subclinical breakdown with infectious bovine rhinotracheitis virus infection in dairy herd of high health status.

Infectious bovine rhinotracheitis (IBR) virus infection was detected by an antibody ELISA in the bulk milk of a large closed dairy herd of high health status in an area of low cattle density in East Anglia. The herd was managed under high standards of biosecurity and was known to have been serologically free of IBR virus for the previous 13 years. Although over 70 per cent of the cows had seroconverted to IBR virus no clinical signs were observed apart from a slight bilateral watery ocular discharge in a few cows, and their performance and productivity were unaffected. The causal virus, which was isolated after it had been reactivated with corticosteroid, had the DNA profile of a bovine herpesvirus type 1 strain normally associated with clinically severe respiratory disease. In spite of extensive enquiries and seroepidemiological investigations the source of the infection was not determined.

Bovine viral diarrhoea, bovine herpesvirus and parainfluenza-3 virus infection in three cattle herds in Egypt in 2000.

This study reported field outbreaks of bovine viral diarrhoea virus (BVDV) infection, either alone or mixed with bovine herpesvirus-1 (BHV-1) and/or parainfluenza-3 virus (PI-3V) in Egypt during 2000. In Lower Egypt, young calves in three cattle herds in El-Minufiya Province, El-Fayoum Province and in governmental quarantine in El-Behira Province, showed symptoms of enteritis, either alone or accompanied by respiratory manifestations. The affected herds were visited and the diseased animals were clinically examined. Many epidemiological aspects, such as morbidities, mortalities and case fatalities, as well as the abortive rate, were calculated. Ethylenediamine tetra-acetic acid-blood samples, sterile nasal swabs and serum samples were obtained for virological and serological diagnosis. The laboratory investigations revealed that the main cause of calf mortalities in the three herds was infection with BVDV, either alone, as on the El-Minufiya farm, or mixed with PI-3V, as on the El-Fayoum farm, or mixed with both BHV-1 and PI-3V, as in the herd in governmental quarantine in El-Behira Province. A total of nine dead calves from the three herds were submitted for thorough post-mortem examination. Tissue samples from recently dead calves were obtained for immunohistochemical and histopathological studies. The most prominent histopathological findings were massive degeneration, necrosis and erosions of the lining epithelium of the alimentary tract. Most of the lymphoreticular organs were depleted of lymphocytes. In pneumonic cases, bronchopneumonia and atypical interstitial pneumonia were evident. The present study suggested that the immunosuppressive effect of BVDV had predisposed the animals to secondary infection with BHV-1 and PI-3V. This study concluded that concurrent infection with BVDV, BHV-1 and PI-3V should be considered as one of the infectious causes of pneumoenteritis and, subsequently, the high morbidities and mortalities among young calves in Egypt. Preventive and control measures against these infectious agents should therefore be adopted. All animals imported into Egypt should be free from BVDV infection. Control programmes for the detection and removal of BVDV-persistent cattle should be applied in cattle herds all over the country.

Comparative pathogenesis of acute and latent infections of calves with bovine herpesvirus types 1 and 5.

This study was conducted to compare the pathogenesis of acute and latent infections with closely related bovine herpesvirus types 1 (BHV-1) and 5 (BHV-5) in their natural host. Two groups of eight calves were inoculated intranasally with BHV-1 or BHV-5. Although BHV-1 and BHV-5 similarly replicate in the nasal mucosa after inoculation, both viruses differ markedly in their ability to cause disease, BHV-5 being responsible of some fatal encephalitis while BHV-1 inducing rhinotracheitis. Virus isolation and immunohistochemistry demonstrated that BHV-5 replicates extensively in neurons of the central nervous system (CNS) and in respiratory cells of lungs, tracheal and nasal mucosae. Invasion of the CNS likely occurs through the trigeminal and olfactory pathways. Both groups developed cross-neutralising antibodies during this experiment suggesting partial clinical cross-protection afforded by the two infections. Three months after primary infection, experimental reactivation showed that BHV-5 was able to establish latency in the trigeminal ganglia but also the CNS of surviving calves. Moreover, laboratory findings suggested that BHV-5 could also persist in the tracheal and nasal mucosae. These results indicate that, after primary infection, BHV-1 and BHV-5 displayed similar biological features and consequently need to be considered together for the control of BHV-1 infection.

