Complete Genome Sequences of Three African Foot-and-Mouth Disease Viruses from Clinical Samples Isolated in 2009 and 2010

The complete genome sequences of three foot-and-mouth disease viruses (one virus of each serotype SAT1, SAT2 and O) were directly sequenced from RNA extracted from clinical bovine samples, demonstrating the feasibility of full-genome sequencing from strong positive samples taken from symptomatic animals.

To evaluate the feasibility of complete genome sequencing from clinical samples using unbiased RNA sequencing (RNA-seq) methods, three FMDV strongly positive epithelial samples from symptomatic cattle were selected using real-time reverse transcription-PCR (RT-PCR) (3). From these samples, originating from Zambia and Namibia, three FMDV isolates (SAT2/ ZAM18/2009, O/ZAM14/2010, and SAT1/NAM01/2010) were obtained and characterized (using virus isolation, antigen enzyme-linked immunosorbent assay [ELISA], and partial sequencing). The original clinical samples were homogenized in phosphate-buffered saline (10% [wt/vol]), pretreated by 0.45 M size selective filtration and nuclease, and RNA was extracted as previously described (4,5). cDNA was synthesized using Super-Script III reverse transcriptase (Thermo Fisher Scientific) and random hexamer primers, according to the manufacturer's instructions. Sequencing libraries were prepared using 1 ng (or the maximum amount available) of cDNA and the Nextera XT kit (Illumina), according to the manufacturer's instructions, quantified with the library quantification kit Illumina platforms (Kapa Biosystems), and fragment length distribution was verified using the Bioanalyzer with the high-sensitivity DNA kit (Agilent Technologies). Sequencing was performed using a MiSeq reagent kit version 3 (Illumina) with 2 ϫ 300-bp paired-end sequencing. Twenty-three libraries were multiplexed using standard Illumina indexing primers.
The complete genome sequences of SAT2/ZAM18/2009 and O/ZAM14/2010 were obtained, with average coverages of 2,151ϫ and 732ϫ, respectively. These FMDV genomes contain a single open reading frame (ORF) of 7,008 (SAT2/ZAM18/2009) and 6,999 nucleotides (nt) (O/ZAM14/2010) encoding a polypeptide precursor protein, and they share high nucleotide homology with AF540910 and HM191257, respectively. The contig representing SAT1/NAM01/2010 contains a single 7,020-nt ORF (polypeptide precursor protein) and shares a high nucleotide homology with AY593842. As only a limited number of FMDV reads were available for the latter sample, two gaps (a 67-nt gap around the poly(C) tract and a 110-nt gap centered around contig position 6000) were closed using PCR amplification and Sanger sequencing, while the average coverage was Ͻ10ϫ.
These data demonstrate the feasibility of direct sequencing of complete FMDV coding sequences from samples from symptomatic animals (real-time RT-PCR threshold cycle [C T ] range of 14.63 to 16.18 for the samples used in this study) using an unbiased cDNA sequencing approach. However, targeted approaches using FMDV-specific cDNA synthesis primers (9) or PCR amplification may result in a better sensitivity for whole-genome sequencing.
(Georgina Tjipura-Zaire) and Zambia (Charles Nyeleti and Tingiya Sikombe) for collection of samples from the field. This work was supported by an internal research grant of CODA-CERVA, European Union FP7 project RAPIDIA-FIELD (grant no. FP7-289364), and Epi-SEQ: a transnational research project supported under the 2nd joint call for transnational research projects by EMIDA ERA-NET (FP7 project no. 219235).

FUNDING INFORMATION
This work, including the efforts of Steven Van Borm, was funded by European Commission (EC) (project RAPIDIA-FIELD FP7-289364), an internal research grant of CODA-CERVA, and Epi-SEQ, a transnational research project supported under the 2nd joint call for transnational research projects by EMIDA ERA-NET (FP7 project nr 219235).