Full genome sequence of bovine alphaherpesvirus 2 (BoHV-2).

We present the complete genome sequence of bovine alphaherpesvirus 2 (BoHV-2), a member of the family Herpesviridae, subfamily Alphaherpesvirinae, genus Simplexvirus. BoHV-2 is the causative agent of bovine ulcerative mammillitis (bovine herpes mammillitis) and pseudo-lumpy skin disease. The genomic architecture of BoHV-2 is typical of most simplexvirus genomes and congruent with that of human alphaherpesvirus 1 (HHV-1). The genome comprises a total of 131,245 base pairs and has an overall G+C content of 64.9 mol%. A total of 75 open reading frames are predicted. The gene repertoire of BoHV-2 is analogous to that of HHV-1, although the coding region of US12 is missing. A phylogenetic analysis supported BoHV-2 as a member of the genus Simplexvirus.

Bovine Alphaherpesvirus 2 infections in Bavaria: an analysis of the current situation - several years after eradicating Bovine Alphaherpesvirus 1.

BACKGROUND: Bavaria, a large federal state in Germany, has been declared free from infections with Bovine Alphaherpesvirus 1 (BoHV-1) in 2011. To maintain this status the cattle population is monitored for antibodies against BoHV-1 regularly. Several years ago, infrequent but recurrent problems in this sero-surveillance were statistically put into correlation with the presence of antibodies against Bovine Alphaherpesvirus 2 (BoHV-2). In Europe, BoHV-2 is primarily known as the agent causing bovine herpes mammillitis. However, very little information about BoHV-2 infections in Bavaria is available so far. Therefore, the aims of this study were to determine BoHV-2 seroprevalences and to detect virus genomes in potential clinical samples. RESULTS: 6801 blood sera of healthy cattle from all over Bavaria were tested for antibodies against BoHV-2, revealing an overall seroprevalence of 5.51%. Interestingly, seroprevalences markedly varied between the North and the South of Bavaria, namely from 0.42 to 11.17%. Concurrently, the previously reported relation between the epidemiologically inexplicable sero-reactivities in BoHV-1 ELISAs and the presence of BoHV-2 infections were statistically corroborated in this study. To detect BoHV-2 genomes a fast and sensitive real time PCR was established. Using a multiple PCR strategy, tissue samples from skin lesions at relevant localizations, corresponding lymph nodes, and trigeminal ganglia from 111 animals, as well as nasal swabs from 918 bovines with respiratory symptoms were tested. However, BoHV-2 genomes were not detected in any of these samples. CONCLUSIONS: BoHV-2 antibodies were found in samples from bovines all over Bavaria, albeit with an explicit South-North-divide. BoHV-2 genomes, however, could not be detected in any of the analyzed samples, indicating that acute clinical cases as well as obvious virus reactivation are relatively rare. Consequently, the future spread of BoHV-2 infections throughout Bavaria, particularly, after eradicating BoHV-1, has to be further monitored.

Isolation and characterization of bovine alphaherpesvirus 2 strain from an outbreak of bovine herpetic mammillitis in a dairy farm.

BACKGROUND: Bovine alphaherpesvirus type 2 (BoHV-2) belongs to family Herpesviridae, subfamily Alphaherpesviridae and can cause two distinct, well-defined conditions: a generalized benign skin infection that somewhat mimics lumpy skin disease (LSD), referred to as Pseudo-Lumpy Skin Disease (PSLD) and a localized ulcerative mammillitis, referred to as Bovine Herpetic Mammillitis (BHM). BHM is a localized form of BoHV-2 infection that causes erosive-ulcerative self-limiting lesions on breast and nipples. BHM is chiefly a disease of lactating dairy cows and has been described sporadically in several countries. In this study we describe an outbreak of bovine herpetic mammillitis caused by BoHV-2 occurred in a dairy farm in Southern Italy. Clinical signs were observed in 26/59 lactating cows with the age ranging between 2 and 6 years. The affected animals were afebrile, showed lesions on the skin of nipples, breast and ventral surface of the abdomen, near the mammary veins and spontaneously recovered within 2 months. RESULTS: BoHV-2 DNA was detected in the crust samples by pan-herpes PCR and real-time quantitative PCR. The virus was isolated on bovine kidney cells and was characterised by deep sequencing technologies. The nucleotide identity to BoHV-2 of the strain ITA/2018/468 retrieved in this study ranged from 98.83 to 100%. Phylogenetic analyses based on three full-length gene (glycoprotein B, thymidine kinase and glycoprotein G) sequences confirmed the close relatedness of the strain ITA/2018/468 to BoHV-2 sequences. CONCLUSIONS: The report represents a significant outbreak of BHM in a dairy farm 50 years after the last description in Italy. However, outbreaks of PLSD have been described in Europe recently, indicating that the virus is present in European territories. Improving the diagnostic algorithms and enacting specific surveillance plans could be useful to understand better the epidemiological and pathogenetic patterns of BoHV-2 infection in livestock animals, and to develop, eventually, effective prophylaxis plans.

