Within the family Geminiviridae, the emergence of new species results from their high mutation and recombination rates. In this study, we report the variability and evolution of digitaria streak virus (DSV), a mastrevirus isolated in 1986 from the grass Digitaria setigera in an island of the Vanuatu archipelago. Viral DNA of DSV samples was amplified from D. setigera specimens, derived from the naturally infected original plant, which were propagated in different laboratories in France and Italy for more than twenty years. From the consensus sequences, the nucleotide substitution rate was estimated for the period between a sample and the original sequence published in 1987, as well as for the period between samples. In addition, the intra-host genetic complexity and diversity of 8 DSV populations with a total of 165 sequenced haplotypes was characterized. The evolutionary rate of DSV was estimated to be between 1.13 × 10^-4 and 9.87 × 10^-4 substitutions/site/year, within the ranges observed in other single-stranded DNA viruses and RNA viruses. Bioinformatic analyses revealed high variability and heterogeneity in DSV populations, which confirmed that mutant spectra are continuously generated and are organized as quasispecies. The analysis of polymorphisms revealed nucleotide substitution biases in viral genomes towards deamination and oxidation of ssDNA. The differences in variability in each of the genomic regions reflected a dynamic and modular evolution in the mutant spectra that was not reflected in the consensus sequences. Strikingly, the most variable region of the DSV genome, encoding the movement protein (MP), showed rapid fixation of the mutations in the consensus sequence and a concomitant dN/dS ratio of 6.130, which suggests strong positive selection in this region. Phylogenetic analyses revealed a possible divergence in three genetic lineages from the original Vanuatu DSV isolate.

For most samples, DNA was extracted by Edwards' method (Edwards et al., 1991) with modifications (Sánchez-Campos et al., 2018), and amplified by rolling circle (RCA), using the TempliPhi kit (GE Healthcare, Chicago, USA) (Mabvakure et al., 2016). The molecular cloning of DSV was performed similarly as described previously. The RCA products were linearized with StuI (Fermentas), which cuts DSV genome at position 1589. The resulting DNA fragments were purified with the QIAquick gel extraction kit (Qiagen N.V, Hilden, Germany) ligated to pBluescript II KS (+/-), previously digested with EcoRV and purified by the same method (Wu et al., 2008), introduced into E. coli DH5a. Finally, the cloned recombinant plasmids were amplified by RCA prior to sequencing. For samples where only consensus sequences were determined, ligation reactions were amplified by RCA prior to sequencing. RCA DNAs were sent to GATC Biotech (Constance, Germany), for Sanger sequencing, using the following primers:

DSV667R (5'-CTGGGTTGTGCGTCATACAC-3'),

DSV2021F (5'-CTCTCCCCAAGAAATGGTGA-3'),

M13FP (5'-CCTCTGGCCCAAGTAGACTT-3') and

M13RP (5'-GCCTAGGTAGACATAATTAC-3').

Some viral samples were obtained using Whatman FTA cards (Owor et al., 2007). Each card was impregnated with plant tissue from D. setigera leaves. Then a 2.5 mm diameter fragment from the card was removed with a Harris punch, washed with the washing solution, and left to dry. The piece of card was used directly in 10 ml RCA reaction (Shepherd et al., 2008). DNA product was cloned and sequenced as above.