David Rowlands collection

We have investigated the interactions of purified radiolabelled hepatitis A virus (HAV) with a variety of continuous cell lines. Virus labelled either in vitro with radiolabelled iodine or in vivo with radiolabelled uridine bound to cells with similar efficiency. Attachment to BS-C-1 cells was calcium ion-dependent and this correlated with infectivity assay results. The cell tropism of HAV attachment was examined using cell suspensions and confluent cell monolayers at both 4 °C and 37 °C. The maximum level of attachment was observed at 4 °C with cells in suspension, but was severely inhibited by 2% foetal calf serum; these results again correlated with infectivity assays. The components of serum which inhibit attachment have been characterized by gel filtration chromatography, sucrose density gradient analysis, immunoprecipitation and Western blotting. The data show that such components are of high M r and that the serum glycoprotein, α2-macroglobulin, can partly mimic the inhibitory effect of whole serum.

We recently reported the enhanced immunogenicity of a peptide epitope when it was presented as a fusion protein with hepatitis B core antigen. In those experiments the fusion protein was expressed in vaccinia virus. We have now refined the system so that large amounts of highly immunogenic particles can be produced using a simple bacterial expression system. We describe the expression of three different viral epitopes as chimeric particles that induce good antibody responses to each epitope after one dose of low amounts of antigen. Finally we demonstrate that the immunogenicity is a reflection of both T helper cell sites within the core protein and also the particulate nature of the immunogens.

Eleven neutralizing monoclonal antibodies (MAbs) were produced to the O1BFS 1860/67 strain of foot-and-mouth disease virus (FMDV), and were characterized for their ability to bind viral and subviral antigens in different ELISA tests and to neutralize heterologous type O isolates. Neutralization escape variants of the homologous virus, isolated under pressure from five of these MAbs, were used in cross-neutralization tests with all of the 11 antibodies. These studies identified three functionally independent, conformational, neutralizing sites. The most conformationally dependent site bound antibody which neutralized a range of type O virus isolates. A second site was less dependent on conformation and was recognized by antibody that was strain-specific. The least conformational site bound MAbs which showed limited cross-neutralization of other type O strains. This latter site appeared to be immunodominant and contained several overlapping epitopes which showed some differences in their specificities. Isoelectrofocusing and sequencing studies of the variants strongly suggested that polypeptide VP2 contributes to the immunodominant site.

The amino acid sequence RGD (arginine-glycine-aspartic acid) is highly conserved in the VP1 protein of foot-and-mouth disease virus (FMDV), despite being situated in the immunodominant hypervariable region between amino acids 135 and 160. RGD-containing proteins are known to be important in promoting cell attachment in several different systems, and we report here that synthetic peptides containing this sequence are able to inhibit attachment of the virus to baby hamster kidney (BHK) cells. Inhibition was dose-dependent and could be reversed on removal of the peptide. A synthetic peptide corresponding to a portion of the same hypervariable region but not containing the RGD sequence did not inhibit virus attachment under the same conditions. Antibody against the RGD region of VP1 blocked attachment of the virus to BHK cells, and neutralizing monoclonal antibodies, which neutralize virus by preventing cell attachment, were blocked by RGD-containing peptides from binding virus in an ELISA test. Cleavage of the C-terminal region of virus VP1 in situ with proteolytic enzymes reduced cell attachment, and antiserum against a peptide corresponding to this region was also able to inhibit attachment of virus to BHK cells. These results indicate that the amino acid sequence RGD at positions 145 to 147 and amino acids from the C-terminal region of VP1 (positions 203 to 213) contribute to the cell attachment site on FMDV for BHK cells.

Foot-and-mouth disease virus (FMDV) A22 Iraq 24/64 adapted to grow in BHK monolayer cells induced antibodies which neutralized many isolates belonging to the A serotype. Plaque-purified virus isolated from this stock also induced broadly reactive antibodies, showing that this property is not due to the combined response to a mixture of variants in the original stock virus. However, viruses obtained by passage in suspension BHK cells of either the monolayer cell-adapted virus or a virus cloned from this stock resulted in the selection of virus which induced antibodies with highly specific neutralizing activity. In addition to their antigenic properties the monolayer and suspension cell-adapted viruses could be distinguished by plaque morphology, tendency to aggregate and ability to attach to BHK cells. Monoclonal antibodies (MAbs) induced with the plaque-purified monolayer-adapted virus had neutralizing activity almost as broad as polyclonal serum, showing that this property can be represented by a single epitope on the virus. These neutralizing MAbs recognize a trypsin-sensitive epitope on the virus. Surprisingly, sequence analysis of the structural protein-coding regions of the genomic RNAs of monolayer and suspension cell-adapted viruses showed no amino acid differences in VP1, the protein known to contain the major neutralization epitope in FMDV and to be the only protein susceptible to cleavage by trypsin in the virus particle. Although three coding differences were found in the capsid protein these were all located in VP2.

