Experimental latent and recrudescent bovine herpesvirus-1 infections in calves

Latent bovine herpesvirus-1 (BHV-1) infection was established in 6 calves and was demonstrated by reinduction of virus shedding after administration of corticosteroids. Latently infected calves failed to transmit BHV-1 during 4 weeks' contact with sentinel calves. Infected calves were killed and necropsied during latency or induced recrudescence. The BHV-1 DNA was demonstrated intranuclearly in trigeminal ganglion neurons by in situ hybridization. The BHV-1 antigen was demonstrated by immunofluorescence in trigeminal ganglion neurons during recrudescence. By electron microscopy, changes in the appearance of the Nissl bodies and a high frequency of nuclear bodies were observed in trigeminal ganglion neurons.     

Establishment of latency associated with glycoprotein E (gE) seroconversion after bovine herpesvirus 1 infection in calves with high levels of passive antibodies lacking gE antibodies

This study was conducted to investigate the glycoprotein E (gE) antibody response raised after inoculation with a low infectious dose of bovine herpesvirus 1 (BHV-1) in six calves possessing high levels of passive immunity from cows repeatedly vaccinated with gE deleted marker vaccine. Four out of the six calves developed gE antibodies 3-5 weeks after infection, whereas the two other ones remained seronegative to gE. After 5 months of infection, the six calves were treated with dexamethasone. Virus was only re-excreted by the four calves which previously seroconverted against gE. The two other calves became seronegative against BHV-1, 30-32 weeks after infection. A second dexamethasone treatment performed 11 months after infection failed to demonstrate a latent infection in these two calves. Moreover, the lack of identification of a cell-mediated immune response, after the two dexamethasone treatments, and the failure to detect BHV-1 DNA sequences in trigeminal ganglia strongly suggest that these two calves were not latently infected. In conclusion, the presence of high levels of maternal immunity lacking gE antibodies does not prevent latency after infection with a low titre of BHV-1. Moreover, latency is associated with a serological response to gE. These results confirm that the gE deletion is a good marker to identify young calves latently infected with a field virus.     

Effects of hypervaccination with bovine herpesvirus type 1 gE-deleted marker vaccines on the serological response and virological status of calves challenged with wild-type virus

Twenty-four calves were immunised four times with gE-deleted infectious bovine rhinotracheitis marker vaccines before being challenged with small doses of wild-type bovine herpesvirus type 1 (BHV-1). The repeated vaccinations induced strong immunity that prevented detectable virus replication and gE-seroconversion after the challenge infection in most of the calves. The hypervaccinated calves that shed virus after the challenge infection showed no delay in gE-seroconversion compared with unvaccinated control calves. Using a sensitive nested PCR, BHV-1 gE sequences could be detected in the trigeminal ganglia of several of the gE-seronegative, challenge-infected calves, possibly indicating the presence of wild-type BHV-1 DNA.     

Attempts to reactivate bovid herpesvirus-2 in experimentally infected calves

A study was carried out to determine whether bovid herpesvirus-2 (BHV-2) is able to induce a recurrent infection in experimentally infected calves. Twelve calves infected with the virus were treated with dexamethasone (DMS) beginning 69 days after the infection, ie, several weeks after the animals had recovered from the disease and were negative for BHV-2. The stress induced by DMS treatment failed to reactivate the clinical condition or to induce shedding of BHV-2. However, treatment with DMS reactivated a latent infectious bovine rhinotracheitis (IBR) virus infection in all calves previously inoculated with BHV-2, and also in 2 noninoculated controls. The reactivation of IBR virus occurred without any clinical evidence of the disease, but the virus was isolated from nasal and pharyngeal swabbings and from the organs. A proliferative ganglionitis of the trigeminal ganglion was also observed. Because of the interference by IBR virus, this study did not resolve the question as to whether BHV-2 can induce a recurrent infection.     

The DNA of an IPV strain of bovid herpesvirus 1 in sacral ganglia during latency after intravaginal infection

Two calves were inoculated intravaginally with a strain of bovid herpesvirus type 1 (BHV-1, IBR/IPV) isolated from a cow with infectious pustular vulvovaginits (IPV). The animals were killed during a latent stage of infection as characterized by seroconversion, absence of virus shedding and recrudescence of virus shedding after dexamethsone treatment.IPV-virus DNA was detected in 9 out of 20 sacral ganglia of the 2 calves. Of the sections, 7.2% (n = 250) contained 1 cell with IPV-virus DNA, which was restricted to the nucleus of neurons. In agreement with findings on herpes simplex virus infections, the viral DNA of BHV-1 is harbored in the local sensory ganglia.Virological and serological implications of the latent IPV infection are discussed.     

Analysis of bovine trigeminal ganglia following infection with bovine herpesvirus 1

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-gamma (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.     

