Foot-and-mouth disease virus assembly: processing of recombinant capsid precursor by exogenous protease induces self-assembly of pentamers in vitro in a myristoylation-dependent manner.

The assembly of foot-and-mouth disease virus (FMDV) particles is poorly understood. In addition, there are important differences in the antigenic and receptor binding properties of virus assembly and dissociation intermediates, and these also remain unexplained. We have established an experimental model in which the antigenicity, receptor binding characteristics, and in vitro assembly of capsid precursor can be studied entirely from purified components.

Mouse models of rhinovirus-induced disease and exacerbation of allergic airway inflammation.

Rhinoviruses cause serious morbidity and mortality as the major etiological agents of asthma exacerbations and the common cold. A major obstacle to understanding disease pathogenesis and to the development of effective therapies has been the lack of a small-animal model for rhinovirus infection. Of the 100 known rhinovirus serotypes, 90% (the major group) use human intercellular adhesion molecule-1 (ICAM-1) as their cellular receptor and do not bind mouse ICAM-1; the remaining 10% (the minor group) use a member of the low-density lipoprotein receptor family and can bind the mouse counterpart.

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.

Discrimination among rhinovirus serotypes for a variant ICAM-1 receptor molecule.

Intercellular adhesion molecule 1 (ICAM-1) is the cellular receptor for the major group of human rhinovirus serotypes, including human rhinovirus 14 (HRV14) and HRV16. A naturally occurring variant of ICAM-1, ICAM-1Kilifi, has altered binding characteristics with respect to different HRV serotypes. HRV14 binds to ICAM-1 only transiently at physiological temperatures but forms a stable complex with ICAM-1Kilifi.

Mouse respiratory epithelial cells support efficient replication of human rhinovirus.

Human rhinoviruses (HRV) are responsible for the majority of virus infections of the upper respiratory tract. Furthermore, HRV infection is associated with acute exacerbation of asthma and other chronic respiratory diseases of the lower respiratory tract. A small animal model of HRV-induced disease is required for the development of new therapies.

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.

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).

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.

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 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.