Sequencing and Phylogenetic Analysis of Viral Genomes

A 4 μL aliquot of the purified amplicon DNA and the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) was used for cycle sequencing according to the manufacturer’s instructions. Besides primers SIN-Reg-B and cSIN-Reg-B (see above), primers Sin-Reg-B-nf: 5’- ATGACATCAAGATTAGCACC-3’ (position 8872–8891) and Sin-Reg-B-nr: 5’- TGATGCGACGGCTAAG –3’ (position 9734–9749) were used in sequencing reactions to reach full redundancy. The reaction was purified using Centri-Sep columns as recommended (Princeton Separations Inc, Adelphia, USA) and analysed by an ABI PRISM 310 Genetic Analyser (Applied Biosystems).
Sequences were aligned using GramAlign v3.0 [33 (link)]. A Maximum clade credibility (MCC) tree with dated tips and internal nodes was inferred using a MCMC Bayesian approach under the GTR model with gamma-distributed rate variation (Γ) and a proportion of invariable sites (I) using a relaxed (uncorrelated lognormal) molecular clock [34 (link)] in BEAST version 1.8.4 [35 (link)]. Four independent MCMC runs of four chains each were run for 10,000,000 states. The First 1,000,000 were used as burning and the MCC was establish from the remaining states. A median joining network [36 (link)] of the sequences was constructed and edited using a 2,190-character set in SPLITSTREE v4.12.3 [37 (link)]