George P. Allen

Equine herpesvirus type 1 unique short fragment encodes glycoproteins with homology to herpes simplex virus type 1 gD, gI and gE

The nucleotide sequence of a 6.4 kbp portion of the 10.6 kbp BamHI fragment D contained in the unique short region of the equine herpesvirus type 1 (EHV-1) genome has been determined. Analysis of this sequence revealed five open reading frames (ORFs), four complete and one incomplete, which were encoded by the same sense strand. Comparison of the EHV-1 DNA sequence with that encoding glycoproteins of other alphaherpesviruses has revealed no significant homologies. Comparison at the amino acid level, however, has demonstrated regions of significant sequence similarity between the three complete EHV-1 ORFs 2, 3 and 4, and the herpes simplex virus type 1 (HSV-1) glycoprotein gD encoded by the US6 gene, the HSV-1 glycoprotein gI encoded by the US7 gene and the HSV-1 glycoprotein gE encoded by the US8 gene, respectively. The interrupted ORF 5 was found to display partial homology with the HSV-1 US9-encoded protein, but no homology was found between the protein encoded by ORF 1 and other proteins. The three collinear EHV-1 ORFs encoding putative glycoproteins with homology to the HSV-1 glycoproteins were therefore designated EHV-1 gD, gI and gE, respectively. Moreover, further similarities were found between EHV-1 gD and pseudorabies virus (PRV) gp50, between EHV-1 gI and PRV gp63 and varicellazoster virus (VZV) gpIV, and between EHV-1 gE and PRV gI and VZV gpI. It is concluded that EHV-1, PRV, HSV-1 and VZV encode homologous glycoprotein genes in the small unique components of their genomes and that the genetic organization of these regions is conserved.

Risk factors for development of neurologic disease after experimental exposure to equine herpesvirus-1 in horses.

OBJECTIVE To identify risk factors associated with development of clinical neurologic signs in horses exposed to equine herpesvirus-1 (EHV-1). ANIMALS 36 adult horses. PROCEDURES Blood samples collected before and after challenge inoculation with nonneuropathogenic or neuropathogenic EHV-1 were analyzed for leukocyte-associated viremia, serum neutralizing antibody, and EHV-1-specific cytotoxic T-lymphocyte precursors (CTLPs). Associations between variables and neurologic disease and correlations between age category or breed and development of neurologic disease were examined. RESULTS 9 horses developed CNS signs (ataxia, hind limb paresis or paralysis, bladder atony, or recumbency). Neurologic deficits were correlated with infection by a neuropathogenic strain of EHV-1, age>20 years, high postexposure viremic load, and low preexposure concentration of CTLPs. No significant correlations were observed between preinfection titers or horse breed and postinfection development of neurologic signs. CONCLUSIONS AND CLINICAL RELEVANCE Horses with high concentrations of preexisting CTLPs, regardless of age, strain of virus, or titer, were more likely to control the magnitude of postinfection leukocyte-associated viremia and subsequent development of neurologic disease; therefore, CTLPs appear to be a critical requirement for protective immunity against EHV-1-induced myeloencephalopathy. The importance of achieving immunity related to high concentrations of vaccine-induced CTLPs in horses at high risk for exposure to neuropathogenic strains of EHV-1 is indicated.

The Increased Prevalence of Neuropathogenic Strains of EHV-1 in Equine Abortions

A panel of 426 archived EHV-1 isolates collected (1951-2006) from equine abortions was analyzed using a real-time Taq-Man((R)) allelic discrimination PCR assay. Based on previous findings, isolates possessing adenine at nucleotide position 2254 (A(2254)) in ORF30 were classified as having a non-neuropathogenic genotype and those with guanine at 2254 (G(2254)) were designated as the neuropathogenic genotype. The resultant data demonstrated that viruses with the neuropathogenic genotype existed in the 1950s and isolates with this genotype increased from 3.3% in the 1960s to 14.4% in the 1990s. The incidence of EHV-1 isolates from 2000 to 2006 with G at position 2254 is 19.4%, suggesting that viruses with the neuropathogenic genotype are continuing to increase in prevalence within the latent reservoir of the virus, leading to greater risks for costly outbreaks of equine herpesvirus neurologic disease. Another highly significant finding was two isolates failed to react with either probe in the allelic discrimination assay. These isolates were found to possess an adenine to cytosine substitution at position 2258 (A(2258)-->C(2258)) in ORF30, in addition to A(2254)-->G(2254). Interestingly, the non-neuropathogenic RAC-H modified live vaccine strain of EHV-1 also contains both A(2254)-->G(2254) and A(2258)-->C(2258) substitutions. This finding clearly suggests that additional research is required before the genetic basis of the neuropathogenic phenotype in EHV-1 is fully understood.

