2.3 Phylogenetic analysis
Chromatographs were analysed and VP2 sequences were edited and assembled
from the three contigs obtained by PCR using CLC Main Workbench
(Qiagen). Assembled sequences were deposited in GenBank under accession
numbers MN258986 - MN259065, MN528597-MN528598 and MN561318 - MN561321
(FAIRsharing.org). Sequences were aligned using the ClustalW algorithm
in Geneious Prime (Biomatters Ltd., Auckland, New Zealand). Sequences
were translated in MEGA X (Kumar, Stecher, Li, Knyaz, & Tamura, 2018),
and aa substitutions compared to the ICTV reference sequence (CPV-N
GenBank accession no. M19296) were identified. To determine the
evolutionary history of the CPV sequences identified in this study, a
phylogenetic analysis of the VP2 region of these sequences and
representative sequences of FPV and CPV taken from GenBank was
performed. This resulted in a total data set of 94 sequences, 1719 nt in
length. Phylogenetic analyses of these was performed using MEGA X.
Evolutionary history was inferred by using the Maximum Likelihood method
and Tamura 3-parameter model with 1000 bootstrap replicates (Tamura,
1992). Initial tree(s) for the heuristic search were obtained
automatically by applying Neighbour-Join and BioNJ algorithms to a
matrix of pairwise distances estimated using the Maximum Composite
Likelihood approach, and then selecting the topology with superior log
likelihood value. The GTR + I model of nucleotide substitution was used.
Mink enteritis virus (MEV) was used as the outgroup to root the tree.