Pathology of bovine abortion and newborn calf death caused by dual infection with Chlamydia psittaci and infectious bovine rhinotracheitis virus.

Nine aborted fetuses and one newborn calf, diagnosed as Chlamydia psittaci (C. psittaci) infection, were pathologically examined. The characteristic lesions in the liver were focal necrosis in 9 aborted fetuses and granulomatous necrotic foci in the newborn calf. Moderate numbers of intranuclear inclusion bodies were found in necrotic foci of the liver, spleen, thymus, lymph nodes, adrenal gland, kidney, lung and forestomach. Immunohistologically, a small number of C. psittaci antigens was demonstrated in necrotic foci of the liver and correlated with distribution of elementary bodies. Moderate numbers of infectious bovine rhinotracheitis (IBR) virus antigens were also detected in degenerating and necrotizing parenchymal cells in various organs and correlated with distribution of intranuclear inclusion bodies. Thus, these aborted bovine fetuses and newborn calf were interpreted as being dually infected with C. psittaci and IBR virus during pregnancy.

Bovine herpesvirus-1-induced pharyngeal tonsil lesions in neonatal and weanling calves.

The potential involvement of the pharyngeal tonsil in the pathogenesis of bovine herpesvirus-1 (BHV-1) infection was examined in neonatal and weanling calves infected by intranasal aerosol. Calves were monitored from days 1 to 5, and on day 6 (neonates) or 8 (weanlings) and, in a second trial at day 4.5, by histology, electron microscopy, immunocytochemistry and virus isolation. Mucosal lesions of neonates were similar to, but less extensive than, those of weanling calves. Loss of microvilli and goblet cells, with minimal epithelial erosions as early as day 1, progressed to necrosis of epithelium and adjacent lymphoid tissue, and leucocyte exudation. Lesions and clinical disease were progressive up to and including day 6 in neonates, but resolving in weanlings on days 5 and 8. By transmission electron microscopy, the physical characteristics of the phagocytic cells appeared similar in both age groups, and viral replication was not identified in leucocytes. Virus was isolated from, or found by immunocytochemistry in, the pharyngeal tonsil of all calves examined, except for two weanlings on days 1 and 8. Virus as detected by immunocytochemistry was restricted to epithelium and superficial lymphoid tissue in neonates, but was found in deep lymphoid tissue around germinal centres in weanlings. The study showed that the pharyngeal tonsil is readily infected with BHV-1 and may be an important lymphoid tissue for early anti-viral responses. The delayed inflammatory response and reduced viral clearance may contribute to the increased susceptibility of neonatal calves to fatal BHV-1 infections.

Histological changes in lungs of calves exposed to an aerosol of Pasteurella haemolytica.

An experiment was designed to study the interaction of Pasteurella haemolytica with an attenuated bovine herpesvirus 1 in calves. Low titre of the virus culture used for aerosol exposure failed to produce measurable interaction. However, the experiment provided the first opportunity to study the light-microscopic changes in lungs of calves (n = 3) to a low-dose exposure (5-min aerosol) of P. haemolytica A1 from a fresh 5-h log-phase culture. The histopathological study was confined to tissue exposed to only P. haemolytica. A limited macroscopic pneumonia was produced in ventral parts of cranial lobes. Four days after exposure, a typical reaction featured four zones. Zone 1a at the centre with acute inflammatory processes and necrosis of phagocytic cells was surrounded by a broad band of compacted, largely necrotic macrophages and polymorphonuclear leucocytes (PMNL) in alveoli of zone 1b. Necrosis was confined to zone 1. Zone 2a frequently occupied the remainder of the lobule with irregular distribution of congestion, oedema with a fibrinous component, and infiltration by numerous PMNL, macrophages and other mononuclear inflammatory cells. The narrow zone 2b was located between zones 1b and 2a and had oedema with a fibrinous component, numerous fibrocytes, few inflammatory cells and empty capillaries. It is suggested that zone 2 served to isolate zone 1 by surrounding it with nonfunctional tissue. The pathogenicity of P. haemolytica is discussed for uncompromised lungs and lungs compromised by virulent BHV1 infection.