Translation of foot-and-mouth disease virus RNA for extended periods in rabbit reticulocyte lysates results in the appearance of a previously undescribed protein. A protein with similar properties can also be detected in BHK cells at late times after virus infection. Specific immunoprecipitation has shown that this protein (Lb′) is closely related to the smaller of the two leader proteins, Lb, although it migrates with an apparently higher M r in SDS-polyacrylamide gels. The conversion of Lb to Lb′ can be mimicked by treatment with carboxypeptidase B. It is suggested that C-terminal trimming of Lb to produce Lb′ results in an increase in negative charge and is responsible for its slower migration in SDS-PAGE.

The surface structure of foot-and-mouth disease virus (FMDV) and the interaction of the individual capsid proteins with the virus RNA have been examined using modification reagents. By measuring the extent of modification of the lysine residues of intact and disrupted virus particles and the 12S protein subunit with Bolton & Hunter reagent it was found that 54 % of the residues of VP 1, 15 % of the residues of VP2 and 37 % of the residues of VP3, equivalent to five, two and four lysine residues respectively, are on the surface of the intact virus particle. Polypeptide VP4 was not modified in intact virus particles, indicating that it has no lysine residues on the surface of the virus. Modification with sodium metabisulphite, which causes a specific transamination reaction between cytidylic acid residues in ssRNA and closely associated basic amino acids, cross-linked all four structural proteins to the virus RNA. Both fragments of VP1, produced by treatment of the virus particle with trypsin, are also cross-linked to the RNA. These observations have been combined with the evidence that the immunogenic activity of VP1 may be contained in two discontinuous sites, at amino acids 141 to 160 and 200 to 213, in proposing a model for the arrangement of this polypeptide in the virus particle.

Typically, the translation of eukaryotic mRNAs into protein is initiated at a single site. However, we have recently shown that not one but two primary products, P20a and P16, are translated from the 5′ end of the coding region of the genome of foot-and-mouth disease virus (FMDV). In this paper we show by partial protease digestion of these proteins that they differ only at their N termini, thus confirming the presence of two initiation sites for translation of FMDV RNA. Sequence analysis of two subtypes of the virus (A10 and A12) confirms the presence of two initiator AUG codons in the expected position on the genome. By correlation with protein synthesis data from these subtypes it appears that the relative use of each initiation site is dependent on its surrounding nucleotide sequence. In addition, the ratio of the two proteins when synthesized in vitro differs markedly from that when they are synthesized in vivo, suggesting the presence of a control mechanism for synthesis of P20a in vivo which may be absent in vitro. We also show that the cleavage site between these two proteins and the structural protein precursor, P88, is located closer to the N terminus of the polyprotein than has previously been reported.

Antibody produced against preparations of VP1, one of the four structural polypeptides of foot-and-mouth disease virus, neutralized the virus and reacted with both full and empty particles in radioimmunoassays (RIA). Antiserum against VP2 reacted with artificial empty particles of the virus but not with full particles. In contrast, none of the individual polypeptides of poliovirus produced antisera which neutralized the virus nor reacted with it in RIA. However, antisera produced with VP1 and VP2 reacted with artificial empty particles in RIA.

Foot-and-mouth disease virus (FMDV) attached to pig kidney cells at 0 °C and could only be recovered in a form with a sedimentation coefficient and buoyant density lower than that of the native virus. Incubation of the virus-cell complex at 37 °C caused disruption of about 80% of the particles into a 12S protein sub-unit that had the same polypeptide composition as that produced by reducing the pH of the virus below pH 7. The remaining 20% had the same polypeptide and RNA composition as the native virus but it had a lower sedimentation coefficient, buoyant density and specific infectivity. These lower values are probably due to the association of the virus with cell membrane components. The 12S subunits were shown to be located inside the cell, indicating that disruption of the virus had occurred within the cell. The results are discussed in relation to the different cell mediated alteration of other picornaviruses.