2.5 Sequence alignment and phylogenetic analyses
Genomic sequences of PBoV strains obtained in this study were screened for sequence similarities using BLAST (http://www.ncbi.nlm.nig.gov/BLAST) against the database in GenBank, and aligned altogether with 35 reference strains (Table 2) using the MegAlign program of the DNAstar software (version 7.1, DNASTAR Inc., USA). Phylogenetic analysis was performed using the minimum-evolution method in the Molecular Evolutionary Genetics Analysis (MEGA) software (version 6.0) with Kimura distances, and a bootstrap of 1,000 replicates.
Seven recombination detection methods (RDP, GENECONV, BOOTSCAN, MaxChi, Chimaera, SiScan and 3Seq) embedded in Recombination detection program (RDP 4.39) were used to detected recombinant events. Only those recombination events supported by more than four programs were deemed to avoid dependence on a single detection method as described previously (Zhai et al., 2010), and the window size was set to 20 bp, the highest acceptable P value was 0.01.