ORF 2 sequence amplification and phylogeny
Sequences of the VP2 protein were PCR amplified using previously
described primers (Lu et al., 2020), and sequences of the amplicons thus
obtained were verified using the Sanger method (Sangon). For
phylogenetic analysis, we constructed a tree based on the
maximum-likelihood (ML) tree method inferred using 144 Carnivore
protoparvovirus 1 VP2 gene sequences (Table S1). These sequences were
aligned using Clustal W (Larkin et al., 2007), and the ML tree was
generated using MEGA X software (Kumar, Stecher, & Tamura, 2016) based
on 3-parameter (T92) model and gamma distribution with invariant sites
(G + I) (Tamura & Nei, 1993). The reliability of the ML tree was
assessed based on 500 bootstrap replicates, and the original tree was
summarized and annotated using Figtree software.