Simple, quick and cost-efficient: A universal RT-PCR and sequencing strategy for genomic characterisation of foot-and-mouth disease viruses
Introduction
Whole-genome sequencing is becoming more and more important in the field of virology, a fact that is also reflected in the fast growth of online nucleotide databases. Sequencing studies can reveal epidemiological aspects such as transmission pathways, deliver new insights into the biology and evolution of a virus and uncover genetic differences that influence host–virus interactions (Mullan et al., 2004, Radford et al., 2012).
In the case of foot-and-mouth disease virus (FMDV), molecular epidemiology, in particular for the tracing of virus transmission pathways, is a central aspect of control strategies and contingency plans (Abdul-Hamid et al., 2011, Cottam et al., 2008). This is of very high importance because FMDV outbreaks very often result in significant economic losses and drastic trade restrictions (Brito et al., 2015).
FMDV is a non-enveloped, positive-sense single-stranded RNA virus, the type species of the genus Aphthovirus within the family Picornaviridae. Its genome is approximately 8400 nucleotides (nt) long and contains one large open reading frame (ORF). The ORF consists of the leader protease (Lpro), four structural proteins (VP1-4), encoded by the genes 1A, 1B, 1C and 1D, as well as seven non-structural proteins (2A-C, 3A-D) (Jackson et al., 2007, Jamal and Belsham, 2013). Furthermore, there is a 5′ untranslated region (UTR) of approximately 1300 nucleotides that contains the internal ribosome entry site (IRES) and other functional elements (Belsham, 2005), as well as a 3′ UTR that folds into a stem-loop structure and contains the poly-A (Dorsch-Hasler et al., 1975).
There are seven antigenically distinct serotypes: O, A, C, Asia-1, SAT1, SAT2 and SAT3. Serotype C occurred in Kenya and Brazil for the last time in 2004, but the other six serotypes still circulate in mixed pools in Africa, the Middle East and Asia (Brito et al., 2015, Rweyemamu et al., 2008). The serotypes can be further divided into different topotypes, genetic lineages and strains. A nucleotide difference of up to 15% in the VP1-coding sequence is necessary to define a topotype within the serotypes A, O, C and Asia-1. This value is raised to 20% for SAT serotypes due to the higher variability within these serotypes (Samuel and Knowles, 2001b). Serotype O is divided into eleven topotypes (WRLFMD, 2017) and serotype A into three major geographically restricted genotypes (Knowles and Samuel, 2003, Reid et al., 2001, Samuel and Knowles, 2001a). Serotype SAT1 and SAT2 are very diverse separated into thirteen and fourteen topotypes, respectively, while SAT 3 comprises five different topotypes (WRLFMD, 2017). No distinct topotypes have been described for Asia 1 (Brito et al., 2015, Reid et al., 2001). Historically, there were three topotypes for serotype C (Knowles and Samuel, 2003, WRLFMD, 2017) but it is unknown if this serotype still exists in the wild.
Traditionally, FMDV molecular epidemiology is based on analyses of the partial or full-length sequence of the genome region encoding the capsid protein VP1 (Knowles and Samuel, 2003, Xu et al., 2013). However, the VP1 coding sequence alone does not reflect all important phenotypic traits of FMDV. The full genome sequence is useful for high resolution phylogenetic analysis, to track virus movements within an outbreak and to reveal additional genomic features of a virus isolate (Cottam et al., 2006, Xu et al., 2013).
Today, several possibilities for whole-genome sequencing are available (Pareek et al., 2011). Next-generation sequencing (NGS) is independent of prior knowledge of target sequences for primer design (Logan et al., 2014), but is still expensive and requires elaborate library preparation and data analysis (Shendure et al., 2008, Wright et al., 2011).
The generation of sequences using RT-PCR and subsequent Sanger sequencing is often labour-and time-intensive because it requires rigorous primer design and testing (Logan et al., 2014). There are several approaches of covering the whole genome of FMDV by generating overlapping PCR fragments (Abdul-Hamid et al., 2011, Cottam et al., 2006, Cottam et al., 2008). These studies either include different primer pairs for different lineages (Abdul-Hamid et al., 2011) or a multitude of primer mixes with unknown applicability for other serotypes (Cottam et al., 2008).
In order to develop a simple, quick and convenient tool to obtain the full coding sequence of FMDV, a universal primer panel for all seven serotypes of FMDV was designed. The genome was amplified and sequenced in overlapping DNA fragments reaching from the IRES to the end of the open reading frame.
Section snippets
Primer design and selection
The complete genomes of 387 FMDV strains representing all seven serotypes were downloaded from the National Center for Biotechnology Information GenBank database (https://www.ncbi.nlm.nih.gov/nucleotide/). Multiple sequence alignments were performed using the MAFFT algorithm implemented in the Geneious software, Version 8 (Biomatters Limited). Primer design was performed according to standard rules (Chuang et al., 2013, Thornton and Basu, 2011) and primers were analysed for annealing
RT-PCR, sequencing and genotyping
A panel of 52 different primer combinations was evaluated with a set of 12 FMDV isolates representing all seven serotypes. In total, the 15 most suitable genome-spanning primer combinations were selected (Table 1). This allowed robust amplification of the entire ORF with a double-amplicon strategy. Most regions of the ORF were covered by more than one primer pair to improve the overall reliability of the amplification (Fig. 1).
All amplicons were sequenced, and consensus sequences of about seven
Discussion
Whole genome sequences or at least sequences covering the whole open reading frame of FMDV are of increasing importance for the molecular epidemiology of FMDV. In the past, the VP1 coding region has been the primary target for epidemiological studies. However, it has been shown that most inter- and intra-serotypic recombination events occur in the non-structural genes of FMDV (Carrillo et al., 2005, Klein, 2009, Lee et al., 2009) and that important evolutionary events can occur outside of VP1 (
Funding
This research was funded by Merck Life Science, Darmstadt, Germany. There is no involvement of the funder in collection, analysis and interpretation of data, in the writing of the report and in the decision to submit the article for publication.
Acknowledgements
We thank Dr Michael Eschbaumer for his support and for editing the manuscript.
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