Construction and manipulation of a full-length infectious bacterial artificial chromosome clone of equine herpesvirus type 3 (EHV-3).

Equine herpesvirus type 3 (EHV-3) is the causal agent of equine coital exanthema, a disease characterized by pox-like lesions on the penis of stallions and the vulva of mares. Although the complete genomic sequence of EHV-3 has been recently made available, its genomic content remains poorly characterized and the molecular mechanisms of disease development not yet elucidated. In an attempt to facilitate genetic manipulation of EHV-3, we describe here the construction of a full-length infectious bacterial artificial chromosome (BAC) clone of EHV-3. Mini-F vector sequences were inserted into the intergenic region between ORF19 and ORF20 (UL41 and UL40, respectively) of EHV-3 strain C175 by homologous recombination in equine dermal cells (NBL-6). DNA of the resulting recombinant virus was electroporated into E. coli and a full-length EHV-3 BAC clone was recovered. Virus reconstituted after transfection of the EHV-3 BAC into NBL-6 cells showed growth properties in vitro that were indistinguishable from those of the parental virus. To assess the feasibility of mutagenesis of the cloned EHV-3 genome, recombinant viruses targeting the glycoprotein E (gE) gene were generated using Red recombination in E. coli and in vitro growth properties of the recombinant viruses were evaluated. We first repaired the gE (ORF74) coding region, since the parental virus used for BAC cloning specifies a truncated version of the gene, and then created gE-tagged and gE-null versions of the virus. Our results demonstrated that: (i) EHV-3 can be efficiently cloned as a BAC allowing easy manipulation of its genome; (ii) gE is dispensable for EHV-3 growth in vitro and is expressed as a product of approximately 110-kDa in infected cells; (iii) viruses having a deletion compromising gE expression or with a truncation of the cytoplasmic and transmembrane domains are significantly compromised with regard cell-to-cell spread. The cloning of EHV-3 as a BAC simplifies future studies to identify the role of its coding genes in viral pathogenesis and host immune responses.

Herpesvirus-associated neurological disease in a donkey.

A 4-year-old donkey was evaluated for progressive neurological abnormalities consisting of depression, stupor, weakness, and recumbency. Diagnostic evaluation for viral involvement identified an asinine herpesvirus in DNA extracted from deep pharyngeal swabs. Specific primers were designed based on comparison with equine herpesviral DNA polymerase sequences and yielded an 875-base pair product from the donkey. This sequence had complete identity with short sequences of asinine herpesvirus previously identified in donkeys with interstitial pneumonia. Amino acid analysis of the entire sequence indicated high similarity with Equid herpesvirus 7 (91%), Zebra herpesvirus 1 (90%), and Equid herpesvirus 2 (89%). With supportive treatment and physical therapy, the donkey gradually recovered over 5 days of hospitalization and returned to normal function. The current case illustrates the potential of a novel asinine herpesvirus to induce neurological disease in donkeys and provides a large viral sequence allowing confident assignment of this virus to the subfamily Gammaherpesvirinae.

Temporal detection of equine herpesvirus infections of a cohort of mares and their foals.

The objectives of this study were to estimate the prevalence of equine herpesviruses (EHV) 1-5 in the nasal secretions (NS) of a cohort of 12 mares and their foals from birth to 6 months of age, estimate the prevalence of EHV-1-5 infection of peripheral blood mononuclear cells (PBMC) of selected foals, and investigate phylogenetic relationships amongst the various strains of EHV-2 and 5. Virus-specific PCR assays were used to detect EHV-1-5 in NS and PBMC. A homologous portion of the glycoprotein B (gB) gene of the various strains of EHV-2 and 5 was sequenced and compared. EHV-2, 4, and 5 were all detected in NS from the horses, but only EHV-4 was associated with respiratory disease (P=0.005). EHV-2 and 5 infections were both common, but foals shed EHV-2 in their NS earlier in life than EHV-5 (P=0.01). Latent EHV-2 and 5 infections were detected in the PBMC of 75 and 88%, respectively, of the foals at approximately 6 months of age. The strains of EHV-2 shed in the NS of individual horses were more genetically heterogeneous than the strains of EHV-5 (95.5-99.3% versus 98.8-99.3% nucleotide identity, respectively). One-month-old foals typically shed strains of EHV-2 that were identical to those infecting their dams whereas older foals often shed virus strains that were different from those of their dams. Although herpesvirus infections were ubiquitous in this cohort of horses, there were distinct clinical consequences and clear epidemiological differences between infections with the different viruses.

