Structures of Foot-and-mouth Disease Virus with neutralizing antibodies derived from recovered natural host reveal a mechanism for cross-serotype neutralization.

The development of a universal vaccine against foot-and-mouth disease virus (FMDV) is hindered by cross-serotype antigenic diversity and by a lack of knowledge regarding neutralization of the virus in natural hosts. In this study, we isolated serotype O-specific neutralizing antibodies (NAbs) (F145 and B77) from recovered natural bovine hosts by using the single B cell antibody isolation technique. We also identified a serotype O/A cross-reacting NAb (R50) and determined virus-NAb complex structures by cryo-electron microscopy at near-atomic resolution. F145 and B77 were shown to engage the capsid of FMDV-O near the icosahedral threefold axis, binding to the BC/HI-loop of VP2. In contrast, R50 engages the capsids of both FMDV-O and FMDV-A between the 2- and 5-fold axes and binds to the BC/EF/GH-loop of VP1 and to the GH-loop of VP3 from two adjacent protomers, revealing a previously unknown antigenic site. The cross-serotype neutralizing epitope recognized by R50 is highly conserved among serotype O/A. These findings help to elucidate FMDV neutralization by natural hosts and provide epitope information for the development of a universal vaccine for cross-serotype protection against FMDV.

The pH stability of foot-and-mouth disease virus.

ᅟ: This review summarized the molecular determinants of the acid stability of FMDV in order to explore the uncoating mechanism of FMDV and improve the acid stability of vaccines. BACKGROUND: The foot-and-mouth disease virus (FMDV) capsid is highly acid labile and tends to dissociate into pentameric subunits at acidic condition to release viral RNA for initiating virus replication. However, the acid stability of virus capsid is greatly required for the maintenance of intact virion during the process of virus culture and vaccine production. The conflict between the acid lability in vivo and acid stability in vitro of FMDV capsid promotes the selection of a series of amino acid substitutions which can confer resistance to acid-induced FMDV inactivation. In order to explore the uncoating activity of FMDV and enhance the acid stability of vaccines, we summarized the available works about the pH stability of FMDV. In this review, we analyzed the intrinsic reasons for the acid instability of FMDV from the structural and functional aspects. We also listed all substitutions obtained by different research methods and showed them in the partial capsid of FMDV. We found that a quadrangle region in the viral capsid was the place where a great many pH-sensitive residues were distributed. As the uncoating event of FMDV is dependent on the pH-sensitive amino acid residues in the capsid, this most pH-sensitive position indicates a potential candidate location for RNA delivery triggered by the acid-induced coat disassociation. SHORT CONCLUSION: This review provided an overview of the pH stability of FMDV. The study of pH stability of FMDV not only contributes to the exploration of molecule and mechanism information for FMDV uncoating, but also enlightens the development of FMDV vaccines, including the traditionally inactivated vaccines and the new VLP (virus-like particle) vaccines.

Novel chimeric foot-and-mouth disease virus-like particles harboring serotype O VP1 protect guinea pigs against challenge.

Foot-and-mouth disease is a highly contagious, acute viral disease of cloven-hoofed animal species causing severe economic losses worldwide. Among the seven serotypes of foot-and-mouth disease virus (FMDV), serotype O is predominant, but its viral capsid is more acid sensitive than other serotypes, making it more difficult to produce empty serotype O VLPs in the low pH insect hemolymph. Therefore, a novel chimeric virus-like particle (VLP)-based candidate vaccine for serotype O FMDV was developed and characterized in the present study. The chimeric VLPs were composed of antigenic VP1 from serotype O and segments of viral capsid proteins from serotype Asia1. These VLPs elicited significantly higher FMDV-specific antibody levels in immunized mice than did the inactivated vaccine. Furthermore, the chimeric VLPs protected guinea pigs from FMDV challenge with an efficacy similar to that of the inactivated vaccine. These results suggest that chimeric VLPs have the potential for use in vaccines against serotype O FMDV infection.

Stabilizing effects of excipients on dissociation of intact (146S) foot-and-mouth disease virions into 12S particles during storage as oil-emulsion vaccine.

Most conventional foot-and-mouth disease virus (FMDV) vaccines contain oil-adjuvant. Their potency decreases upon prolonged storage. Intact (146S) FMDV particles can dissociate into 12S degradation products with a concomitant decrease in immunogenicity. We therefore measured virion stability in vaccines using two previously developed ELISAs to separately quantify 12S and 146S particles. Virions completely dissociated into 12S particles within 3 months after oil-emulsification. Dissociation occurred at a much lower rate in a comparable aqueous solution that was not oil-emulsified. Thus, oil-emulsification stimulates virion dissociation, presumably due to the protein denaturing effect of the oil-water interface. In real-time stability studies the stability of oil-adjuvanted virions of four different FMDV strains was significantly increased by addition of sucrose and BSA in a synergistic manner. Contrary to BSA addition, the effect of sucrose addition was concentration dependent. This study illustrates the importance of analysing antigen integrity after oil-emulsification and provides methods for FMDV vaccine stabilization.

