Sequence analysis of the 4.7-kb BamHI-EcoRI fragment of the equine herpesvirus type-1 short unique region.

To localize gene that may encode immunogens potentially important for recombinant vaccine design, we have analysed a region of the equine herpesvirus type-1 (EHV-1) genome where a glycoprotein-encoding gene had previously been mapped. The 4707-bp BamHI-EcoRI fragment from the short unique region of the EHV-1 genome was sequenced. This sequence contains three entire open reading frames (ORFs), and portions of two more. ORF1 codes for 161 amino acids (aa), and represents the C terminus of a possible membrane-bound protein. ORF2 (424 aa) and ORF3 (550 aa) are potential glycoprotein-encoding genes; the predicted aa sequences contain possible signal sequences, N-linked glycosylation sites and transmembrane domains; they also show homology to the glycoproteins gI and gE of herpes simplex virus type-1 (HSV-1), and the related proteins of pseudorabies virus and varicella-zoster virus. The predicted aa sequence of ORF4 shares no homology with other known herpesvirus proteins, but the nucleotide sequence shows a high level of homology with the corresponding region of the EHV-4 genome. ORF5 may be related to US9 of HSV-1.

High level expression of equine herpesvirus 1 glycoproteins D and H and their role in protection against virus challenge in the C3H (H-2Kk) murine model.

N and C-terminal truncated forms of equine herpesvirus 1 (EHV 1) glycoproteins gD and gH were expressed in baculovirus resulting in the production of secreted recombinant proteins. A carboxy-terminal histidine tag was included on each of the genes for protein isolation by nickel affinity chromatography. Recombinant gD was recognized by three gD specific monoclonal antibodies, 20C4, 5H6 and F3132. F3132 is a conformationally dependent monoclonal antibody with virus neutralizing activity. Expression of gH was confirmed by reacting the protein with the gH peptide specific antiserum R319. The truncated gD gene was also expressed as a beta-galactosidase fusion protein which was purified from E. coli by nickel affinity chromatography. C3H mice were inoculated with purified recombinant gD or gH or insect cells which had been infected with recombinant baculoviruses. Mice were subsequently challenged with EHV 1. Purified recombinant baculovirus gD provided the most protection and produced high levels of virus neutralizing antibodies. The gD fusion protein was less effective at protecting mice and insect cells infected with either of the recombinant baculoviruses or purified recombinant gH were poor at conferring protection. The results emphasize the importance of using purified proteins in vaccine formulations and of including EHV 1 gD as a component of a subunit vaccine.

The production of a truncated form of baculovirus expressed EHV-1 glycoprotein C and its role in protection of C3H (H-2Kk) mice against virus challenge.

A truncated form of the equine herpesvirus 1 (EHV-1) glycoprotein C (gC) gene was expressed in baculovirus. The gC signal sequence was substituted with the honeybee melittin signal sequence and the transmembrane region was replaced with a histidine tag. The recombinant virus produced high levels of gC in both the cells and supernatants of infected cells. The protein was present by 24 h and maximal secretion occurred at 96 h post-infection. The recombinant protein was antigenically authentic as shown by its reaction with each of a panel of individual monoclonal antibodies specific for the five distinct antigenic sites on EHV-1 gC. Recombinant gC was purified from the supernatant of infected cells by immuno-affinity chromatography and used to immunize C3H (H-2Kk haplotype) mice. This incurred a gC specific antibody response against both the recombinant protein and EHV-1 gC. 'Pepscan' analysis showed that the gC specific antibodies in serum from these mice reacted with the same epitopes on gC as those recognized by antibodies in convalescent equine sera (i.e. antibodies were specific to antigenic sites one and five). A third previously unrecognized antibody binding site at the carboxyl terminus was also detected (Antibody binding domain I). A T-cell proliferative response against EHV-1 was detected in splenocyte populations taken from vaccinated mice. Further, the recovery of virus from the lungs and turbinates following challenge of mice with EHV-1 was significantly reduced. These findings indicate that baculovirus expressed gC may contribute significantly to a subunit vaccine preparation aimed at protecting horses from EHV-1 infection.

The expression of the proteins of equine herpesvirus 1 which share homology with herpes simplex virus 1 glycoproteins H and L.