Detection of equine herpesvirus 3 in equine skin lesions by polymerase chain reaction.

During a recent breeding season, ulcerative, pustular skin lesions were observed on the external genitalia of 2 mares and 1 stallion within a small herd. Based on the location and description of the skin lesions plus the clinical history, equine coital exanthema, caused by equine herpesvirus 3 (EHV3), was the primary differential diagnosis. Scrapings of skin lesions from the perineum of 2 mares were submitted for diagnostic evaluation. Virus isolation was attempted by inoculation of several cell lines of equine origin, but no cytopathic agent was detected. The skin scrapings were processed for DNA extraction, and polymerase chain reaction (PCR) amplification was performed for herpesvirus DNA polymerase and DNA-packaging protein (terminase) genes using nested, degenerate primers targeted to conserved regions of the herpesvirus genome. Products of the expected sizes were generated for both assays, and subsequent nucleotide sequencing of the amplification products established that EHV3 had been detected in DNA extracted from the skin lesions. Detection of EHV3 was confirmed using an EHV3-specific PCR assay targeted to the gC gene. Using the novel EHV3 nucleotide sequence identified in this report, a sensitive and specific PCR assay targeted to the highly conserved DNA polymerase gene was developed.

Identification of equine herpesvirus 3 (equine coital exanthema virus), equine gammaherpesviruses 2 and 5, equine adenoviruses 1 and 2, equine arteritis virus and equine rhinitis A virus by polymerase chain reaction.

OBJECTIVE: To develop rapid (< 8 hour) tests using polymerase chain reaction (PCR) for the diagnosis of equine herpesvirus 3 (EHV3; equine coital exanthema virus), equine gammaherpesviruses 2 (EHV2) and EHV5, equine adenovirus 1 (EAdV1), EAdV2, equine arteritis virus (EAV), equine rhinitis A virus (ERAV; formerly equine rhinovirus 1) DESIGN: Either single round or second round (seminested) PCRs were developed and validated. METHODS: Oligonucleotide primers were designed that were specific for each virus, PCR conditions were defined and the specificity and sensitivity of the assays were determined. The application of the tests was validated using a number of independent virus isolates for most of the viruses studied. The PCRs were applied directly to clinical samples where samples were available. RESULTS: We developed a single round PCR for the diagnosis of EHV3, a seminested PCR for EHV2 and single round PCRs for EHV5, EAdV1, EAdV2 and RT-PCRs for EAV and ERAV. The PCR primer sets for each virus were designed and shown to be highly specific (did not amplify any recognised non-target template) and sensitive (detection of minimal amounts of virus) and, where multiple virus isolates were available all isolates were detected. CONCLUSION: The development and validation of a comprehensive panel of PCR diagnostic tests, predominantly for viruses causing equine respiratory disease, that can be completed within 8 hours from receipt of clinical samples, provides a major advance in the rapid diagnosis or exclusion diagnosis of these endemic equine virus diseases in Australia.

The nucleotide sequence of the glycoprotein G homologue of equine herpesvirus 3 (EHV3) indicates EHV3 is a distinct equid alphaherpesvirus.

EHV3 causes equine coital exanthema and has been classified as an alphaherpesvirus on the basis of its biological properties; however due to the absence of any sequence information the phylogenetic relationship has not previously been examined. The complete nucleotide sequence of the EHV3 glycoprotein G (gG) gene was determined and showed that this virus is most closely related to the alphaherpesviruses equine herpesviruses type 1 (EHV 1) and type 4 (EHV4). EHV3 gG contains conserved and variable regions which are homologous to those previously defined for EHV1 and EHV4 gG proteins. Consistent with EHV1 and EHV4 gG, the variable region of EHV3 gG was found to elicit a strong antibody response in experimentally and naturally infected horses and could be exploited for use as a diagnostic reagent.

Temporal regulation of equine herpesvirus type 3 transcription.

