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Equid herpesvirus 3 (EHV-3) is a member of the subfamily Alphaherpesvirinae that causes equine coital exanthema. Here, we report the first complete genome sequence of EHV-3. The 151,601-nt genome encodes 76 distinct genes like other equine alphaherpesviruses, but genetically, EHV-3 is significantly more divergent., (Copyright © 2014 Sijmons et al.)

Cervid herpesvirus 2 (CvHV2) has been isolated from reindeer (Rangifer tarandus tarandus), and serological data indicate that in reindeer this virus is endemic in Fennoscandia, Alaska, Canada, and Greenland. CvHV2 has been described as a cause of subclinical genital infections in reindeer, but little information on primary infections exists. In this study, six seronegative and presumably pregnant reindeer were allocated to one of two groups. Two animals were inoculated with CvHV2 intratracheally, and two animals intravaginally, with one control animal in each group receiving sterile water. Mild hyperthermia and serous discharges from the vagina and nose were observed. No abortions were recorded, but one calf died shortly after birth. Inoculated animals seroconverted and had neutralizing antibodies after days 7 to 10 postinfection. CvHV2 was detected by PCR in nasal and vaginal swabs from animals in both groups but could be isolated only from nasal swabs in the respiratory group and from vaginal swabs in the genital group. CvHV2 was detected by PCR in various organs and tissues postmortem. In control animals, the virus could not be isolated in spite of PCR-positive nasal and vaginal swab samples and some degree of positive immunostaining. One of the animals that were inoculated intratracheally developed a hemorrhagic, necrotizing bronchopneumonia, which was CvHV2 positive by PCR and immunohistochemistry. We conclude that CvHV2 can cause systemic infection, that both genital and respiratory inoculations can lead to virus shedding, and that the virus can infect the fetus in utero.

Phylogenetic studies of the emergence and spread of natural recombinants in herpesviruses infecting humans and animals have been reported recently. However, despite an ever-increasing amount of evidence of recombination in herpesvirus history, the recombination process and the consequences on the genetic diversity of the progeny remain poorly characterized. We addressed this issue by using multiple single-nucleotide polymorphisms (SNPs) differentiating the two subtypes of an alphaherpesvirus, bovine herpesvirus 1 (BoHV-1). Analysis of a large sample of progeny virions obtained in a single growth cycle of coinfected BoHV-1 strains provided a prospective investigation of the recombination dynamics by using SNPs as recombination markers. We found that the simultaneous infection with two closely related herpesviruses results in a highly diversified recombination mosaic. From the analysis of multiple recombinants arising in the progeny, we provide the first evidence of genetic interference influencing the recombination process in herpesviruses. In addition, we report striking differences in the levels of recombination frequency observed along the BoHV-1 genome. With particular emphasis on the genetic structure of a progeny virus population rising in vitro, our data show to which extent recombination participates to the genetic diversification of herpesviruses.

Homologous recombination between different species of alphaherpesviruses has been described between herpes simplex viruses 1 and 2 but has not yet been observed between other alphaherpesviruses. In the present study we chose to assess to what extent in vitro recombination can occur between members of a well-defined group of closely related viruses such as ruminant alphaherpesviruses. At 24 h after infection of epithelial bovine kidney cells with a double-deleted mutant of bovine herpesvirus 1 (BoHV-1) (containing green fluorescent protein and red fluorescent protein genes) and different ruminant alphaherpesviruses, four types of progeny viruses were detected and distinguished according to their phenotype. Frequent recombination events between identical or different strains of BoHV-1 were observed (up to 30%), whereas only two BoHV-1/BoHV-5 recombinants were identified, and no recombinants between BoHV-1 and less closely related caprine and cervine herpesviruses were detected. Restriction analysis of the genomes of the two BoHV-1/BoHV-5 recombinants showed different genetic backgrounds. One possessed a restriction pattern close to BoHV-1, whereas the other one was close to BoHV-5. This exhaustive analysis of each combination of coinfection in a unique situation of five closely related alphaherpesviruses revealed the importance of a high degree of genetic relatedness and similar parental virus growth kinetics for successful interspecific recombination., (Copyright 2004 American Society for Microbiology)

Homologous recombination between strains of the same alphaherpesvirus species occurs frequently both in vitro and in vivo. This process has been described between strains of herpes simplex virus type 1, herpes simplex virus type 2, pseudorabies virus, feline herpesvirus 1, varicella-zoster virus, and bovine herpesvirus 1 (BoHV-1). In vivo, the rise of recombinant viruses can be modulated by different factors, such as the dose of the inoculated viruses, the distance between inoculation sites, the time interval between inoculation of the first and the second virus, and the genes in which the mutations are located. The effect of the time interval between infections with two distinguishable BoHV-1 on recombination was studied in three ways: (i) recombination at the level of progeny viruses, (ii) interference induced by the first virus infection on beta-galactosidase gene expression of a superinfecting virus, and (iii) recombination at the level of concatemeric DNA. A time interval of 2 to 8 h between two successive infections allows the establishment of a barrier, which reduces or prevents any successful superinfection needed to generate recombinant viruses. The dramatic effect of the time interval on the rise of recombinant viruses is particularly important for the risk assessment of recombination between glycoprotein E-negative marker vaccine and field strains that could threaten BoHV-1 control and eradication programs.