Three-dimensional structure of foot-and-mouth disease virus and its biological functions.

Foot-and-mouth disease (FMD), an acute, violent, infectious disease of cloven-hoofed animals, remains widespread in most parts of the world. It can lead to a major plague of livestock and an economical catastrophe. Structural studies of FMD virus (FMDV) have greatly contributed to our understanding of the virus life cycle and provided new horizons for the control and eradication of FMDV. To examine host-FMDV interactions and viral pathogenesis from a structural perspective, the structures of viral structural and non-structural proteins are reviewed in the context of their relevance for virus assembly and dissociation, formation of capsid-like particles and virus-receptor complexes, and viral penetration and uncoating. Moreover, possibilities for devising novel antiviral treatments are discussed.

Foot-and-mouth disease virus particles inactivated with binary ethylenimine are efficiently internalized into cultured cells.

Conventional foot-and-mouth disease (FMD) vaccines are produced from virus grown in cell culture that is chemically inactivated by using binary ethylenimide (BEI). Here, we show that BEI treatment preserves both the architecture of FMDV particles, as inactivated viral particles showed by electron microscopy characteristics similar to those of infectious virions, as well as the general features of infectious virus internalization. Binding of inactivated particles to BHK-21 cells was blocked by preincubation with either a FMDV-specific monoclonal antibody or a synthetic peptide spanning the integrin-binding viral motif Arg-Gly-Asp (RGD). In addition, these particles were internalized into cultured cells through endocytosis, being directed to early endosomes, as indicated by their colocalization with the marker protein Rab5. When purified BEI-inactivated virions were labelled and their interaction with live cultured cells analyzed by time-lapse fluorescence microscopy, a major subpopulation of virus particles, about 80%, was shown to undergo internalization into a static endosome population, insensitive to the microtubule depolymerization exerted by nocodazole, while the remaining subpopulation (about 20%) was dynamic and sensitive to this drug. Thus, BEI-inactivated particles provide an interesting tool to study early steps in FMDV-cell interactions enabling a distinction between FMDV internalization and productive infection. Possible implications for FMDV immune response elicited following vaccine administration are discussed.

[Transmission risk for foot-and-mouth-disease in an animal-densed region in Germany--results from an expert survey]. / Risiken der Ubertragung der Maul- und Klauenseuche in einer viehdichten Region in Deutschland--Ergebnisse einer empirischen Expertenbefragung.

Due to its strong impact on economics and trading the Foot-and-Mouth-Disease (FMD) is one of the most important animal diseases within animal husbandry. Because no recent specific field observation for FMD exists in Germany, the risk assessment needs validated epidemiological models to prepare decision tools for FMD-outbreak management. The aim of this investigation was therefore to prepare a risk assessment for different transmission pathways to use for FMD-models in future. To prepare a FMD-transmission model the risk was assessed within a highly animal densed region in Germany by means of an expert survey. For each transmission pathway an assessment was given in the categories low, medium, high and severe. Some pathways were rated homogenously between the experts, but some were rated heterogeneously. Therefore areas were identified with common rating as well as areas, where further investigations to specify FMD-models are necessary.

Production of FMDV virus-like particles by a SUMO fusion protein approach in Escherichia coli.

Virus-like particles (VLPs) are formed by the self-assembly of envelope and/or capsid proteins from many viruses. Some VLPs have been proven successful as vaccines, and others have recently found applications as carriers for foreign antigens or as scaffolds in nanoparticle biotechnology. However, production of VLP was usually impeded due to low water-solubility of recombinant virus capsid proteins. Previous studies revealed that virus capsid and envelope proteins were often posttranslationally modified by SUMO in vivo, leading into a hypothesis that SUMO modification might be a common mechanism for virus proteins to retain water-solubility or prevent improper self-aggregation before virus assembly. We then propose a simple approach to produce VLPs of viruses, e.g., foot-and-mouth disease virus (FMDV). An improved SUMO fusion protein system we developed recently was applied to the simultaneous expression of three capsid proteins of FMDV in E. coli. The three SUMO fusion proteins formed a stable heterotrimeric complex. Proteolytic removal of SUMO moieties from the ternary complexes resulted in VLPs with size and shape resembling the authentic FMDV. The method described here can also apply to produce capsid/envelope protein complexes or VLPs of other disease-causing viruses.

