Summary auto-generated
This study examined genetic heterogeneity within foot-and-mouth disease virus (FMDV) isolates by using PCR amplification and molecular cloning of viral genome segments encoding capsid proteins. The researchers analyzed two FMDV type O isolates: O1Lombardy and O3Venezuela. They amplified approximately 1 kilobase of genomic RNA encoding parts of VP3, VP1, and peptide 2A, then cloned and sequenced the products. Analysis of up to 11 O3Venezuela sequences revealed six silent nucleotide changes and six amino acid substitutions in VP1 at positions 45, 83, 141, 145, 170, and 178. The O1Lombardy isolate showed seven silent changes and amino acid variations at positions 85 and 134. Control experiments confirmed that sequence variation arose from viral genetic heterogeneity rather than PCR errors. Seven distinct VP1 isotypes of O3Venezuela were identified, differing at multiple amino acid positions. Notably, some isotypes contained isoleucine instead of arginine at position 145, a site potentially involved in viral receptor binding. The findings demonstrate that individual FMDV isolates consist of multiple co-propagating viral isotypes with potentially different antigenic properties, confirming the quasispecies nature of FMDV and having important implications for vaccine development.
Key findings
- Seven distinct VP1 isotypes of O3Venezuela coexist in serially passaged virus, differing at positions 45, 83, 141, 145, and 170, indicating substantial antigenic heterogeneity within a single isolate
- Sequence heterogeneity in VP1 predominantly occurs in loop regions connecting beta-sheets, suggesting possible selection pressure during viral circulation in the field
- Some O3Venezuela variants contain isoleucine at VP1 position 145 instead of arginine, which may be involved in cell receptor binding, yet these genomes remain infectious
- The PCR-based cloning method can reliably detect and distinguish multiple viral isotypes, confirmed by control experiments showing Taq polymerase does not introduce artificial mutations
- Coexistence of multiple FMDV isotypes has significant implications for subunit vaccine design, as single-sequence vaccines may not protect against all naturally occurring variants
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Abstract
Genome segments of the foot-and-mouth disease virus isolates O1Lombardy and O3Venezuela that encode, among other products, capsid protein VP1 were amplified using PCR, and the products were cloned and sequenced. The alignment of up to 11 O3-specific sequences revealed six silent nucleotide changes as well as six changes that cause amino acid substitutions in capsid protein VP1 at positions 45, 83, 141, 145, 170 and 178. The heterogeneity of three O1-specific sequences consisted of seven silent exchanges and amino acid changes at positions 85 and 134 on VP1. Amplification, subcloning and sequencing of cloned O3-specific cDNA was performed to examine the nature of the sequence heterogeneity. As no difference was found among five subcloned sequences, we conclude that the Taq polymerase copied the DNA correctly. The sequence heterogeneity observed with both virus isolates is, therefore, consistent with the quasispecies structure of foot-and-mouth disease virus. Furthermore, amino acid changes at a number of sites have been found to be involved in the formation or modulation of neutralizing epitopes. The novel aspect of this study is the ability to estimate, by cloning of PCR products, the number of virus isotypes, possibly varying in antigenicity, that are able to co-propagate. Seven isotypes of O3Venezuela were identified. Some are of particular interest because they exhibit a change at VP1 codon 145 that causes the replacement of arginine, possibly essential for virus attachment to cells, by isoleucine.
† Present address: Max-Planck-Institut für Züchtungsforschung, Köln, Germany.