Detection of OvHV-2 from an outbreak of sheep associated malignant catarrhal fever from crossbred cattle of Southern India.

An outbreak of sheep associated malignant catarrhal fever in crossbred cattle in a village of Andhra Pradesh, southern India, affected thirteen adult cows and two calves from a population of forty animals. All the affected animals were died between December and January 2013-14. The clinical and gross postmortem findings were typical of MCF in Indian crossbred cattle. Migrating sheep flocks were suspected source of infection for the cattle. The diagnosis was confirmed by heminested PCR in all the affected cattle and the suspected sheep flock. The PCR provided evidence of ovine herpes virus type 2.

Detection of bovine herpesvirus 2 and bovine herpesvirus 4 DNA in trigeminal ganglia of naturally infected cattle by polymerase chain reaction.

Establishment of latent infection within specific tissues in the host is a common biological feature of the herpesviruses. In the case of bovine herpesvirus 2 (BoHV-2), latency is established in neuronal tissues, while bovine herpesvirus 4 (BoHV-4) and ovine herpesvirus 2 (OvHV-2) latent virus targets on cells of the monocytic lineage. This study was conducted in quest of BoHV-2, BoHV-4 and OvHV-2 DNA in two hundred trigeminal ganglia (TG) specimens, derived from one hundred clinically healthy cattle, majority of them naturally infected with bovine herpesvirus 1 (BoHV-1) and bovine herpesvirus 5 (BoHV-5). Total DNA extracted from ganglia was analyzed by polymerase chain reaction (PCR) designed to amplify part of the genes coding for BoHV-2, and BoHV-4 glycoprotein B and, for OvHV-2, the gene coding for phosphoribosylformylglycinamidine synthase-like protein. BoHV-2 DNA was detected in TG samples of two (2%) and BoHV-4 DNA in nine (9%) of the animals, whereas OvHV-2 DNA could not be detected in any of the TG DNA. The two animals in which BoHV-2 DNA was identified were also co-infected with BoHV-1 and BoHV-5. Within the nine animals in which BoHV-4 DNA was detected, six were also co-infected with BoHV-1 and BoHV-5. This report provides for the first time evidence that viral DNA from BoHV-2 and BoHV-4 can be occasionally detected in TG of naturally infected cattle. Likewise, in this report we provided for the first time evidence that the co-infection of cattle with three distinct bovine herpesviruses might be a naturally occurring phenomenon.

Acute and latent infection by bovine herpesvirus type 2 in a guinea pig model.

Bovine herpetic mammillits is a self-limiting cutaneous disease of the udder and teats of cows associated with bovine herpesvirus 2 (BoHV-2) whose pathogenesis is poorly understood. This article describes the use of guinea pigs (Cavia porcellus) to study the pathogenesis of BoHV-2 infection. Twelve weanling female guinea pigs inoculated subcutaneously with BoHV-2 in the genitalia and teats developed local hyperemia, edema, vesicles, ulcers and scabs. Infectious virus was recovered between days 3 and 7 post-infection (pi) from the genital area (9/12) and teats (1/12); and all inoculated animals seroconverted (virus-neutralizing titers of 16-128). Histological examination of lesions revealed lymphoplasmacytic perivascular infiltrates and intranuclear inclusion bodies in keratinocytes. PCR examination of tissues collected at day 35 pi detected latent viral DNA predominantly in lumbosacral spinal segments. In another experiment, eight females inoculated with BoHV-2 in the genitalia and treated with dexamethasone (Dx) at day 35 pi developed mild to moderate local signs, yet no virus could be recovered from lesions. PCR examination of spinal segments from these animals confirmed the presence of latent viral DNA. These results demonstrate that guinea pigs are susceptible to BoHV-2 infection and therefore may be used to study selected aspects of BoHV-2 biology.

Distribution of latent bovine herpesvirus 2 DNA in tissues of experimentally infected sheep.

