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.