Recovery of complete genomes of canine parvovirus from clinical samples

Canine parvovirus (CPV) is a highly pathogenic virus that affects dogs, especially puppies. CPV is believed to have evolved from feline panleukopenia virus (FPV), eventually giving rise to three antigenic types, CPV-2a, 2b, and 2c. CPV-2 is recognized for its resilience in contaminated environments, ease of transmission among dogs, and pathogenicity for puppies. Despite the relevance of the virus, complete genome sequences of CPV available at GenBank, to date, are scarce. In the current study, we have developed a methodology to allow the recovery of complete CPV-2 genomes directly from clinical samples. For this, seven fecal samples from Gurupi, Tocantins, North Brazil, were collected from puppies with clinical signals of viral enteritis, and submitted to viral DNA isolation and amplification. Two multiplex PCR strategies were designed including primers targeting fragments of 400 base pairs (bp) and 1,000 bp along the complete genome. Sequencing was performed with the Nanopore(R) technology and results obtained with the two approaches were compared. Genome assembly revealed that the 400 bp amplicons generated larger numbers of reads, allowing a more reliable coverage of the whole genome than those attained with primers targeting the larger (1000 bp) amplicons. Nevertheless, both enrichment methodologies were efficient in amplification and sequencing. Viral genome sequences were of high quality and allowed more precise typing and subtyping of viral genomes compared to the commonly employed strategy relying solely on the analysis of the VP2 region, which is limited in scope. The CPV-2 genomes recovered in this study belong to the CPV2a and CPV-2c subtypes, closely related to isolates from the neighboring Amazonian region. In conclusion, the technique reported here may contribute to increase the number of full CPV genomes available, which is essential for understanding the genetic mechanisms underlying the evolution and spread of CPV-2.