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.

Ultraviolet disinfection with a novel microwave-powered device.

AIMS: To evaluate the antimicrobial efficacy of a novel u.v. beaker, powered in a domestic microwave oven. METHODS AND RESULTS: Three beakers were compared, with most rapid killing obtained in the Neutra Plasma 50. Ultraviolet light generated within the beakers efficiently killed planktonic and surface-associated Streptococcus mutans, Pseudomonas aeruginosa, vegetative Bacillus stearothermophilus, herpes simplex and polio viruses. Candida albicans and Mycobacterium phleii were less rapidly killed, and only 70% inactivation of B. stearothermophilus endospores was achieved. Irradiation for 45 s reduced viable bacterial counts in saliva by > 99%. CONCLUSIONS: The u.v.-generating beakers efficiently reduced viable counts of bacteria, yeast and viruses. Kinetics of killing varied, reflecting the fact that lethal mechanisms are complex, and probably depend on interplay between u.v. and heat. SIGNIFICANCE AND IMPACT OF THE STUDY: This novel method of generating u.v., using a cheap and widely available power source, provides a rapid, inexpensive and non-toxic method of disinfection with a wide range of applications in hospitals, clinics and the home.

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.

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.

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.

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.

Identification of the equine herpesvirus type 1 glycoprotein 17/18 as a homologue of herpes simplex virus glycoprotein D.

The DNA sequence of the equine herpesvirus type 1 (EHV-1) gD gene homologue has been determined for the strain Ab1 and compared with previously published sequences. A portion of the gene has been located to a region of the genome which also encodes homologues of the herpes simplex virus type 1 genes for gE and gI and is known to encode an epitope of the virion protein gp17/18. Analysis of the EHV-1 strain Kentucky A (KyA) by DNA hybridization showed the presence of a gD gene homologue and established the absence of genes for gI and gE. Western blot analysis, however, showed that KyA virus particles contain gp17/18, thus indicating that this protein is encoded by the gD gene homologue. The KyA gp17/18 was found to be smaller than that detected in other strains and this is accounted for by a frameshift mutation in the KyA sequence relative to Ab1. The mutation in the KyA strain results in an altered C-terminal sequence and could explain the apparent structural differences suggested by the reactivities with monoclonal antibodies (MAbs). We have also expressed part of the Ab1 gD gene as a fusion protein with glutathione S-transferase in Escherichia coli and shown that this reacts with the MAb 5H6 originally used to map gp17/18. These experiments establish that gp17/18 is encoded by the gD gene homologue.

Glycoprotein 60 of equine herpesvirus type 1 is a homologue of herpes simplex virus glycoprotein D and plays a major role in penetration of cells.

Monoclonal antibodies (MAbs) specific for equine herpesvirus type 1 (EHV-1) glycoprotein 60 (gp60) and gp 17/18 (F3132 and 5H6 respectively) were found to react with the same protein, which was identified as a homologue of herpes simplex virus type 1 gD. MAb F3132 strongly neutralized virus infectivity and inhibited the penetration of the virus into the cell. The effects on penetration were shared with three other MAbs against this protein (P68, F3116 and F3129), but no effect on virus penetration was found with any other anti-EHV-1 MAb tested. The level of glycosylation of gp60 was analysed using glycanase enzymes and glycosylation inhibitors, and consisted of mainly N-linked carbohydrate. The M(r) of non-N-glycosylated gp60 was 50K.

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.

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.

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.

Studies on glycoprotein 13 (gp13) of equid herpesvirus 1 using affinity-purified gp13, glycoprotein-specific monoclonal antibodies and synthetic peptides in a hamster model.

Hamsters were immunized with either an affinity-purified preparation of equid herpesvirus 1 (EHV-1) glycoprotein 13 (gp13) or synthetic peptides representing three sequences within the homologous glycoprotein of EHV-4, resulting in the production of anti-peptide (in the case of peptide-immunized animals) or antivirus antibodies. The sera from gp13-immunized hamsters contained antibodies which showed virus-neutralizing activity and complement-mediated antibody lysis of EHV-1-infected target cells. These hamsters were protected from EHV-1 challenge. The characteristics of a panel of anti-gp13 monoclonal antibodies (P28, P17, 14H7, 16E4 and 16H9) were assessed both in vivo and in vitro. 16E4 and P28 showed high levels of complement-mediated neutralization of virus, complement-mediated lysis of virus-infected target cells and passive protection of hamsters. Furthermore, epitope mapping studies demonstrated that this glycoprotein contains a neutralizing epitope recognized by EHV-1-immune horse serum. The data imply that gp13 has potential as a candidate antigen for a molecular vaccine.

