Canine Parvovirus and Vaccine-Origin Feline Panleukopenia Virus in Wastewater, Arizona, USA: July 2022-June 2023

Canine parvovirus (CPV) is a virus of veterinary health significance and a member of the Parvoviridae family. Despite its clinical significance and global distribution, surveillance is often limited to cases serious enough to result in veterinary visit and/or hospitalization, thereby limiting our understanding of its evolution and diversity. In this study, we coupled wastewater surveillance (WWS), long-range polymerase chain reaction (PCR) and long-read sequencing and demonstrate the utility of this approach for community-level monitoring of parvovirus diversity. We screened archived viral concentrates from wastewater (WW) collected monthly from July 2022 to June 2023 as part of a previous virus surveillance study from a population of ~500,000 people in Maricopa County, Arizona, USA. Using long-range PCR, the coding-complete sequences (~4.5 kb) were amplified as single contigs and sequenced on a long-read sequencer (MinION). Reads were trimmed, assembled, and contigs subjected to a bioinformatics workflow that includes phylogenetics, immuno-informatics and protein structure modelling. The ~4.5 kb amplicons were amplified from all the samples and sequenced. Twelve contigs (length: 4555 nt to 4675 nt: GC%: 35% to 36%) were assembled from 86,858 trimmed and size-selected reads (length 4400 nt-4900 nt) and all typed as parvoviruses. Overall, there were 11 CPV variants (2a, 2b and 2c) and 1 feline panleukopenia virus (FPV) variant. The FPV was 100% similar in the VP2 genomic region to the 1964 Johnson snow leopard strain present in the Felocell vaccine, suggesting recent shedding post-vaccination. For the CPVs, our analysis showed multiple amino acid substitutions in the VP2 and NS1 proteins, suggestive of host immune pressure and viral adaptation, respectively. The CPV variants clustered predominantly with North and South American variants, suggesting transboundary viral movement and multiple CPV-2c transmission chains seem evident. To the best of our knowledge, we here document the first detection of vaccine-origin FPV in WW. We show the presence of CPV-2a, 2b and 2c in the population sampled and provide evidence that suggests transmission of CPVs across the Americas. Our results also show that WWS coupled with long-range PCR and long-read sequencing is a feasible population-level complement to clinical case surveillance that also facilitates detection of vaccine-origin virus variants. The model we demonstrate here for tracking parvoviruses can also be easily extended to other DNA viruses of human and veterinary health significance.