Bovine herpesvirus-1 and parainfluenza-3 virus interactions: clinical and immunological response in calves.

Calves infected with bovine herpesvirus-1 (BHV-1) or both BHV-1 and parainfluenza-3 virus (PIV-3) developed clinical signs including fever, cough, and nasal and ocular discharges. Animals infected with both viruses appeared more depressed and showed higher rectal temperature, while calves inoculated with PIV-3 alone had a very mild clinical disease. Both BHV-1 and PIV-3 were recovered from nasal secretions up to six to eight days postinoculation. However, the virus titers were lower in calves with mixed infection. An increase in serum antibodies to both BHV-1 and PIV-3 was detected by serum neutralization and enzyme-linked immunosorbent assay. Antibody responses were delayed and significantly lower in calves given mixed infection than in calves infected with a single virus. Interleukin-2 activity in cultures of lymphocytes from BHV-1 and BHV-1 plus PIV-3 infected calves was higher compared to control calves.     

Construction and characterization of a glycoprotein E gene-deleted bovine herpesvirus type 1 recombinant

OBJECTIVE: To construct and characterize a recombinant glycoprotein (g)E gene-deleted bovine herpesvirus (BHV) type 1 (BHV-1). PROCEDURE: The BHV-1 gEgene-coding region and the flanking upstream and downstream sequences were cloned. The aforementioned cloned DNA was digested with suitable enzymes to release the amino terminal two thirds of that region, and was ligated to the beta-galactosidase (beta-gal) gene. The resulting plasmid DNA was cotransfected with DNA from full-length, wild-type (WT), BHV-1 Cooper strain of the virus. Recombinant viruses expressing beta-gal (blue plaques) were plaque purified and assayed further by blot hybridization for genetic characterization and by immunoblotting for reactivity against BHV-1 gE peptide-specific rabbit polyclonal antibody. One recombinant virus, gEdelta3.1IBR, was characterized in vitro and in vivo. The ability of the recombinant virus to induce BHV-1 neutralizing antibodies in infected calves was investigated by plaque-reduction tests. RESULTS AND CONCLUSIONS: The gEdelta3.1IBR virus contained a deletion in the viral gE gene-coding sequences where a stable chimeric reporter (beta-gal) gene was inserted. One-step growth kinetics and virus yield of the recombinant and parent viruses were similar, but early after infection, the recombinant virus yield was comparatively less. After intranasal inoculation, the recombinant gEdelta3.1IBR virus replicated in the upper respiratory tract of calves, but the amount of progeny viruses produced was hundredsfold reduced, and duration of virus shedding was shorter. Results of in vivo calf experiments and serum neutralization tests indicated that deleting the gE gene has little effect on inducing neutralizing antibodies against BHV-1, but is sufficient to reduce BHV-1 virulence in calves.     

Encephalitis induced by bovine herpesvirus 5 and protection by prior vaccination or infection with bovine herpesvirus 1.

Calves were intranasally challenged with bovine herpesvirus 5 (BHV5) and followed for the development of viral infection, clinical encephalitis, histologic lesions in the brain, and viral sequences in the trigeminal ganglia. Calves that were previously vaccinated with bovine herepesvirus 1 (BHV1, n = 4) or previously infected with BHV1 (n = 5) or that had not been exposed to either virus (n = 4) were compared. No calf developed signs of encephalitis, although all calves developed an infection as indicated by nasal secretion of BHV5 and seroconversion to the virus. Histologic lesions of encephalitis consisting of multifocal gliosis and perivascular cuffs of lymphocytes were observed in calves not previously exposed to BHV1. BHV5 sequences were amplified from the trigeminal ganglia of calves previously vaccinated and from calves not previously exposed to BHV1; calves sequentially challenged with BHV1 and later BHV5 had exclusively BHV1 sequences in their trigeminal ganglia. Administration of dexamethasone 28 days after BHV5 challenge did not influence clinical disease or histologic lesions in either previously unexposed calves (n = 2) or previously immunized calves (n = 2), although it did cause recrudescence of BHV5, as detected by nasal virus secretion.     

Presence of bovine herpesvirus 1 gB-seropositive but gE-seronegative Dutch cattle with no apparent virus exposure

Two hundred and thirty-seven of 2052 cattle which had not been vaccinated against bovine herpesvirus 1 (BHV-1) were seropositive in a glycoprotein B (gB)-blocking ELISA, but seronegative in a glycoprotein E (gE)-blocking ELISA. In order to detect whether they were latently infected with BHV-1, 10 of them were treated with corticosteroids in an attempt to reactivate putatively latent virus. After successive treatments with dexamethasone and prednisolone, no virus excretion was detected and they showed no increase in antibody titres. In contrast, one gE-seropositive animal re-excreted BHV-1 and had a four-fold increase in antibody titre after the corticosteroid treatments. After slaughter, no BHV-1 DNA could be detected with a sensitive PCR in samples of the trigeminal, cervical and sacral ganglia and spinal cords of the gE-seronegative cattle.     

