The first step in the replication of the plus-stranded poliovirus RNA is the synthesis of a complementary minus strand. This process is initiated by the covalent attachment of UMP to the terminal protein VPg, yielding VPgpU and VPgpUpU. We have previously shown that these products can be made in vitro in a reaction that requires only synthetic VPg, UTP, poly ( The Picornaviridae family of plus-strand RNA viruses includes a large number of pathogens with widely different host range and disease symptoms (38). At the same time, picornaviruses show a strong similarity in their gene organization and in the mechanism by which they replicate their genomes (58). An unusual feature of their genomes is the presence of a small protein VPg, covalently linked to the 5Ј end of the RNA. Virus replication in the infected host cell is a two-step process, carried out primarily by the viral RNA polymerase, in conjunction with other viral and possibly also cellular proteins. It takes place in small vesicles that are derived from the host's cellular membranes and with which the nonstructural proteins of the virus are associated. First, the incoming viral RNA is transcribed into complementary minus strands which are then used as templates for the synthesis of the progeny plus strands. Although the basic steps of replication are well known, very little is understood about the details of these processes and in particular about the exact functions of the cis-acting RNA structures contained within picornaviral RNAs (1). One of the important unanswered questions about minus-strand synthesis is how the viral RNA polymerase recognizes and selects only its own RNA as template among the many polyadenylated mRNAs that are present in the host cell (45).Poliovirus is perhaps the best known member of the Picornaviridae. Its RNA genome of about 7,500 nucleotides (nt) is composed of a long 5Ј-nontranslated region (NTR), a single open reading frame, a short 3ЈNTR, and a poly(A) tail (Fig. 1A) (32). The 5Ј-terminal UMP of the viral RNA is linked to the hydroxyl group of VPg by a phosphodiester bond (Fig. 1A) (2, 56). The 5ЈNTR consists of two independent domains. The first is a cloverleaf-like structure which is involved both in plus-strand RNA synthesis (3,4,24,70) and in the process of switching from translation to replication (18). The second is a large and complex structure, the internal ribosomal entry site (IRES) (28, 47), which promotes translation of a polyprotein. This polyprotein (Fig. 1A) contains a capsid region (P1) and two nonstructural domains (P2 and P3) (32). The initial cleavage of the polyprotein is carried out by proteinase 2A pro at the P1/P2 site (64). Most other cleavages are mediated by the activities of proteinase 3C pro and its precursor, 3CD pro (22,23,29,73). The proteins of the P2 domain are predominantly involved in inducing the biochemical and structural changes that occur in the infected cell (8), but 2CATPase is also essential for viral genome replication (49). Those of the P3 region are the ones most directly invol...

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Investigation into the pathogenesis of foot-and-mouth disease (FMD) has focused on the study of the disease in cattle with less emphasis on pigs, small ruminants and wildlife. 'Atypical' FMD-associated syndromes such as myocarditis, reproductive losses and chronic heat intolerance have also received little attention. Yet, all of these manifestations of FMD are reflections of distinct pathogenesis events. For example, naturally occurring porcinophilic strains and unique virus-host combinations that result in high-mortality outbreaks surely have their basis in molecular-, cellular- and tissue-level interactions between host and virus (i.e. pathogenesis). The goal of this review is to emphasize how the less commonly studied FMD syndromes and host species contribute to the overall understanding of pathogenesis and how extensive in vitro studies have contributed to our understanding of disease processes in live animals.