The pathology of concurrent bovine viral diarrhoea and infectious bovine rhinotracheitis virus infection in newborn calves.

Concurrent bovine viral diarrhoea (BVD) and systemic infectious bovine rhinotracheitis (IBR) are reported from two neonatal (11 and 15 days old) calves. The diseases occurred sporadically in a large-scale herd which may have been due to the calves' heterogeneous immunobiological status. Gross pathological and histopathological examinations revealed focal interstitial pneumonia with acidophilic intranuclear inclusions in the alveolar epithelial cells and necrotic foci in the liver with a few intranuclear inclusions in the hepatocytes. There were subserous haemorrhages in the forestomachs and intestine, necrotic changes in the rumen, enteritis, lymphocytic necrosis in the Peyer's patches, and fibrinoid necrosis in the wall of some of the neighbouring blood vessels. BVD virus was demonstrated by immunofluorescence (IF), whereas IBR virus by electron microscopy, immunofluorescence and virus isolation.

Protection of newborn calves against fatal multisystemic infectious bovine rhinotracheitis by feeding colostrum from vaccinated cows.

To determine whether consumption of colostrum with high levels of serum neutralizing antibody to bovine herpesvirus 1 would protect neonatal calves from the frequently fatal multisystemic form of infectious bovine rhinotracheitis, Holstein calves were fed for 48 h after birth with either pooled colostrum from seropositive vaccinated cows or colostrum from seronegative unvaccinated cows. The serum neutralizing antibody achieved in the former calves was between 64 and 256 and the titer in the latter calves was below 8. At 48 h of age the calves were challenged by aerosolization with bovine herpesvirus 1. All five seronegative calves died or were euthanized in a moribund state between days 5 and 7 of the trial, whereas all five seropositive animals remained healthy throughout the study. Twice daily clinical examination revealed significantly lower scores in the seronegative group from 60 h postinfection. Relative lung weights were greater in the seronegative group, associated with a severe acute necrotizing bronchiolitis with fibrin exudation. The seronegative group of calves also demonstrated an acute necrotizing rumenitis, pharyngitis, glossitis, esophagitis, laryngitis and tracheitis. The seropositive animals had only small areas of subacute necrotizing fibrinopurulent rhinitis. Bovine herpesvirus 1 virus was isolated from all nasal passages of all calves but isolation of virus in the seronegative calves was made from the trachea (5/5), lung (4/5), bronchial lymph nodes (4/5), spleen (4/5), thymus (3/5), liver (2/5), rumen (2/5) and brain (1/5).(ABSTRACT TRUNCATED AT 250 WORDS)

[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.

Bovine herpesvirus-1 and Pasteurella haemolytica aerobiology in experimentally infected calves.

Eight calves (2 calves in each of 4 groups) were exposed to an aerosol of bovine herpesvirus-1 (BHV-1) and 4 days later to an aerosol of Pasteurella haemolytica. Samples of tracheal and exhaled air were taken simultaneously beginning 1 day before viral exposure and once a day up to 3 to 4 days after the bacterial exposure. Samples were also taken during the period of aerosol exposure. Only 0.04% to 0.42% of P haemolytica-carrying droplets of the bacterial aerosol passed beyond the cranial part of the respiratory tract to the trachea. Nevertheless, numbers of bacteria as few as 1 bacterium/L of tracheal air were sufficient to produce fatal disease in the lungs of BHV-1-infected calves. In 1 of 4 groups, BHV-1 was isolated from most daily samples of exhaled and tracheal air. Pasteurella haemolytica was isolated 7 times more frequently from air when calves were kept at 1 C than when calves were kept at 23 C. The number of P haemolytica-carrying droplets in exhaled air was low (less than 1/L of air); however, samples obtained during the time that calves were coughing contained up to 10 P haemolytica-carrying droplets/L of air. It was learned that the cranial part of the respiratory tract serves as an efficient filter on inhalation and exhalation, but this filter is deficient in the animal when coughing occurs. This process expels infective droplets of size suitable for inhalation by other cattle in close proximity.