Several expression systems were used in studies aimed at characterizing the equine herpesvirus 1 (EHV-1) glycoprotein H and L homologues of HSV-1 (EHV-1 gH and gL) and the products were compared to the authentic proteins synthesized in virus infected cells. Using an in vitro transcription/translation system two gH species were detected (an unprocessed 89 kDa and a processed 116 kDa product). Three low molecular weight proteins were found in the case of gL (21.8 kDa, 22.9 kDa and 26.9 kDa) and these showed a slight reduction in mobility on the addition of microsomal membranes to the reactions. A gL fusion protein was produced in pGEX-2T, expression being confirmed by Western blotting using a gL-specific antiserum raised against a peptide incorporating the 13 carboxyl terminal amino acids of the protein. A gH specific peptide antiserum precipitated both gH and two smaller proteins from EHV-1 infected cells thought to be two forms of gL. Insect cells infected with gH or gL baculovirus recombinants were used to vaccinate C3H (H-2k) mice. Some protection against EHV-1 infection was conferred to the gH inoculated mice. The results will enable further studies on the importance of the gH and gL interaction in the pathogenesis of EHV-1 to be evaluated and their potential in contributing to a subunit vaccine to be assessed.

Production of hybrid cell lines secreting antibodies to herpes simplex virus type 2.

Following different immunization schedules we have produced 102 myeloma--spleen cell hybrid lines (30.8% of the total) secreting antibodies to HSV-2 infected cells. The highest yield of hybrids was derived from fusion of myeloma cells with spleen cells from mice sensitized by footpad injection of formalin-inactivated virus and boosted intravenously with the same antigen. To date, twelve of the antibody-producing hybrids have been cloned by limiting dilutions to generate over 100 monoclonal lines reacting specifically with HSV-infected but not with mock-infected cells. Several hybrid lines have also been injected into mice to generate tumors and high titers of anti-HSV-specific antibodies have been detected in ascitic fluids. All of the hybrids so far tested have retained the capacity to secrete anti-HSV antibodies after long-term in culture or repeated passages through mice. Antibodies reacting with cytoplasmic or nuclear antigens in infected cells as well as type-specific and type common antibodies have been detected.

Role of T-cells, virus neutralising antibodies and complement-mediated antibody lysis in the immune response against equine herpesvirus type-1 (EHV-1) infection of C3H (H-2k) and BALB/c (H-2d) mice.

The suitability of C3H (H-2k) and BALB/c (H-2d) mice for use as small animal models to study immunity to EHV-1 was assessed. An in vitro T cell response mediated by both CD4+ and CD8+ T cells was detected both during the acute phase of infection and after challenge with a second dose of EHV-1 at two months in lymphocyte populations taken from the spleens of both types of mouse. The responses were apparent until at least 61 days after the primary inoculation. After challenge, T cells from mice previously infected with EHV-1 responded by as early as day 3 after infection and higher levels of T cell proliferation were reached than in mice undergoing a primary infection. Immunological cross-reactivity with the closely related virus, EHV-4 was detected and some activity against herpes simplex virus type-1 (HSV-1) was observed during the acute phase of infection. T cell responses were detected in the draining cervical lymph nodes but not in the inguinal lymph nodes of the mice and these were the primary sites of T cell activation. Complement-dependent virus neutralising antibodies were present by day 8 after infection. These antibodies were also able to lyse EHV-1 infected target cells in vitro. Complement-independent virus neutralising antibodies were found before challenge only in C3H mice. The clinical signs and duration of virus shedding were reduced after challenge. The time course of the appearance of the different immune effector mechanisms is discussed in relation to the clearance of virus from the infected mice. The results suggest that C3H mice provide a better model in which to study potential vaccine candidates against EHV-1 infections of the horse than BALB/c mice.

Location of open reading frames coding for equine herpesvirus type-1 glycoproteins with homology to gE and gI of herpes simplex virus.

