2.7 Phylogenetic, recombination and statistical analysis
Nucleotide and amino acid sequences downloaded from NCBI GenBank were used for phylogenetic analysis and aligned employing the E-INS-I algorithm in MAFFT version 7 (Katoh et al., 2013). IQ-TREE version 1.6.7 (Nguyen et al., 2015) was used to determine the best-fit nucleotide and amino acid substitution models for each alignment using the ModelFinder program and the Akaike information criterion (AIC) (Kalyaanamoorthy et al., 2017). Phylogenetic trees were inferred using the maximum likelihood method in IQ-TREE employing the SH-like approximate likelihood ratio test (SH-aLRT) and ultrafast bootstrap with 1000 replicates to assess nodal support (Hoang et al., 2018). Branch support of >70 SH-aLRT and >70% UFBoot is shown as a grey circle on the branch node of all phylogenetic trees. Nucleotide alignments using MAFFT version 7 were screened for recombination events using RDP4 and possible recombination events were then confirmed using Simplot. To compare the virus composition in FPV-cases and healthy controls, a two-tailed Fisher’s Exact Test was used and a p-value of <0.05 was considered significant.