The biology of latent infection by bovine herpesvirus 2 (BoHV-2), the agent of mammillitis in cows, remains largely unknown. We herein report attempts to reactivate the latent infection and investigated the sites of BoHV-2 latency in experimentally infected sheep. Ewes inoculated with BoHV-2 in the udder's skin shed virus for up to five days, developed mammillitis and seroconverted. However, attempts to reactivate latent infection by dexamethasone administration at day 40 pi failed. Nevertheless, viral DNA--and not infectious virus--was detected by PCR in several nerve ganglia and/or regional lymph nodes (LNs) of all animals at day 40 post-reactivation. Likewise, lambs previously inoculated with BoHV-2 in the nose harbored latent viral DNA in trigeminal ganglia, tonsils and regional LNs. These results demonstrate that BoHV-2 establishes latent infection in nerve ganglia and in regional lymphoid tissues, yet virus reactivation is not easily achieved by standard protocols used.

Use of a polymerase chain reaction assay to detect bovine herpesvirus type 2 DNA in skin lesions from cattle suspected to have pseudo-lumpy skin disease.

Beef cattle from a herd in north Alabama were examined because of an outbreak of nonfatal skin disease characterized by discrete circumscribed areas of inflammation that developed on the skin from the neck to the hips. Areas of inflammation, which tended to be superficial, underwent necrosis and scabbed over. The scabs eventually dropped off leaving discrete, round, whitish, hairless lesions that were 1.2 to 2.5 cm diameter. Because clinical signs were consistent with those expected with pseudo-lumpy skin disease (PLSD) caused by bovine herpesvirus type 2 (BHV-2), samples from 16 representative animals were submitted for BHV-2 testing. All 16 animals were seropositive for BHV-2, but the virus could not be isolated from skin biopsy specimens or buffy coat samples. Results of a polymerase chain reaction assay incorporating primers designed to amplify 2 DNA sequences from BHV-2 were positive for 3 of the 10 cattle, suggesting that skin lesions in these cattle were a result of PLSD. Our findings suggest that PLSD may be more common and widespread in the United States than suggested by the frequency with which BHV-2 has been isolated from cattle with PLSD-like skin lesions.

Development of a shuttle polymerase chain reaction for the detection of bovine herpesvirus 2.

Three different polymerase chain reaction (PCR) protocols were evaluated for their ability to detect bovine herpesvirus 2 (BoHV-2): single-step PCR with 3 reaction stages (denaturation, annealing and extension), 2 reaction stages (denaturation and annealing/extension; shuttle PCR), and semi-nested PCR with 3 reaction stages. All the PCR protocols showed the same sensitivity (detection limit of 0.4 TCID(50)). A non-specific band sometimes appeared in mock cell DNA at annealing temperatures below 64 degrees C. The shuttle PCR was found to be superior to the other protocols under consideration because of the speed of its application. Furthermore, no non-specific band was detected in DNAs of eight other DNA viruses. Thus, the shuttle PCR seems to be an excellent diagnostic tool for BoHV-2 infections.

An unusual outbreak of malignant catarrhal fever in a beef herd in Israel.

Malignant catarrhal fever (MCF. corrizza contagiosa) is an invariably fatal communicable disease in cattle, whose causative agent is the ovine herpes virus-2, or the alcelaphine herpes virus-1. In one feed-lot family farm, 34 calves out of 100 became ill at the rate of one to four calves per week, and all of them subsequently died over a period of 4 months. Most of the initial cases were manifested clinically as the head and eye form, but most of the entire clinical spectrum of forms (the respiratory, intestinal and nervous forms) characteristic for MCF were observed as this epidemic progressed. Very few calves died without showing any specific signs of MCF. Pathological examinations revealed characteristic obliterative arteriovasculitis in the brain of calves with nervous signs, typical of MCF. Polymerase chain reaction (PCR) testing revealed 100% homology between the 238 bp hemi-nested PCR fragment and the ovine herpes virus-2 sequences. Based on the clinical signs, epidemiological data, pathological, and histopathological findings, and the PCR results, it was concluded that MCF occurred on the farm. The fact that sheep and goats were housed in close proximity on the same farm reinforced this diagnosis.

Development of a polymerase chain reaction and restriction typing assay for the diagnosis of bovine herpesvirus 1, bovine herpesvirus 2, and bovine herpesvirus 4 infections.

A multiplex polymerase chain reaction (PCR) method coupled with a restriction analysis of PCR products (PCR with restriction fragment length polymorphism) was developed for the simultaneous detection of bovine herpesvirus 1, bovine herpesvirus 2, and bovine herpesvirus 4 infections. The specificity, sensitivity, and practical diagnostic applicability of this method were evaluated. This assay may be also adapted to the diagnosis of suid herpesvirus 1 and equine herpesviruses 1 and 3 and could become a powerful diagnostic tool.