The DNA fragments representing the entire short unique region and part of the repeat sequences of the equine herpesvirus type-1 genome were cloned into plasmid vectors. The approximate positions of the junctions between the short unique region and the inverted repeats were then located by restriction endonuclease mapping. Two open reading frames coding for potential glycoproteins have been identified within the short unique region, using DNA sequence analysis. The predicted amino acid sequences of these open reading frames had extensive homology to the herpes simplex virus glycoproteins gE and gI and the related glycoproteins of pseudorabies virus and varicella-zoster virus.

Inhibition of biofilms associated with dentures and toothbrushes by tetrasodium EDTA.

AIMS: We examined the efficacy of tetrasodium EDTA in eradicating biofilms derived from salivary inocula or pure cultures of Candida albicans on discs of polymethyl methacrylate (PMMA) denture base or on toothbrushes that had been used normally for 4-8 weeks. Its efficiency in virus neutralization was also determined. METHODS AND RESULTS: Overnight (16 h) treatment with 4% (w/v) tetrasodium EDTA solution reduced salivary and C. albicans biofilm viable counts by > or =99%. Biofilm removal was confirmed using confocal laser scanning microscopy. Presence/absence of sucrose during biofilm formation had no effect on killing efficacy. Prolonged treatment of PMMA with tetrasodium EDTA did not influence subsequent formation of C. albicans biofilms or affect surface roughness of the PMMA, but it reduced subsequent biofilm formation from a salivary inoculum. Infectivities of herpes simplex virus and polio virus suspensions were reduced by >99.99% by treatment for 1 and 2 h, respectively. CONCLUSIONS: Tetrasodium EDTA solution efficiently disinfected toothbrushes and PMMA discs, with the detachment of biofilms, and rapidly neutralized both nonenveloped and enveloped viruses. SIGNIFICANCE AND IMPACT OF THE STUDY: Dentures and toothbrushes become contaminated by bacterial biofilms and by viruses. There is a need for disinfection methods that are rapidly effective, cost-effective, nontoxic and easily implemented. These studies indicate that tetrasodium EDTA solution has disinfection applications in the oral care field.

Glycoproteins with type common and type specific antigenic sites excreted from cells infected with herpes simplex virus types 1 and 2.

Agar immunodiffusion tests demonstrated that BHK 21 cells infected with either HSV I or HSV 2 release only a few HSV-specified antigens into the extracellular fluid (infected cell released polypeptides-ICRP). Neutralization blocking experiments showed that the majority of antigens/(including the Band II common antigen) involved as target sites in antibody-mediated virus neutralization are present in the ICRP of both HSV I and HSV 2.SDS-PAGE identified six regions of virus-specified proteins in the ICRP from both HSV I- and HSV 2-infected BHK cells. All these specifically released proteins are glycosylated, although to varying degrees. The SDS-PAGE profiles of HSV I and HSV 2 ICRP are different but do show some similarities, the most notable being a highly glycosylated protein with an estimated mol. wt. of 50,000 to 54,000 in HSV I ICRP and 52,000 to 56,000 in HSV 2 ICRP. Immune precipitation demonstrated that these two proteins contain the Band II antigenic site. Similar studies showed that the major type I specific antigenic site, which is involved as a target site in the neutralization of virus infectivity, is located in the highest mol. wt. glycoprotein region of HSV I ICRP and has a similar mobility to the VP7/8 region of purified enveloped virus.

The effect of temperature on the synthesis of rhinovirus type 2 RNA.

The reduced yields of rhinovirus type 2 at temperatures above 37 degrees C were shown to result from the degradation of virus-induced RNA, leaving little RNA available for inclusion into mature infectious virions. The degradation occurred about 6 h p.i., and appeared to be selectively effecting the single-stranded species. Lysosomal nucleases do not appear to have a role in this supra-optimal degradation.

Virulence is not conserved in recombinants between herpes simplex virus types 1 and 2.

The virulence of 31 herpes simplex virus type 1 X herpes simplex virus type 2 intertypic recombinants was determined following intraperitoneal inoculation into CBA mice. Only eight of the recombinants killed any of the mice and of these, only one recombinant was as virulent as its type 2 parent, the other seven recombinants being intermediate between their type 1 and type 2 parental viruses in virulence. These results indicate that most heterotypic combinations are attenuated independently of the virulence of the parental viruses and therefore that the virulence of HSV is controlled multigenically.