Herpesviral Diseases of the Horse

Equidae are susceptible to infection by three viruses of the family Herpetoviridae. Equine herpesvirus 1 (EHV-1), an alphaherpesvirus which exists as two subtypes, is responsible for respiratory disease of young animals as well as for sporadic and epizootic abortigenic or neurologic disease. Epizootic abortion caused by EHV-1 infection can cause economically devastating losses.

Identification of the envelope surface glycoproteins of equine herpesvirus type 1.

The structural polypeptides of purified enveloped virions of the Army 183 strain of equine herpesvirus type 1 (EHV-1) were examined by different analytical techniques to identify the envelope glycoproteins. Glycoproteins were identified by electrophoretic analysis in polyacrylamide slab gels of virus labelled in vivo with [3H]glucosamine or labelled enzymically in vitro with either UDP-[14C]galactose or sodium [3H]borohydride. Fluorograms revealed eleven glycoproteins (mol. wt. 260000, 150000, 138000, 90000, 87000, 65000, 62000, 60000, 50000, 46000, and 24000). These glycoproteins probably correspond to virion protein (VP) 1-2, 9b, 10, 13, 14, 16, 17, 18, 21, 22a and 25 respectively, as designated in two other EHV-1 strains. In addition, a poorly resolved glucosamine-rich region (mol. wt. 250000 to 200000) corresponded to VP 3 to 8. The two isotopic surface labelling methods revealed that all the virus glycoproteins were exposed on the envelope surface.

Characterization of the major glycoproteins of equine herpesviruses 4 and 1 and asinine herpesvirus 3 using monoclonal antibodies.

A panel of 14 monoclonal antibodies (MAbs) was used to characterize the high abundance glycoproteins of equine herpesviruses 4 (EHV-4) and 1 (EHV-1), and asinine herpesvirus 3 (AHV-3). The specificities of the MAbs, which had been determined previously for strains of EHV-4 and -1 from the U.S.A., in general were confirmed by ELISA for Australian strains of these viruses. Of the 14 MAbs seven were EHV-4 and -1 type-common and cross-reacted with AHV-3. Of the five MAbs that were EHV-1 type-specific, four cross-reacted with AHV-3, whereas neither of the EHV-4 type-specific MAbs reacted with AHV-3, providing further evidence for a closer evolutionary relationship between EHV-1 and AHV-3 than that between either of these viruses and EHV-4. By Western blot and immunoprecipitation analyses, the identity of the six major glycoproteins, gp2, gp10, gp13, gp14, gp18 and gp21/22a, of an Australian EHV-1 isolate was verified, and it was shown that AHV-3 had cross-reactive glycoproteins of very similar Mr to those of EHV-1; five homologous glycoproteins of EHV-4 were also identified. It was determined that the EHV-4 gp13 homologue had a much reduced Mr (67K) when the virus was grown in a continuous cell line than when grown in equine foetal kidney cells (95K). It is suggested that altered glycosylation by the cell line is responsible for this change in Mr. Those glycoproteins acting as major immunogens in the naturally infected host, at least in their ability to elicit antibody, were identified. It was found that gp2, gp13, gp14, gp18 and a glycoprotein at 120K (EHV-1) or 116K (EHV-4) were all important immunogens in mares following EHV-1-induced abortion, and in a specific pathogen-free foal experimentally infected with EHV-1 and later cross-challenged with EHV-4. Gp2, gp14 and gp18 were the major immunogens in the donkey in response to AHV-3 infection. The type specificity associated with these glycoproteins was also examined and it was found that although most if not all contain type-specific epitopes, gp2 and a glycoprotein at 120K, and to a lesser extent gp13 and gp18, were significantly type-specific in the serum from a mare following natural EHV-1 infection and abortion.