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. Recombinant capsid precursor protein (P1 region) was expressed in Escherichia coli as myristoylated or unmyristoylated protein. The protein sedimented in sucrose gradients at 5S and reacted with monoclonal antibodies which recognize conformational or linear antigen determinants on the virion surface. In addition, it bound the integrin alpha(v)beta(6), a cellular receptor for FMDV, indicating that unprocessed recombinant capsid precursor is both structurally and antigenically similar to native virus capsid. These characteristics were not dependent on the presence of 2A at the C terminus but were altered by N-terminal myristoylation and in mutant precursors which lacked VP4. Proteolytic processing of myristoylated precursor by recombinant FMDV 3C(pro) in vitro induced a shift in sedimentation from 5S to 12S, indicating assembly into pentameric capsid subunits. Nonmyristoylated precursor still assembled into higher-order structures after processing with 3C(pro), but these particles sedimented in sucrose gradients at approximately 17S. In contrast, mutant precursors lacking VP4 were antigenically distinct, were unable to form pentamers, and had reduced capacity for binding integrin receptor. These studies demonstrate the utility of recombinant capsid precursor protein for investigating the initial stages of assembly of FMDV and other picornaviruses.

Internalization of swine vesicular disease virus into cultured cells: a comparative study with foot-and-mouth disease virus.

We performed a comparative analysis of the internalization mechanisms used by three viruses causing important vesicular diseases in animals. Swine vesicular disease virus (SVDV) internalization was inhibited by treatments that affected clathrin-mediated endocytosis and required traffic through an endosomal compartment. SVDV particles were found in clathrin-coated pits by electron microscopy and colocalized with markers of early endosomes by confocal microscopy. SVDV infectivity was significantly inhibited by drugs that raised endosomal pH. When compared to foot-and-mouth disease virus (FMDV), which uses clathrin-mediated endocytosis, the early step of SVDV was dependent on the integrity of microtubules. SVDV-productive endocytosis was more sensitive to plasma membrane cholesterol extraction than that of FMDV, and differential cell signaling requirements for virus infection were also found. Vesicular stomatitis virus, a model virus internalized by clathrin-mediated endocytosis, was included as a control of drug treatments. These results suggest that different clathrin-mediated routes are responsible for the internalization of these viruses.

Structure of Foot-and-mouth disease virus serotype A10 61 alone and complexed with oligosaccharide receptor: receptor conservation in the face of antigenic variation.

Foot-and-mouth disease viruses (FMDVs) target epithelial cells via integrin receptors, but can acquire the capacity to bind cell-surface heparan sulphate (or alternative receptors) on passage in cell culture. Vaccine viruses must be propagated in cell culture and, hence, some rationale for the selection of variants in this process is important. Crystal structures are available for type O, A and C viruses and also for a complex of type O strain O(1)BFS with heparin. The structure of FMDV A10(61) (a cell culture-adapted strain) complexed with heparin has now been determined. This virus has an RGSD motif in place of the otherwise conserved RGD integrin-binding motif and the potential to bind heparan sulphate (suggested by sequence analyses). FMDV A10(61) was closely similar in structure to other serotypes, deviating most in antigenic sites. The VP1 GH loop comprising the integrin-binding motif was disordered. Heparin bound at a similar site and in a similar conformation to that seen in the analogous complex with O(1)BFS, although the binding had a lower affinity and was more ionic.

Early events in integrin alphavbeta6-mediated cell entry of foot-and-mouth disease virus.

We have shown that foot-and-mouth disease virus (FMDV) infection mediated by the integrin alphavbeta6 takes place through clathrin-dependent endocytosis but not caveolae or other endocytic pathways that depend on lipid rafts. Inhibition of clathrin-dependent endocytosis by sucrose treatment or expression of a dominant-negative version of AP180 inhibited virus entry and infection. Similarly, inhibition of endosomal acidification inhibited an early step in infection. Blocking endosomal acidification did not interfere with surface expression of alphavbeta6, virus binding to the cells, uptake of the virus into endosomes, or cytoplasmic virus replication, suggesting that the low pH within endosomes is a prerequisite for delivery of viral RNA into the cytosol. Using immunofluorescence confocal microscopy, FMDV colocalized with alphavbeta6 at the cell surface but not with the B subunit of cholera toxin, a marker for lipid rafts. At 37 degrees C, virus was rapidly taken up into the cells and colocalized with markers for early and recycling endosomes but not with a marker for lysosomes, suggesting that infection occurs from within the early or recycling endosomal compartments. This conclusion was supported by the observation that FMDV infection is not inhibited by nocodazole, a reagent that inhibits vesicular trafficking between early and late endosomes (and hence trafficking to lysosomes). The integrin alphavbeta6 was also seen to accumulate in early and recycling endosomes on virus entry, suggesting that the integrin serves not only as an attachment receptor but also to deliver the virus to the acidic endosomes. These findings are all consistent with FMDV infection proceeding via clathrin-dependent endocytosis.