Isolates of foot-and-mouth disease virus (FMDV) exist as complex mixtures of variants. Two different serotype O1 Campos preparations that we examined contained two variants with distinct plaque morphologies on BHK cells: a small, clear-plaque virus that replicates in BHK and CHO cells, and a large, turbid-plaque virus that only grows in BHK cells. cDNAs encoding the capsids of these two variants were inserted into a genome-length FMDV type A12 infectious cDNA and used to produce chimeric viruses that exhibited the phenotype of the original variants. Analyses of these viruses, and hybrids created by exchanging portions of the capsid gene, identified codon 56 in VP3 (3056) as the critical determinant of both cell tropism and plaque phenotype. Specifically, the CHO growth/clear-plaque phenotype is dependent on the presence of the highly charged Arg residue at 3056, and viruses with this phenotype and genotype were selected during propagation in tissue culture. The genetically engineered Arg 3056 virus was highly attenuated in bovines, but viruses recovered from animals inoculated with high doses of this virus had lost the ability to grow in CHO cells and contained either an uncharged residue at 3056 or a negatively charged Glu substituted for a Lys at a spatially and antigenically related position on VP2 (2134). Comparison of these animal-derived viruses to other natural and engineered viruses demonstrated that positively charged residues are required at both 2134 and 3056 for binding to heparin. Taken together, these results indicate that in vitro cultivation of FMDV type O selects viruses that bind to heparin and that viruses with the heparin-binding phenotype are attenuated in the natural host.

Identifying when past exposure to an infectious disease will protect against newly emerging strains is central to understanding the spread and the severity of epidemics, but the prediction of viral cross-protection remains an important unsolved problem. For foot-and-mouth disease virus (FMDV) research in particular, improved methods for predicting this cross-protection are critical for predicting the severity of outbreaks within endemic settings where multiple serotypes and subtypes commonly co-circulate, as well as for deciding whether appropriate vaccine(s) exist and how much they could mitigate the effects of any outbreak. To identify antigenic relationships and their predictors, we used linear mixed effects models to account for variation in pairwise cross-neutralization titres using only viral sequences and structural data. We identified those substitutions in surface-exposed structural proteins that are correlates of loss of cross-reactivity. These allowed prediction of both the best vaccine match for any single virus and the breadth of coverage of new vaccine candidates from their capsid sequences as effectively as or better than serology. Sub-sequences chosen by the model-building process all contained sites that are known epitopes on other serotypes. Furthermore, for the SAT1 serotype, for which epitopes have never previously been identified, we provide strong evidence – by controlling for phylogenetic structure – for the presence of three epitopes across a panel of viruses and quantify the relative significance of some individual residues in determining cross-neutralization. Identifying and quantifying the importance of sites that predict viral strain cross-reactivity not just for single viruses but across entire serotypes can help in the design of vaccines with better targeting and broader coverage. These techniques can be generalized to any infectious agents where cross-reactivity assays have been carried out. As the parameterization uses pre-existing datasets, this approach quickly and cheaply increases both our understanding of antigenic relationships and our power to control disease.

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To determine the role of the poly(C) tract found at the 5' end of the genome of foot-and-mouth disease virus, synthetic RNAs (in vitro transcripts) with poly(C) tracts of different lengths have been produced and evaluated. RNAs with poly(C) tracts of 35, 25, 16, 6, or 2 residues displayed similar specific infectivities in baby hamster kidney (BHK) cells. Vwruses recovered from cells transfected with in vitro transcripts containing 6 to 35 Cs had properties similar to those of the wild-type virus in cell culture, and poly(C) tracts present in the synthetic RNA-derived viruses ranged from 75 to 140 bases in length. Viruses recovered from transcripts containing only two Cs showed very different properties. Specifically, viruses grew to much lower levels in cell culture and maintained a poly(C) tract of only two residues. The pool of viruses harvested from cells transfected with the synthetic C2 RNA also contained a small amount of a virus with a 42-base deletion in the region of the poly(C) tract, which appeared to have arisen by recombination. Taken together, these data suggest that recombination provides the mechanism of poly(C) elongation and that viruses with poly(C) tracts over 75 bases in length have a selective advantage in cell culture. Interestingly, all of the in vitro transcript-derived viruses [including viruses with poly(C) tracts of only two residues] were equally virulent in mice, indicating that poly(C) tract length has no effect on virulence in this animal model.