Characterization of the high Mr glycoprotein (gP300) of equine herpesvirus type 1 as a novel glycoprotein with extensive O-linked carbohydrate.

The high Mr glycoprotein (gp300) of equine herpesvirus type 1 was found to have an Mr, estimated by SDS-PAGE, of over 400,000 and was confirmed as being a surface glycoprotein by 125I-labelling. In contrast to [3H]glucosamine, gp300 showed very low levels of [3H]glucosamine, gp300 showed very low levels of [3H]mannose incorporation. The Mr of gp300 showed no detectable change upon treatment of purified virus with N-glycanase, and showed only a small change in virus-infected cells treated with tunicamycin. In addition, gp300 failed to bind the lectin concanavalin A. Taken together, these results indicate a lack of N-linked carbohydrate on gp300. The major carbohydrate species were found to be composed primarily of O-linked chains, as indicated by the sensitivity of the protein to monensin, to exoglycanase enzymes specific for sugars present in O-linked chains and to mild alkaline borohydride treatment, which revealed three species of carbohydrate of Mr of greater than 10,000, 2400 and 1100, respectively. Neuraminidase treatment and binding of Helix pomatia lectin indicated the presence of alpha-N-acetylglucosamine and sialic acid as terminal sugars. Immunological cross-reactivity of gp300 with a high Mr protein of equine herpesvirus type 4 was shown and it also exhibited a marked Mr variation in the vaccine strain Rhinomune.

Identification of the gB homologues of equine herpesvirus types 1 and 4 as disulphide-linked heterodimers and their characterization using monoclonal antibodies.

Equine herpesvirus types 1 and 4 (EHV-1 and EHV-4) labelled with [14C]glucosamine were purified from infected cell culture medium and profiles of their structural proteins were obtained that enabled identification of the major glycoproteins. Nine glycosylated polypeptides were identified for each virus. Preparations of the purified viruses each contained a glycoprotein which was linked by disulphide bonds, as determined by diagonal gel electrophoresis under reducing/non-reducing conditions. High Mr forms of this glycoprotein were detected for EHV-1 when the sample was not heated. The EHV-1 protein consisted of three polypeptides of Mr 108K, 76K and 58K and the EHV-4 protein consisted of three polypeptides of Mr 112K, 74K and 61K. Western blotting and immunoprecipitation with monoclonal antibodies confirmed that the EHV-1 gB homologue migrates with an apparent Mr of 108K (140K under non-reducing conditions) but is cleaved to give glycoproteins of 76K and 58K which are held together by disulphide bonds. The EHV-4 gB homologue consists of a 112K glycoprotein which is cleaved to give glycoproteins of 74K and 61K which are also linked by disulphide bonds.

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.

Studies on the herpes simplex virus alkaline nuclease: detection of type-common and type-specific epitopes on the enzyme.

Five monoclonal antibodies to the alkaline nuclease of herpes simplex virus (HSV) types 1 and 2 have been used in immunoperoxidase tests to demonstrate the nuclear localization of the enzyme within HSV-1- and HSV-2-infected cells and to purify the enzyme from cells infected with either virus by immunoadsorbant chromatography. Affinity chromatography with a 32P-labelled extract of HSV-2-infected cells has enabled us to demonstrate that the nuclease eluting from the immunoadsorbant is a phosphoprotein, hence confirming the nuclease to be identical to the phosphorylated polypeptide previously referred to as ICSP 22 (HSV-2) or ICP 19 (HSV-1). In addition, the results clearly demonstrate that monoclonal antibodies Q1, CC1 and CH2 are directed against HSV type-common epitopes while V1 and T2T1 antibodies are against HSV-2-specific epitopes on the enzyme. Using the type-specific monoclonal antibodies in an immunoperoxidase test, the enzyme specified in cells infected with intertypic recombinants has been typed; correlation of these data with restriction endonuclease maps of the recombinants has enabled us to map the position of the active site of the nuclease gene to map units 0.168 to 0.184 on the genomes of both HSV-1 and HSV-2. Taken with the data mapping the mRNA encoding this enzyme, the nuclease active site can be mapped to 0.168 to 0.175 on the genome. Finally, the use of monoclonal antibodies in immunofluorescence tests on infected cells has demonstrated that the nuclease is synthesized within 2 h post-infection.

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.