The control of infectious bovine rhinotracheitis induced by bovine herpesvirus 1 (BoHV-1) requires sensitive and specific diagnostic assays. As BoHV-1 is antigenically and genetically related to four other alphaherpesviruses of ruminants-namely, BoHV-5, caprine herpesvirus 1 (CpHV-1), cervine herpesvirus 1 (CvHV-1) and CvHV-2-diagnostic tests able to discriminate BoHV-1 from these related viruses are needed to avoid misdiagnosis, especially because some of these viruses are able to cross the species barrier. In this study, murine monoclonal antibodies (MAbs) specific for BoHV-1, BoHV-5, CpHV-1, CvHV-1, and CvHV-2 were produced with the aim of setting up an immunofluorescence assay able to discriminate between these related herpesviruses. Produced MAbs were selected for their viral specificity by enzyme-linked immunosorbent assay and indirect immunofluorescence staining of virus-infected cells. Radioimmunoprecipitation characterization of the selected MAbs revealed that four of them are directed against glycoprotein C (gC) and one of them is directed against gD of these related viruses. The obtained results demonstrate that the antibodies produced allow an unambiguous discrimination of each of the four alphaherpesviruses related to BoHV-1.

Recombination is thought to be an important source of genetic variation in herpesviruses. Several studies, performed in vitro or in vivo, detected recombinant viruses after the coinoculation of two distinguishable strains of the same herpesvirus species. However, none of these studies investigated the evolution of the relative proportions of parental versus recombinant progeny populations after coinoculation of the natural host, both during the excretion and the reexcretion period. In the present study, we address this by studying the infection of cattle with bovine herpesvirus 1 (BoHV-1). The recombination of two BoHV-1 mutants lacking either glycoprotein C (gC(-)/gE(+)) or E (gC(+)/gE(-)) was investigated after inoculation of cattle by the natural route of infection. The results demonstrated that (i) recombination is a frequent event in vivo since recombinants (gC(+)/gE(+) and gC(-)/gE(-)) were detected in all coinoculated calves, (ii) relative proportions of progeny populations evolved during the excretion period toward a situation where two populations (gC(+)/gE(+) and gC(-)/gE(+)) predominated without fully outcompeting the presence of the two other detected populations (gC(+)/gE(-) and gC(-)/gE(-)), and (iii) after reactivation from latency, no gC(+)/gE(-) and gC(-)/gE(-) progeny viruses were detected, although gC(+)/gE(-) mutants, when inoculated alone, were detected after reactivation treatment. In view of these data, the importance of gE in the biology of BoHV-1 infection and the role of recombination in herpesvirus evolution are discussed.

The consequences of the vaccination of neonatal calves with the widely used live-attenuated temperature-sensitive (ts) bovine herpesvirus type 1 (BHV-1) were investigated. The ts strain established acute and latent infections in all vaccinated calves either with or without passive immunity. Four of seven calves vaccinated under passive immunity became clearly BHV-1 seronegative by different serological tests, as did uninfected control calves after the disappearance of maternal antibodies, and they remained so for long periods. A cell-mediated immune response was detected by a BHV-1 gamma interferon assay, but this test failed to detect the seronegative latent carriers (SNLCs). While they are not detected, SNLCs represent a threat for BHV-1-free herds or countries. This study demonstrates that SNLCs can be easily obtained by inoculation with a live-attenuated BHV-1 under passive immunity and that latent carrier animals without any antibody do exist. Consequently, this situation could represent a good model to experimentally produce SNLCs.

The presence of maternally derived antibodies can interfere with the development of an active antibody response to antigen. Infection of seven passively immunized young calves with a virulent strain of bovine herpesvirus type 1 (BHV-1) was performed to determine whether they could become seronegative after the disappearance of maternal antibodies while latently infected with BHV-1. Four uninfected calves were controls. All calves were monitored serologically for 13 to 18 months. In addition, the development of a cell-mediated immune response was assessed by an in vitro antigen-specific gamma interferon (IFN-gamma) production assay. All calves had positive IFN-gamma responses as early as 7 days until at least 10 weeks after infection. However, no antibody rise was observed after infection in the three calves with the highest titers of maternal antibodies. One of the three became seronegative by virus neutralization test at 7 months of age like the control animals. This calf presented negative IFN-gamma results at the same time and was classified seronegative by enzyme-linked immunosorbent assay at around 10 months of age. This calf was latently infected, as proven by virus reexcretion after dexamethasone treatment at the end of the experiment. In conclusion, this study demonstrated that BHV-1-seronegative latent carriers can be obtained experimentally. In addition, the IFN-gamma assay was able to discriminate calves possessing only passively acquired antibodies from those latently infected by BHV-1, but it could not detect seronegative latent carriers. The failure to easily detect such animals presents an epidemiological threat for the control of BHV-1 infection.

Bovine herpesvirus 1 (BHV-1) induces apoptotic cell death in bovine peripheral blood mononuclear cells and B-lymphoma cells. Using a BHV-1 glycoprotein H null mutant, we have demonstrated that although penetration of BHV-1 is not required, attachment of BHV-1 viral particles is essential for the induction of apoptosis.