The bovine herpesvirus type 1 envelope protein Us9 acidic domain is crucial for anterograde axonal transport

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.     

Antibody response to glycoprotein E after bovine herpesvirus type 1 infection in passively immunised, glycoprotein E-negative calves

This study was conducted to determine whether young calves with maternal antibodies against bovine herpesvirus type 1 (BHV-1) but without antibodies against glycoprotein E (gE) can produce an active antibody response to gE after a BHV-1 infection. Five calves received at birth colostrum from gE-seronegative cows which had been vaccinated two or three times with an inactivated BHV-1, gE-deleted marker vaccine. After inoculation with a wild-type virulent strain of BHV-1, all the passively immunised gE-negative calves shed virus in large amounts in their nasal secretions. All the calves seroconverted to gE within two to four weeks after inoculation and then had high levels of gE antibodies for at least four months. The development of an active cell-mediated immune response was also detected by in vitro BHV-1-specific interferon-gamma assays. All the calves were latently infected, because one of them re-excreted the virus spontaneously and the other four did so after being treated with dexamethasone. The results showed that under the conditions of this work the gE-negative marker could also distinguish between passively immunised and latently infected calves.     

Intrapreputial infection of young bulls with bovine herpesvirus type 1.2 (BHV-1.2): acute balanoposthitis, latent infection and detection of viral DNA in regional neural and non-neural tissues 50 days after experimental reactivation

Venereal infection of bulls with bovine herpesvirus type 1.2 (BHV-1.2) may result in acute balanoposthitis followed by the establishment of latent infection, presumably in dorsal root nerve ganglia. We herein report the characterization of the acute and latent infection of young bulls with a Brazilian BHV-1.2 isolate and the investigation of neural and non-neural sites in which viral DNA persists during latent infection, i.e. 110 days after inoculation and 50 days after experimental reactivation. Intrapreputial inoculation of BHV-1.2 isolate SV-56/90 (10(6.5)pfu per animal) resulted in severe balanoposthitis, characterized by redness of the penis and preputial mucosa, coalescent vesicles and fibrinous exsudate in all four infected bulls. Virus shedding was detected in preputial secretions and semen up to days 14 and 13 pi, respectively. Dexamethasone administration at day 60 pi led to reactivation of the infection in all animals, resulting in virus shedding in preputial secretions and/or in semen. At day 50 post-reactivation (pr), the animals were euthanized and regional tissues were collected for PCR and virus isolation. Viral DNA was consistently detected in the dorsal root ganglia of nerves genito-femoral (4/4) and obturator (4/4); frequently in the pudendal (3/4), sciatic (3/4) and rectal caudal nerve ganglia (2/3). In addition, viral DNA was detected in the pelvic sympathetic plexus of one bull and in regional lymph nodes (deep inguinal (2/4); sacral (1/4); medial iliac (1/4)) of two bulls. No infectious virus could be recovered from homogenates of DNA positive tissues, indicating the absence of actively replicating virus. These results demonstrate that BHV-1.2 DNA may persist in several sacral nerve ganglia and in regional lymph nodes as well during latent infection, i.e. 50 days after experimental reactivation. These findings may help in understanding the pathogenesis of acute and latent genital infection by BHV-1.2.     

Recrudescence of bovine herpesvirus-5 in experimentally infected calves

A latent infection of bovine herpesvirus-5 (BHV-5) was established in 4 calves. These calves, plus 2 controls, were given dexamethasone (DM) to reactivate the latent virus. The 4 principal calves developed antibodies to BHV-5 by postinoculation day (PID) 21. Antibody titers increased until PID 42 before decreasing to low levels of PID 75. After the first DM treatment (started on PID 76), an anamnestic antibody response was demonstrated in the 4 principal calves. Calves, 2, 3, and 4 were euthanatized and necropsied at PID 121, and their antibody titers were again decreasing. The virus BHV-5 was not isolated from the tissues by conventional techniques of viral isolation but was isolated from the trigeminal ganglion and spinal cord of calf 3 by explantation techniques. The BHV-5 was isolated, using conventional viral isolation techniques, from a nasal swab sample of calf 1 on PID 91 (15 days after the first DM treatment) and from the thoracic lymph node 6 days after the start of a 2nd DM treatment. Seemingly, BHV-5 may be latently harbored in the nerve tissues or calves and this virus may be reactivated from the upper respiratory tract following subsequent DM treatment.     

Periodic reappearance of bovine herpesvirus type 4 DNA in the sera of naturally and experimentally infected rabbits and calves.