The structure of foot-and-mouth disease virus.

Structural studies of foot-and-mouth disease virus (FMDV) have largely focused on the mature viral particle, providing atomic resolution images of the spherical protein capsid for a number of sero- and sub-types, structures of the highly immunogenic surface loop, Fab and GAG receptor complexes. Additionally, structures are available for a few non-structural proteins. The chapter reviews our current structural knowledge and its impact on our understanding of the virus life cycle proceeding from the mature virus through immune evasion/inactivation, cell-receptor binding and replication and alludes to future structural targets.

Pressure-inactivated FMDV: a potential vaccine.

Foot-and-mouth disease virus (FMDV) is the causative agent of the foot-and-mouth disease (FMD). Alternative FMD vaccines have been pursued due to important disadvantages of the one currently in use. High hydrostatic pressure (HP) has been observed to inactivate some viruses. Here, we investigated the effects of HP on FMDV O1 Campos-Vallée (CVa) infectivity. A treatment consisting of 2.5 kbar at -15 degrees C and 1M urea, completely abolished FMDV infectivity, maintaining the integrity of its capsid structure. Moreover, its ability to elicit neutralizing antibody production in rabbits was preserved. Taken together, our results suggest that HP could be a safe, simple, cheap and reproducible way for viral vaccine production.

The ultrastructure of the developing replication site in foot-and-mouth disease virus-infected BHK-38 cells.

Foot-and-mouth disease virus (FMDV) is the type species of the Aphthovirus genus of the Picornaviridae: Infection by picornaviruses results in a major rearrangement of the host cell membranes to create vesicular structures where virus genome replication takes place. In this report, using fluorescence and electron microscopy, membrane rearrangements in the cytoplasm of FMDV-infected BHK-38 cells are documented. At 1.5-2.0 h post-infection, free ribosomes, fragmented rough endoplasmic reticulum, Golgi and smooth membrane-bound vesicles accumulated on one side of the nucleus. Newly synthesized viral RNA was localized to this region of the cell. The changes seen in FMDV-infected cells distinguish this virus from other members of the Picornaviridae, such as poliovirus. Firstly, the collapse of cellular organelles to one side of the cell has not previously been observed for other picornaviruses. Secondly, the membrane vesicles, induced by FMDV, appear distinct from those induced by other picornaviruses such as poliovirus and echovirus 11 since they are relatively few in number and do not aggregate into densely packed clusters. Additionally, the proportion of vesicles with double membranes is considerably lower in FMDV-infected cells. These differences did not result from the use of BHK-38 cells in this study, as infection of these cells by another picornavirus, bovine enterovirus (a close relative of poliovirus), resulted in morphological changes similar to those reported for poliovirus-infected cells. With conventional fixation, FMDV particles were not seen; however, following high-pressure freezing and freeze-substitution, many clusters of virus-like particles were seen.

High-pressure freezing in the study of animal pathogens.

High-pressure freezing is applicable to both morphological and immunocytochemical studies. We are investigating the morphogenesis of foot-and-mouth disease virus and African swine fever virus by the use of high-pressure freezing of infected cells. Foot-and-mouth disease virus particles are not detected in sections of conventionally immersion-fixed infected cells, but when the cells are prepared by high-pressure freezing, newly formed virions are readily seen throughout the cell. We report two methods for high-pressure freezing of virally infected cells: first, two sapphire discs frozen 'face to face' with a narrow spacer to prevent cell damage and, second, a fibrous filter substrate that can be easily cut into discs to fit into the freezing planchettes. Cells readily adhere to the fibres in vitro, and the complete disc can be rapidly transferred to the planchettes for freezing. Immunolabelling studies of the microneme proteins of the parasite Eimeria tenella indicate that high-pressure freezing followed by freeze-substitution in acetone with uranyl acetate allows high-sensitivity immunolabelling for these proteins.

A novel three-dimensional variant of the watershed transform for segmentation of electron density maps.

Electron density maps at moderate resolution are often difficult to interpret due to the lack of recognizable features. This is especially true for electron tomograms that suffer in addition to the resolution limitation from low signal-to-noise ratios. Reliable segmentation of such maps into smaller, manageable units can greatly facilitate interpretation. Here, we present a segmentation approach targeting three-dimensional electron density maps derived by electron microscopy. The approach consists of a novel three-dimensional variant of the immersion-based watershed algorithm. We tested the algorithm on calculated data and applied it to a wide variety of electron density maps ranging from reconstructions of single macromolecules to tomograms of subcellular structures. The results indicate that the algorithm is reliable, efficient, accurate, and applicable to a wide variety of biological problems.