4.2. Parvovirus PCR and Molecular Characterization of CPV Strains

Organ homogenates were obtained as previously described [74]. DNA was extracted from homogenates using the DNeasy Blood & Tissue Kit (Qiagen S.p.A., Hilden, Germany), according to the manufacturer’s instructions. The presence of CPV DNA was confirmed using a primer pair [75] in a PCR protocol amplifying a 700 bp fragment of the VP2 gene, as previously described [28]. Briefly, PCR was carried out using the GoTaq G2 DNA Polymerase (Promega Italia s.r.l., Milan, Italy) in a 50 μL reaction mix consisting of 10 µL of 5× GoTaq^® Reaction Buffer, 1 µL of MgCl₂ (25 mM), 1 µL of dNTP mix (10 mM), 0.5 µL of each primer VP2-850-Forward and VP2-1550-Reverse (0.5 µM), 0.25 µL of GoTaq^® G2 DNA Polymerase, 31.75 μL of nuclease-free water, and 5 μL of DNA extract. Amplification was conducted under the following thermal conditions: 94 °C for 2 min to activate TaqPol followed by 40 cycles of 94 °C for 30 s, 55 °C for 1 min, and 72 °C for 1 min as well as a final extension of 72 °C for 10 min.

The nearly complete VP2 gene sequence (a 1745-bp fragment) was assessed using a primer pair [76] and direct sequencing [77].

Sequencing encompassing both CPV ORFs (including NS and VP genes) was carried out using the primer pairs developed by Pérez et al. [78], as previously described, and the amplicons were directly sequenced using forward, reverse, and internal primers [29].

The nucleotide VP2 coding sequences were obtained using the ClustalW program and analyzed using the BioEdit software. The sequences were submitted to nBLAST to search related sequences in public domain databases. The CPV antigenic variants (CPV-2a, 2b and 2c) were deduced based on the 426-VP2 amino acid residue [79].

To elucidate the genetic relationships between the obtained CPV strains and the dataset of sequences obtained from the NCBI database, a phylogenetic tree was constructed with the MEGA-X software, using the maximum-likelihood (ML) method according to the Tamura 3-parameter (T92) model with discrete Gamma distribution (+G) employing five rate categories, assuming that a certain fraction of the sites were evolutionarily invariable (+I), and employing bootstrap analyses with 1000 replicates. The phylogeny is depicted in Supplementary Materials Figure S2, showing a representative CPV strain for each genetic and antigenic variant.

These sequence data have been previously or newly submitted to the DDBJ/EMBL/GenBank databases under accession numbers reported in Supplementary Materials Table S1.