The transcription of equine herpesvirus type 3 (EHV-3; equine coital exanthema virus) has been examined and found to be temporally regulated into three classes: immediate early (IE), early (E), and late (L). Hybridization of in vivo 32PO4-labeled transcripts revealed that IE transcript(s) are derived exclusively from the inverted repeat segments (IRs) of the viral genome, while E and L transcripts are not restricted to any specific region of the genome. Northern blot analysis of EHV-3 IE RNA revealed a single transcript of approximately 5.7 kb (3.8 MDa). We have previously shown that transcription of equine herpesvirus type 1 (EHV-1) DNA is temporally regulated and produces a single 6 kb IE RNA which is derived from the IRs segments. In this paper, we show that the EHV-1 and EHV-3 IE RNA species are homologous, reflecting the colinearity of the genomes of these two related viruses. While four IE polypeptides are synthesized in EHV-1 infected cells in the presence of actinomycin D following the removal of a cycloheximide block, only one major IE polypeptide (180 kDa) is detectable in EHV-3 infected cells under these conditions. However, immunoprecipitation of EHV-3 infected cell extracts with polyvalent rabbit antisera to IE1 of EHV-1 revealed at least two other viral specific IE polypeptides.

Temperature sensitivity of equine herpesvirus isolates: a brief review.

This article reviews the findings on temperature sensitivity of equine herpesvirus isolates with an emphasis on equine herpesvirus 3, etiological agent of equine coital exanthema. The hypothesis is presented that the relative apathogenic nature of this herpesvirus may be an indirect result of its inability to synthesize and/or process glycoproteins needed by the virus to produce infectious virions at the normal body temperature of its natural host. It is suggested that equine herpesvirus 3 is the more evolved and naturally attenuated member of the equine herpesviruses.

Molecular pathogenesis of equine coital exanthema: identification and expression of infected cell polypeptides at the restricted temperature during equine herpesvirus 3 infection.

Equine herpesvirus 3 (EHV-3)-infected equine cells display a kinetics of infected cell polypeptide (ICP) synthesis at 34 degrees C that is typical of coordinate cascade gene regulation of herpesviruses. In contrast, when infected cell cultures are incubated at the restricted temperature of 39 degrees C, the shift from early (beta) gene expression to late (gamma) gene expression is perturbed, i.e., there is an accumulation of early (beta) gene products and a decrease in, or absence of, late (gamma) gene products. Some of the affected late (gamma) gene products may be glycoproteins since these ICPs co-migrated with radiolabeled bands from infected cells incubated with [3H] glucosamine, separated by polyacrylamide gel electrophoresis. These findings are consistent with previous findings (Jacob, 1986), indicating that the growth restriction is in a late viral function(s) and possibly involves envelopment of nucleocapsids into infectious virions.

Molecular pathogenesis of equine coital exanthema (ECE): temperature sensitivity (TS) and restriction endonuclease (RE) fragment profiles of several field isolates.

Examination of six field isolates of equine herpesvirus 3, the causative agent of equine coital exanthema, indicates that all were temperature sensitive (ts) at the body temperature, 39 degrees C, of their host (Equine asinus and callabus) when grown in cell culture. The isolates were characterized by fingerprint analysis with the restriction endonucleases XbaI, EcoRI, BamHI and Hind III to establish possible epidemiologic relatedness. Three of the six isolates may be considered related. Variation in the mobility of the BamHI-A and Hind III-K fragments indicates that a small plaque isolate may contain a 5.7 kb insert of DNA in the unique short region of the genome.

Molecular pathogenesis of equine coital exanthema: restriction endonuclease digestions of EHV-3 DNA and indications of a unique XbaI cleavage site.

Equine herpesvirus type 3 (EHV-3) DNA, isolated from purified virions of the large-plaque strain, was digested with the restriction endonucleases XbaI, Bg/II, EcoRI, and HindIII. Several lines of evidence indicated that the DNA extracted from purified virions was composed of long (L) and short (S) components and was present as two isomeric forms, P and IS. The evidence included: (i) after electrophoresis on agarose gels, the summed molecular weights of the digestion products exceeded that expected from intact, unit size DNA; (ii) quantitative measurements of radioactivity (molar ratios) indicated 'minor bands' (0.5 M) interspersed among the major (1.0 M) bands; and (iii) a brief digestion with lambda-5'-exonuclease, prior to digestion with restriction endonuclease, resulted in the loss of some submolar and molar ratio bands, indicative of three termini. A preliminary fragment linkage map of the XbaI digestion products revealed EHV-3 DNA to contain only one recognition site in the unique sequence of the S component. From this linkage map, the size of the S component was deduced to be (22.3 +/- 5) X 10(6) molecular weight.