Solid matrix-antibody-antigen complexes incorporating equine herpesvirus 1 glycoproteins C and D elicit anti-viral immune responses in BALB/c (H-2K(d)) and C3H (H-2K(k)) mice.

Glycoproteins C and D (gC and gD) derived from equine herpesvirus 1 (EHV-1)-infected cells were incorporated into individual solid matrix-antibody-antigen (SMAA) complexes and administered to BALB/c (H-2K(d)) and C3H (H-2K(k)) mice. Antibodies against each of the glycoproteins were produced that neutralised virus infectivity and mediated the lysis of EHV-1-infected target cells in the presence of complement. Immunoglobulin (Ig)G2b was the predominant antibody isotype produced in BALB/c mice against gC, while equal amounts of IgG2a/2b were found in the serum of C3H mice (indicative of a T-helper(1) response). Glycoprotein D immunisation elicited predominantly an IgG1 response in BALB/c mice (indicative of a T-helper(2) response) and an IgG2a/2b response in C3H mice. EHV-1-specific local and systemic T-cell proliferative responses were detected in vitro following administration of SMAA complexes. Suppression of the local T-cell response was seen following virus challenge of mice immunised with SMAA gC. SMAA gD provided some protection against intranasal EHV-1 challenge. These data show that the SMAA system is an effective way of presenting subviral components to the immune system and further emphasises the importance of including glycoprotein D as a component of a subunit EHV-1 vaccine.

Monoclonal antibodies to two glycoproteins of herpes simplex virus type 2.

Monoclonal antibodies to herpes simplex virus type 2 were found to precipitate different numbers of radiolabeled polypeptides from lysates of virus-infected cells. Antibodies directed against two viral glycoproteins were characterized. Antibodies from hybridoma 17 alpha A2 precipitated a 60,000-molecular-weight polypeptide which chased into a 66,000- and 79,000-molecular-weight polypeptide. All three polypeptides labeled in the presence of [3H]glucosamine and had similar tryptic digest maps. The 60,000-molecular-weight polypeptide also chased into a 31,000-molecular-weight species which did not label with [3H]glucosamine. Antibodies from hybridoma 17 beta C2 precipitated a 50,000-molecular-weight polypeptide which chased into a 56,000- and 80,000-molecular weight polypeptide. These polypeptides also shared a similar tryptic digest map and labeled with [3H]glucosamine. Both monoclonal antibodies were herpes simplex virus type 2 specific. The viral proteins precipitated by 17 alpha A2 antibodies had characteristics similar to those reported for glycoprotein E, whereas the proteins precipitated by 17 beta C2 antibodies appeared to represent a glycoprotein not previously described. This glycoprotein should be tentatively designated glycoprotein F.

Antigenic and structural conservation of herpesvirus DNA-binding proteins.

Previously, we have shown a common antigen of several herpesviruses (pseudorabies virus, equine abortion virus and bovine mammillitis virus) to be antigenically related to the major DNA-binding proteins of herpes simplex virus types 1 and 2. In this study we have purified the cross-reacting polypeptide from cells infected with pseudorabies virus, equine abortion virus and bovine mammillitis virus and shown the cross-reacting protein to be a major DNA-binding protein for each virus. Tryptic peptide analysis of the cross-reacting DNA-binding proteins of all five viruses has shown structural similarities. The proteins thus were shown to share common antigenic sites, to have similar biological properties and to have a highly conserved amino acid sequence. This unexpected similarity between proteins from diverse herpes viruses suggests an essential and fundamental role of the major DNA-binding protein in herpes virus replication.

Hypoglycaemia and malignancy differences of closely related sublines of a rat tumour.

A possible basis for the differences in malignancy between three closely related sublines of the WBP1 ascites tumour of the rat has been studied by examining the biochemical changes in rat sera during tumour growth in vivo. Death appeared to be due to hypoglycaemia and the ability to induce this condition correlated with the differences in malignancy between the sublines; WBP1 (X) and WBP1 (V), the more malignant sublines, inducing hypoglycaemia earlier and more rapidly than the least malignant subline WBP1 (A). Possible mechanisms whereby the tumour produces these effects are discussed.

