Asymmetric patterns of reassortment and concerted evolution in Cardamom bushy dwarf virus
Introduction
Genetic exchange plays a key role in the evolution of many viruses, and can have important epidemiological implications. For instance, by generating novel combinations of nucleotide polymorphisms, genetic exchange may lead to the emergence of highly virulent strains (Hou and Gilbertson, 1996, Li et al., 2010) or new species (Padidam et al., 1999, Gibbs and Weiller, 1999), or facilitate adaptation to different hosts (Scholtissek et al., 1985, Idris et al., 2008) or vectors (Beaty et al., 1981). Three forms of genetic exchange can occur when multiple species or genetically distinct strains co-infect the same host cell. These include homologous recombination, whereby a nucleic acid sequence from a donor genome replaces a homologous sequence within a recipient genome, and non-homologous recombination, during which genetic exchange occurs between unrelated sequences. The third form, known as reassortment (or pseudo-recombination), occurs only in multipartite viruses with segmented genomes, and involves the exchange of discrete genome components between different species or strains. Whilst the genome segments of most multipartite viruses are contained within a single virion, multipartite viruses which infect plants have individually encapsidated genome components. This strategy, combined with vector mediated transmission, may lead to high rates of reassortment in multipartite plant viruses (Roossnick, 2005, Martin et al., 2011a).
Genetic exchange appears to occur frequently in single-stranded DNA (ssDNA) plant viruses belonging to the Geminiviridae and Nanoviridae families (Zhou et al., 1997, Padidam et al., 1999, Hughes, 2004, Hu et al., 2007, Fu et al., 2009, Lefeuvre et al., 2009, Hyder et al., 2011, Stainton et al., 2012, Wang et al., 2013, Grigoras et al., 2014). These viruses have circular genome components and replicate via a rolling circle mechanism which may facilitate recombination (Preiss and Jeske, 2003, Martin et al., 2011a). Whilst geminiviruses have monopartite or bipartite genomes, multipartite nanoviruses can contain up to 12 individually encapsidated genome components (Mandal, 2010). Each nanovirus genome component is approximately 1.0 kb in length, and contains a single gene, a common stem-loop region (CR-SL) and a major common region (CR-M). Although the function of the CR-M is unclear, the CR-SL contains sequence motifs that may be involved in recognition specificity of the master replication (M-Rep) protein (Herrera-Valencia et al., 2006), which initiates replication of all genome components (Timchenko et al., 2000). Homogenization of common regions by repeated inter-component homologous recombination is thought to be an important mechanism by which M-Rep proteins maintain replicational control over distinct genome components (Hughes, 2004, Martin et al., 2011a). This process may also promote the efficient replication of novel components which are introduced into a genome through reassortment (Hou and Gilbertson, 1996, Martin et al., 2011a).
Given the large number of discrete genome components that nanoviruses contain, there is huge potential for reassortants to arise when multiple genotypes co-infect the same host cell. However, there have been few reports of reassortment in nanoviruses (Horser et al., 2001, Bell et al., 2002, Hu et al., 2007, Stainton et al., 2012, Grigoras et al., 2014). In this study, we examined the role of reassortment and recombination in the evolutionary dynamics of Cardamom bushy dwarf virus (CBDV), an aphid-borne nanovirus belonging to the Babuvirus genus (Mandal et al., 2004, Mandal et al., 2013). CBDV infects large cardamom, Amomum subulatum, an important cash crop in sub-Himalayan regions of Northeast India, Nepal and Bhutan. It is the causal agent of ‘foorkey’ disease, which is characterised by excessive sprouting of dwarf tillers, reduced yield and mortality, and severely constrains large cardamom production (Mandal et al., 2013). We obtained sequence data for six discrete genome components from 163 CBDV isolates. We expect that our data will provide a valuable source of information for future analyses of genetic diversity and genetic exchange in nanoviruses, as well as comparative studies with other ssDNA viruses.
Section snippets
Sample collection
Leaf samples were collected in 2011 and 2012 from CBDV infected plants at a range of locations and altitudes throughout Sikkim and the Darjeeling district of West Bengal, Northeast India (Fig. 1; Supplementary Table 1). Samples were dried with silica gel and maintained in Ziplock bags until DNA extraction.
DNA extraction and PCR amplification of CBDV genome components
Approximately 100 mg of leaf tissue from each infected plant sample was ground to a fine paste with a mortar and pestle using liquid nitrogen. Total genomic plant DNA and viral DNA were then
Nucleotide diversity and phylogenetic analyses
DNA fragments corresponding to six discrete genome components were sequenced for 163 CBDV isolates collected at a range of locations and altitudes throughout Sikkim and the Darjeeling district of West Bengal, Northeast India (Fig. 1; Supplementary Table 1). The genome components included DNA-R, which encodes the CBDV M-Rep protein, DNA-U3, which has an unknown function, DNA-S, which encodes the CBDV coat protein, DNA-M, which encodes the CBDV movement protein, DNA-C, which encodes the CBDV cell
Conclusions
We have demonstrated that both reassortment and recombination play important roles in CBDV evolution. However, different CBDV genome components clearly vary in their propensity to reassort and to recombine. The observed patterns of genetic exchange may reflect different levels of purifying selection acting on specific genome components and/or intra-genome interactions. Our study highlights the importance of characterising multiple discrete genome components from a large number of isolates to
Acknowledgements
We thank Dr. Darren Martin for helpful advice regarding RDP4 analyses, Dr. Bikash Mandal (Indian Agricultural Research Institute, New Delhi) for providing a CBDV DNA-M sequence to facilitate primer design, and the Spice Board (Government of India) for allowing us to access their large cardamom research stations in Pangthang (East Sikkim) and Kabi (North Sikkim). This work was funded by a Department of Biotechnology (DBT, Government of India) grant entitled ‘Technological Innovations and
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2018, Current Opinion in VirologyCitation Excerpt :The main problems with both the ancestral component category state mapping approach and the simpler phylogenetic incongruence testing approaches are, firstly, that they become impractical when analysing either large numbers of sequences (over a few hundred) or genomes with large numbers of components, and, secondly, that their accuracy can be seriously undermined by intra-component genetic recombination. Reassortment can also be analysed using recombination detection methods [35–44]. When alignments of concatenated genome segments are loaded into recombination detection computer programmes like RDP4 [45] or GARD [46], the sequences can be simultaneously analysed for reassortment and intra-segment/component recombination.
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2015, Infection, Genetics and EvolutionCitation Excerpt :The infection status of the plants was visually assessed based on the expression of foorkey disease symptoms. CBDV DNA had also been extracted from a subset of the plants (approximately 10%) during previous studies (Savory and Ramakrishnan, 2014; Savory et al., 2014). Aphid specimens from each infected plant were collected using a paintbrush and were preserved in 95% ethanol until DNA extraction.