Near-complete genome sequencing of swine vesicular disease virus using the Roche GS FLX sequencing platform.

Sandra C Abel Nielsen,Jose Alfredo Samaniego,M Thomas P Gilbert,Christian A W Bruhn,Nick J Knowles,Jemma Wadsworth,Junwen Wang

doi:10.1371/journal.pone.0097180

Sandra C Abel Nielsen, Jose Alfredo Samaniego + Show 5 more

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https://doi.org/10.1371/journal.pone.0097180

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Abstract

Swine vesicular disease virus (SVDV) is an enterovirus that is both genetically and antigenically closely related to human coxsackievirus B5 within the Picornaviridae family. SVDV is the causative agent of a highly contagious (though rarely fatal) vesicular disease in pigs. We report a rapid method that is suitable for sequencing the complete protein-encoding sequences of SVDV isolates in which the RNA is relatively intact. The approach couples a single PCR amplification reaction, using only a single PCR primer set to amplify the near-complete SVDV genome, with deep-sequencing using a small fraction of the capacity of a Roche GS FLX sequencing platform. Sequences were initially verified through one of two criteria; either a match between a de novo assembly and a reference mapping, or a match between all of five different reference mappings performed against a fixed set of starting reference genomes with significant genetic distances within the same species of viruses. All reference mappings used an iterative method to avoid bias. Further verification was achieved through phylogenetic analysis against published SVDV genomes and additional Enterovirus B sequences. This approach allows high confidence in the obtained consensus sequences, as well as provides sufficiently high and evenly dispersed sequence coverage to allow future studies of intra-host variation.

Highlights

Swine vesicular disease (SVD) is a highly contagious viral disease of pigs
The first outbreak of SVD was observed in 1966 in Italy, at the time, it was misidentified as foot-and-mouth disease (FMD) [1]
Previous studies have confirmed that SVD virus (SVDV) is antigenically related to the human pathogen coxsackievirus B5 (CV-B5) [6,7], leading Graves to propose that SVD and its causative agent most likely originated from infection of pigs with a human CV-B5

Summary

Introduction

Swine vesicular disease (SVD) is a highly contagious viral disease of pigs. The first outbreak of SVD was observed in 1966 in Italy, at the time, it was misidentified as foot-and-mouth disease (FMD) [1]. The causative agent, SVD virus (SVDV), was shown to be an enterovirus [1,2,3] and classified as a member of the Enterovirus B species within the Picornaviridae family. It is a non-enveloped virus with a single-stranded RNA genome of positive polarity of approximately 7.4 kb in length [4,5], and shares the same physico-chemical properties of other enteroviruses [1,3]. They analyzed sequence data from 42 SVDV isolates and seven CV-

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