Synthesis and processing of equine herpesvirus type 1 glycoprotein 14

Glycoprotein 14 (gp14) of equine herpesvirus type 1 (EHV-1), the homolog of herpes simplex virus (HSV) glycoprotein B (gB), was investigated employing a panel of monoclonal antibodies to ascertain the regulatory class, rate of synthesis, and type of glycosylation of this polypeptide. Application of immunoprecipitation, Western blot, and SDS-PAGE analysis in conjunction with the use of metabolic inhibitors (cycloheximide, antinomycin D, phosphonoacetic acid, tunicamycin, and monensin), and time-course and pulse-chase experiments revealed the following information: (1) Three gp14-related polypeptides with molecular weights of 138 kilodaltons (K), 77-75K, and 55-53K are present in EHV-1-infected cell extracts. (2) All three species are synthesized in the presence of the DNA synthesis inhibitor phosphonoacetic acid although their synthesis is enhanced by DNA replication, indicative of a beta-gamma class molecule. (3) The 138K species is synthesized first as a precursor of the smaller species of gp14, the 77-75K and 55-53K forms. (4) Use of glycosylation inhibitors and digestion of immunoprecipitated gp14 with endoglycosidases indicate that the primary translation product is a 118K molecule which is cotranslationally glycosylated to the 138K form by the addition of high mannose oligosaccharides. (5) The 77-75K species contains both high mannose and hybrid oligosaccharides while the 55-53K form of gp14 contains some complex oligosaccharides. (6) In the absence of a reducing agent, the 138K polypeptide and a large 145K species are observed in both infected cell extracts and purified virions. Thus, EHV-1 gp14 appears to be synthesized as a large precursor molecule of 138K and is proteolytically cleaved to two smaller forms, 77-75K and 55-53K, which are linked by a disulfide bond(s) to form a 145K complex. This model of gp14 synthesis and maturation is similar to those proposed for a number of HSV gB equivalents found in the Alphaherpesvirnae.

Genetic relatedness and pathogenicity of equine herpesvirus 1 isolated from onager, zebra and gazelle.

Summary.Equine herpesvirus 1 was isolated from an onager in 1985, a zebra in 1986 and a Thomson’s gazelle in 1996 in USA. The genetic relatedness and pathogenicity of these three viruses were investigated based on the nucleotide sequences of the glycoprotein G (gG) gene, experimental infection in hamsters, and comparison with horse isolates. The gG gene sequences of EHV-1 from onager and zebra were identical. The gG gene sequences of the gazelle isolate showed 99.5% identity to those of onager and zebra isolates. The gG gene sequences of EHV-1 isolated from horses were 99.9–100% identical and 98, 98 and 97.8% similar to gG from onager, zebra and gazelle isolates, respectively. Hamsters inoculated with onager, zebra and gazelle isolates had severe weight loss, compared with hamsters inoculated with horse isolates. The histopathological findings were related to the virulence of each isolate. The results indicated that EHV-1 isolates from onager, zebra and gazelle differ from horse EHV-1 and are much more virulent in hamsters.

Polymorphism of open reading frame 71 of equine herpesvirus-4 (EHV-4) and EHV-1

Open reading frame (ORF) 71 genes of both equine herpesvirus-1 (EHV-1) and EHV-4 encode a unique glycoprotein, which has been described to vary in molecular mass from 200 to 450 kDa. Using PCR and nucleotide sequence analysis, it was shown that the ORF 71 genes of EHV-1 and EHV-4 are polymorphic due to a variable number of reiterated sequences in two regions, designated regions A and B. Region A was threonine-rich and was located near the N terminus. Region B comprised a 38 amino acid repeat near the C terminus that expanded following cell culture adaptation. Western blot analysis of viruses showed that EHV-4 gp2 was modified by glycosylation and that variation in region A resulted in the marked differences in the molecular mass of EHV-4 gp2.

Cell-free synthesis of equine herpesvirus type 3 nucleocapsid polypeptides.

Abstract Rate-velocity centrifugation in sucrose gradients of nuclear lysates from cells infected with equine herpesvirus type 3 (EHV-3) demonstrated two major species of viral nucleocapsids that differed in buoyant density, sedimentation velocity, and DNA content. Nucleocapsids present in the bottom sucrose band (B capsids) comprised 62% of the total nucleocapsid protein and possessed a higher density and seven times more DNA than nucleocapsids isolated from the top sucrose band (A capsids). Analysis of B-capsid proteins in SDS-polyacrylamide gels revealed the presence of five major polypeptides with molecular weights of 151,000, 59,000, 48,000, 40,000, and 30,000. In vitro synthesis of EHV-3 nucleocapsid polypeptides was demonstrated in a cell-free translation system consisting of (1) 150,000 g supernatant from rabbit reticulocytes; (2) preincubated ribosomes from the cells of a continuous equine cell line (ETCC); (3) factors dissociated from infected ETCC cell ribosomes with high salt; (4) RNA extracted from the cytoplasm of cells infected with EHV-3. EHV-3 nucleocapsid polypeptides were selected from the total in vitro synthesized products by indirect immunoprecipitation with specific antiviral rabbit serum and antiglobulin goat serum. Polyacrylamide-gel electrophoretic analysis of the cell-free products synthesized in response to poly(A)-rich RNA from EHV-3 infected cells and precipitated with immune serum prepared against purified viral nucleocapsids indicated that four of the five major nucleocapsid polypeptides were made in vitro .