Nylon membrane-immobilized PCR for detection of bovine viruses.

Bridge Technology is an amplification technique in which pairs of primers are immobilized on a solid support, allowing amplification only at the location of the primer pair spot. The technique has diagnostic potential since an array of primer pairs, each specific for a different pathogen, can be used with a diagnostic sample without inter-pair interactions that plague the development of multiplex PCRs. As a result, one assay should be able to determine which of multiple pathogens are present and which are absent in each sample. As test material, we examined the specificity of detection of the RNA-containing bovine viral diarrhea virus (BVDV) and two DNA-containing bovine herpesviruses 1 and 2 (BHV-1 and BHV-2). Nylon membranes with two spots of UV-immobilized primer pairs--one for BVDV and one for BHV--were used in amplification with both corresponding templates, with each template singly and with no template. When amplification was assayed by chemiluminescent detection of incorporated DIG-nucleotides, the expected amplification patterns were obtained.

Comparison of six Australian isolates of Bovine herpes virus 2 based on UL24 gene after a passage in MDBK cells.

A region of the UL24 gene of six Australian field isolates of Bovine herpesvirus 2 (BHV-2) was sequenced after a passage in Madin-Darby bovine kidney (MDBK) cells by polymerase chain reaction (PCR). While the PCR product covered the first half of the UL24 gene, a particular interest was focused on the 274-297 nucleotide (nt) region in which a two nt deletion had previously been detected in the BHM-1 strain of BHV-2. Most isolates tested did not generate any defective UL24 genes during the passage. However, a third of the UL24 genes of BHM-1 strain contained the two nts deletion, but only when a high multiplicity of infection (MOI) was used. Also in the isolate 554 at least a half of the UL24 genes were found to be altered independently of the MOT used. These UL24 genes had an insertion of four nts within the 274-297 nt region. The predicted truncation of the UL24 protein of both viruses occurred at the same stop codon. The region of the gene in which these mutations of the UL24 gene occurred is common to all herpesviruses.

Bovine herpesvirus type 2 is closely related to the primate alphaherpesviruses.

Bovine herpesvirus type 2 (BoHV-2), also known as bovine mammillitis virus, is classified in the Family Herpesviridae, Subfamily Alphaherpesvirinae, and Genus Simplexvirus along with herpes simplex viruses type 1 and 2 (HSV-1 and HSV-2) and other primate simplexviruses on the basis of similarities in 4 genes within the 15 kb U(L) 23-29 cluster. This could be explained either by a global similarity or a recombination event that brought primate herpesviral sequences into a bovine virus. Our sequences for DNA polymerase (U(L)30), a large gene adjacent to the previously identified conserved cluster, and glycoprotein G (U(S)4), a gene as distant from the cluster as possible on the circularized genome, confirm the close relationship between BoHV-2 and the primate simplexviruses, and argue for a global similarity and probably a close evolutionary relationship. Thus one can speculate that BoHV-2 may represent a greater hazard to humans than has been appreciated previously.

Loss of thymidine kinase activity due to a base deletion in a candidate vaccine strain of bovine herpesvirus 2.

The nucleotide (nt) sequence of the thymidine kinase (TK) gene (a 918 nt long coding region) of two TK-deficient (TK) strains of bovine herpesvirus 2 (BHV-2) was determined. The candidate vaccine strain C290BU5, which was no longer able to cause disease, was found to have an A deletion after nt 887 of the TK gene with a predicted change of His 296 to Pro, altering the last 10 amino acids (aa) and extending the gene by another 34 aa. The strain which still caused disease, C290BU3, had a T insertion after nt 16 causing a predicted chain termination after only 16 aa.

Glycoprotein E of bovine herpesvirus type 1 is involved in virus transmission by direct cell-to-cell spread.

In order to identify the role of the bovine herpesvirus type 1 (BHV-1) glycoprotein E (gE) in the viral infection cycle, we have constructed a BHV-1 gE deletion mutant strain (BHV-1 gE-). This strain was assayed in vitro by comparing its growth kinetics with the wild type strain used as a host of the deletion. Our results indicate that those conditions which prevent the infection by direct adsorption to the cells (presence of a semi-solid medium or presence of neutralizing antibodies in the medium) selectively inhibit the growth of the gE- strain, suggesting that gE plays a central role in the BHV-1 spread by direct cell-to-cell transmission, a major mechanism of the BHV-1 in vivo virulence.