Herpes simplex virus non-structural proteins. IV. Purification of the virus-induced deoxyribonuclease and characterization of the enzyme using monoclonal antibodies.

The alkaline nucleases induced by herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) have been purified from high salt extracts of virus-infected cells. The purification used three types of column chromatography and resulted in apparently homogeneous DNase preparations with good recovery. The enzyme from HSV-2-infected cells has been characterized. It had both exonuclease and endonuclease activity, each with an unusually high pH optimum. The enzyme had an absolute requirement for magnesium which could not be replaced by other divalent cations. Analysis of the sedimentation characteristics and electrophoretic properties of the purified enzyme indicated that it was composed of a single subunit of mol. wt. 85 000. The purified HSV-2 enzyme was used as an immunogen to prime BALB/c mice which were used to prepare monoclonal antibodies. Three monoclonal antibodies were shown by several criteria to react with the enzyme. Thus, we were able to confirm that the 85K polypeptide did indeed have nuclease activity. This polypeptide was designated ICSP 22 in earlier studies and is a major polypeptide of virus-infected cells.

Some properties of recombinants between type 1 and type 2 herpes simplex viruses.

Four intertypic recombinants of herpes simplex virus have been shown to possess genetic information for functions characteristic of each of the two parental types. The functions were identified by (a) polyacrylamide gel electrophoresis of purified virus particles and of polypeptides synthesized in cells infected with the recombinants and (b) analysis of antigenic sites interacting with type specific neutralizing antibody. The analysis shows that each recombinant possess a different combination of these type specific markers. Finally we have been unable to detect recombination between herpes simplex type 1 and pseudorabies viruses.

Antigenic and protein sequence homology between VP13/14, a herpes simplex virus type 1 tegument protein, and gp10, a glycoprotein of equine herpesvirus 1 and 4.

Monospecific polyclonal antisera raised against VP13/14, a major tegument protein of herpes simplex virus type 1 cross-reacted with structural equine herpesvirus 1 and 4 proteins of Mr 120,000 and 123,000, respectively; these proteins are identical in molecular weight to the corresponding glycoprotein 10 (gp10) of each virus. Using a combination of immune precipitation and Western immunoblotting techniques, we confirmed that anti-VP13/14 and a monoclonal antibody to gp10 reacted with the same protein. Sequence analysis of a lambda gt11 insert of equine herpesvirus 1 gp10 identified an open reading frame in equine herpesvirus 4 with which it showed strong homology; this open reading frame also shared homology with gene UL47 of herpes simplex virus type 1 and gene 11 of varicella-zoster virus. This showed that, in addition to immunological cross-reactivity, VP13/14 and gp10 have protein sequence homology; it also allowed identification of VP13/14 as the gene product of UL47.

Inter- and intra-strain genomic variation in equine herpesvirus type 1 isolates.

Restriction enzyme digests of DNA from 22 unselected isolates of EHV-1 were analysed by hybridization with cloned DNA fragments covering the genome. In addition to a small amount of inter-strain variation, heterogeneity within strains was observed, caused by loss of specific restriction endonuclease sites in the DNA of a proportion of the virus particles of any one stock. Fifteen strains demonstrated the same intra-strain variation involving loss of the BamHI L-M site which was shown to lie within coding sequence for the large subunit of ribonucleotide reductase. This particular mutation may therefore be selected for by passage in RK13 cells.

Glycoprotein 300 is encoded by gene 28 of equine herpesvirus type 1: a new family of herpesvirus membrane proteins?

A portion of equine herpesvirus type 1 (EHV-1) gene 28, which is homologous to herpes simplex virus type 1 gene UL32, was expressed using a prokaryotic system to yield a fusion protein which reacted on Western blots with P19, a monoclonal antibody (MAb) that reacts with EHV-1 glycoprotein 300 (gp300), confirming that this gene encodes gp300. Hydrophobicity analysis showed that gp300 is a glycoprotein with multiple hydrophobic domains that might interact with, or span, the membrane several times. As such, it may represent the first member of a new family of herpesvirus glycoproteins to be identified as a virus structural component. Gp300 was also shown to be modified by palmitic acid residues, and a second MAb (1G12) directed against gp300 inhibited fusion between EHV-1-infected cells.