Full sequencing of viral genomes: practical strategies used for the amplification and characterization of foot-and-mouth disease virus

Abstract

Nucleic acid sequencing is now commonplace in most research and diagnostic virology laboratories. The data generated can be used to compare novel strains with other viruses and allow the genetic basis of important phenotypic characteristics, such as antigenic determinants, to be elucidated. Furthermore, virus sequence data can also be used to address more fundamental questions relating to the evolution of viruses. Recent advances in laboratory methodologies allow rapid sequencing of virus genomes. For the first time, this opens up the potential for using genome sequencing to reconstruct virus transmission trees with extremely high resolution and to quickly reveal and identify the origin of unresolved transmission events within discrete infection clusters. Using foot-and-mouth disease virus as an example, this chapter describes strategies that can be successfully used to amplify and sequence the full genomes of RNA viruses. Practical considerations for protocol design and optimization are discussed, with particular emphasis on the software programs used to assemble large contigs and analyze the sequence data for high-resolution epidemiology.

Fig. 1.

Outline of RT-PCR strategies that have been used to amplify the complete genome sequences of FMDV. (a) Long overlapping products (∼3 kb) are generated by PCR using full-length cDNA as a template. Sequences are obtained using a panel of specific sequencing primers (see ref. 3). (b) Short products (∼700 bp) are generated using FMDV-specific primers, which also incorporate regions (labelled F and R) targeted by the sequencing primers (10). (c) Long-range RT-PCR is used to amplify a product comprising the complete L fragment of FMDV. This may either be sequenced using many specific primers (11) or can be fragmented by restriction digest and cloned into a bacterial plasmid vector (pilot studies using this method have been undertaken by IAH in collaboration with the Wellcome Trust Sanger Institute, Cambridge).

Fig. 2.

Amplification of full-FMDV genomes by RT-PCR. Agarose gel electrophoresis shows RT-PCR fragments representing amplification of the entire FMDV genome. (a) amplification of O/UKG/2001 genome using 5 RT-PCR fragments and (b) amplification of O/UKG/2007 genomes using 24 RT-PCR fragments.

Fig. 3.

Phylogenetic analysis of FMDV genomes from the 2001 outbreak in the United Kingdom. (a) Maximum likelihood phylogenetic tree representing 14 FMDV complete genomes rooted to sequence “1,” constructed using PhyloWIN95 (33), incorporating the HKY model of nucleotide substitution with gamma distributed rate heterogeneity. Bootstrap values from 1,000 replicates are shown. (b) Statistical parsimony representation of the same 14 complete FMDV sequences, constructed using TCS (Version 1.21; 34). Each line represents a nucleotide substitution and each dot a putative ancestor virus.

Fig. 4.

Physical separation of the laboratory activities: outline of the separate steps required for the amplification and sequencing of FMDV genomes.