Structure of the genome of equine herpesvirus type 3.

Restriction endonuclease mapping studies were performed to determine the molecular structure of the genome of equine herpesvirus type 3 (EHV-3). Purified EHV-3 DNA, either unlabeled or 32P-labeled, was analyzed using the restriction enzymes BamHI, BclI, BglII, EcoRI, and HindIII. The findings that four 0.5 M (molar) fragments were present, that two of these were terminal fragments, and that all 0.5 M fragments contained homologous DNA sequences as judged by DNA hybridization analyses indicated that DNA sequences located at one terminus are repeated within the molecule and that two populations of molecules exist with regard to the arrangement of this pair of shared sequences. Mapping of BamHI, BclI, BglII, EcoRI, and HindIII fragments by double digestion of intact EHV-3 DNA, reciprocal digestion of isolated restriction enzyme fragments, and blot hybridization experiments revealed that the EHV-3 genome is a linear, double-stranded DNA molecule with a molecular size of 96.2 +/- 0.48 MDa and is comprised of two covalently linked segments, designated L (long) and S (short). The S region is approximately 22.9 MDa in size and consists of a unique segment (Us) of approximately 5.8 MDa bracketed by 8.5 MDa inverted repeat sequences that allow the S region to invert relative to the fixed L region which is approximately 73.3 MDa in size and consists only of unique sequences. Thus, these data confirm that EHV-3 DNA exists in two isomeric forms and has a molecular structure similar to that of the genomes of EHV-1 (B. E. Henry, S. A. Robinson, S. A. Dauenhauer, S. S. Atherton, G. S. Hayward, and D. J. O'Callaghan, Virology 115, 97-114, 1981; D. J. O'Callaghan, G. A. Gentry, and C. C. Randall, "The Herpesvirus," Vol. 2, pp. 215-318, Plenum, New York, 1983; D. J. O'Callaghan, B. E. Henry, J. H. Wharton, S. A. Dauenhauer, R. B. Vance, J. Staczek, and R. A. Robinson, "Developments in Molecular Virology," Vol. 1, pp. 387-418, Nijhoff, The Hague, 1981; W. T. Ruyechan, S. A. Dauenhauer, and D. J. O'Callaghan, J. Virol., 42, 297-300, 1982), pseudorabies virus (W. Stevely, J. Virol., 22, 232-234, 1977; T. Ben-Porat, F. J. Rixon, and M. L. Blankenship, Virology, 95, 285-294, 1979), varicella-zoster virus (A. M. Dumas, J. L. Geelen, M. W. Weststrate, P. Wertheim, and J. Van Der Noordaa, J. Virol., 39, 390-400, 1981; S. E. Straus, H. S. Aulakh, W. T. Ruyechan, J. Hay, T. A. Casey, G. F. Vande Woude, J. Owens, and H. A. Smith, J. Virol., 40, 516-525, 1981.(ABSTRACT TRUNCATED AT 400 WORDS)

Analysis of small and large plaque variants of equine herpesvirus type 3 by restriction endonucleases. Brief report.

Six of 6 equine herpesvirus type 3 (EHV3) isolates, 5 of which were epidemiologically unrelated, produced a mixture of small and large plaque variants in equine foetal kidney cells under methylcellulose. In 4 of 4 instances the cleavage site(s) generating the Bam HI A fragment of large plaque variants was distinct from the site(s) for the same fragment of small plaque variants.

Genetic relatedness of equine herpesvirus types 1 and 3.

The genetic relatedness of two types of equine herpesviruses (EHVs), 1 (EHV-1) and 3 (EHV-3), was determined by DNA-DNA reassociation kinetics. Denatured, labeled viral DNA probe was allowed to reassociate in the presence or absence of the second unlabeled viral DNA. The initial rate of reassociation of either labeled viral DNA was increased by the presence of the heterologous viral DNA to an extent indicating only 2 to 5% homology between the two EHV genomes. Moreover, labeled RNA extracted from EHV-3-infected cells hybridized to filter-immobilized EHV-1 DNA only 2 to 3 percent as efficiently as to the homologous EHV-3 DNA. These results demonstrate that the genital (EHV-3) and nongenital (EHV-1) types of EHVs exhibit very little genetic homology.