A BHV-4 specific nested PCR was used for the detection of viral DNA in serum samples of rabbits and calves. All animals were followed up for 62 days, blood samples were taken for PCR studies every second day. Maternal infection of calves resulted in the repeated regular reappearance (10-14 days) of the virus (DNA) in serum samples. When PCR positive five-day-old calves were infected with tissue culture adapted virus, the reappearance of the DNA in the serum was shown to be irregular, nevertheless, DNA peaks reappeared during the whole observation period. A PCR negative calf infected at the age of 60 days was found to possess viraemia until p.i.d. 32. In rabbits treated intravenously with BHV-4 the inoculum or a primary viraemia was detected at p.i.d. 2-3 and p.i.d. 14-16. Published data on human herpesviruses suggest, that the target cells might be a pluripotent stem cell population of the bone marrow and differentiated virus-infected cells destroyed by the immune system might be the source of viral DNA detected in the serum. Frequency of DNA reappearance was depended on the age of the infected animals but not on the inoculated amount of BHV-4. The described phenomenon might be part of BHV-4 infection of very young animals.     

Dynamics of Moraxella bovis infection and humoral immune response to bovine herpes virus type 1 during a natural outbreak of infectious bovine keratoconjunctivitis in beef calves

Infectious bovine keratoconjunctivitis (IBK) is an acute disease caused by Moraxella bovis (Mb). Several factors may predispose animals to an IBK outbreak; one commonly observed is infection with bovine herpes virus type 1 (BHV-1). The aim of this study was to investigate the dynamics of BHV-1 virus infection and its relation with clinical cases of IBK in weaned calves from a beef herd with a high prevalence of lesions caused by Mb. Sampling was carried out in six stages and included conjunctival swabs for isolating Mb as well as blood samples for identifying antibodies specific for BHV-1. A score for IBK lesions after observing each eye was determined. The findings of this study showed a high prevalence of BHV-1 virus infection (100% of animals were infected at the end of the trial); 67% of animals were culture-positive for Mb, but low rates of clinical IBK (19% of calves affected) were detected at the end of the trial. These results suggest that infection with BHV-1 did not predispose these animals to IBK, and that Mb infection produced clinical and subclinical disease in the absence of BHV-1 co-infection.     

Effects in calves of mixed infections with bovine viral diarrhea virus and several other bovine viruses

The objective of this study was to verify whether a mixed infection in calves with bovine viral diarrhea virus (BVDV) and other bovine viruses, such as bovid herpesvirus-4 (BHV-4), parainfluenza-3 (PI-3) and infectious bovine rhinotracheitis (IBR) virus, would influence the pathogenesis of the BVDV infection sufficiently to result in the typical form of mucosal disease being produced.

Accordingly, two experiments were undertaken. In one experiment calves were first infected with BVDV and subsequently with BHV-4 and IBR virus, respectively. The second experiment consisted in a simultaneous infection of calves with BVDV and PI-3 virus or BVDV and IBR virus.

From the first experiment it seems that BVDV infection can be reactivated in calves by BHV-4 and IBR virus. Evidence of this is that BVDV, at least the cytopathic (CP) strain, was recovered from calves following superinfection. Moreover, following such superinfection the calves showed signs which could most likely be ascribed to the pathogenetic activity of BVDV. Superinfection, especially by IBR virus, created a more severe clinical response in calves that were initially infected with CP BVDV, than in those previously given the non-cytopathic (NCP) biotype of the virus. Simultaneous infection with PI-3 virus did not seem to modify to any significant extent the pathogenesis of the experimentally induced BVDV infection whereas a severe clinical response was observed in calves when simultaneous infection was made with BVDV and IBR virus.     

The use of immunomodulators in the control of infectious bovine rhinotracheitis

Three experiments have been carried out to verify the effectiveness of an immunomodulator, Baypamun (Bayer AG) in limiting the spread of Bovine herpesvirus-1 (BHV-1), the causal agent of infectious bovine rhinotracheitis (IBR). In the first experiment, four calves infected with BHV-1 developed severe disease whereas four calves given Baypamun simultaneously with the virus had less severe disease. Four other calves in contact with the infected calves became severely ill but another four given Baypamun were only mildly affected. In the second experiment three calves infected with BHV-1, which reacted with typical disease, were allowed to remain in contact with six calves. All six calves were given Baypamun at various times following the exposure to BHV-1 infection and all showed a much reduced reaction with two treated for 4 days developing no clinical disease. Finally, in the third experiment one calf vaccinated one month before the start of the experiment did not develop any signs of disease when housed together with a calf experimentally infected with BHV-1. Of four other calves, vaccinated when the infected calf showed the first signs of disease, only the two given Baypamun in addition to the vaccine, were protected from clinical disease whereas the two given vaccine only developed classical signs of IBR. In the three experiments the virus shedding by the Baypamun-treated calves resulted to be significantly reduced.