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In October 2020, a dead pig was submitted to South Dakota State University (Brookings, SD, USA) for diagnostic testing. Histopathologic examination revealed mild to moderate enteritis, hepatitis, and visceral edema. Hemolytic Escherichia coli was isolated. No significant lung lesions were noted. Approximately 8 months later, we performed viral metagenomic sequencing on archived lung tissue for an unrelated research project and unexpectedly identified CPV-2. Using a Taqman PCR (Applied Biosystems, https://www.thermofisher.com), we confirmed that the sample was CPV-2 positive; cycle threshold (Ct) was 24.4. Sanger sequencing of overlapping amplicons confirmed the CPV-2 genome sequence determined by metagenomic sequencing. We submitted the strain SDS21601 sequence to GenBank (accession no. MZ666397).
We used a TaqMan PCR to test 90 archived porcine lung samples submitted for respiratory disease diagnostic testing for CPV-2. Of the 90 samples, 9 (10%) were positive for CPV-2, including those with strain SDS21601, and Ct values were 22.4–36.3. The samples were collected September–November 2020 from swine farms within 150 miles of Brookings. We sequenced the genome from a second strongly positive sample (Ct 22.4) and submitted strain SDS21608 to GenBank (accession no. MZ666398). An amplicon from 4 of the remaining 7 samples positive by PCR was generated by PCR and confirmed as CPV-2 by Sanger sequencing. The 3 samples that failed to yield a CPV-2–specific amplicon had Ct values >32. Sequence comparison showed 99.9% nt identity between SDS21601 and SDS21608. blastp (https://blast.ncbi.nlm.nih.gov) analysis of SDS21601 virus capsid protein (VP) 2 found 100% identity to CPV-2 from a coyote sampled in Montana in 2012. Analysis of the VP2 amino acid sequences identified an F212I mutation previously identified only from US wildlife, mainly coyotes.
We performed in situ hybridization on archived formalin-fixed paraffin-embedded tissues from SDS21608 by using a commercially available CPV-2 probe. CPV-2 nucleic acids were hybridized sporadically as intracytoplasmic punctate signals in few monocyte–macrophage lineage cells in the medullary and subcapsular sinuses of a bronchial lymph node (Figure). However, the primary anatomic site of CPV-2 infection and replication was not determined. In other examined tissues, we observed neither typical virus-associated microscopic lesions as seen in carnivores nor obvious CPV-2 hybridization signals.
To further investigate the extent of CPV-2 circulation among swine, 8 months after collection of the CPV-2–positive lung tissue, we collected 20 serum samples from multiparous sows on the farm where strain SDS21601 originated. Of the 20 samples, 13 (65%) were positive for CPV-2–specific antibodies by hemagglutination inhibition (HI) assay; titers were 10–40 (Table). Nearly all serum samples (19 of 20) had antibody titers to porcine parvovirus 1 (PPV-1), ranging from 16 to 4,096. This result was expected given that pigs on the farm received commercial PPV-1 vaccine before farrowing. There was no correlation between CPV-2 and PPV-1 HI titers, indicating a lack of cross-reactivity between CPV-2 and PPV-1 antibodies in the HI assay.
To further investigate seroprevalence of CPV-2 in South Dakota, we randomly selected 25 sow serum samples from unrelated submissions collected at 5 farms (5 samples/farm) and analyzed them by HI for CPV-2. Of the 25 samples, 23 (92%) were positive for CPV-2; titers were 10–80. Together with the 10% positivity detected by quantitative PCR, these results suggest widespread CPV-2 infection of swine in South Dakota.
Members of Carnivore protoparvovirus 1 display >98% identity. Amino acid residue 300 of VP2 has been shown to be a critical determinant for the cross-species transfer of CPV-2 between carnivores of different species (6). Glycine 300 and tyrosine 305, observed in the VP2 of both swine CPV-2 strains (SDS21601 and SDS21608), are diagnostic of CPV-2 isolates from canids (7). The F212I mutation present in both swine CPV-2 strains, which was previously found only in wildlife, suggests a sylvatic origin. Of the species in which F212I has been identified, only coyotes are common in the agricultural areas of the upper US Midwest and are peridomestic. We hypothesize that the source of swine CPV-2 infection is CPV-2–positive coyote feces.
Our results demonstrate spillover of CPV-2 to swine. CPV-2 has been associated with severe enteritis in insectivorous Taiwanese pangolin (Manis pentadactyla pentadactyla), further demonstrating the propensity of CPV-2 to overcome host barriers (8). The ability of CPV-2 to cause disease in swine remains unknown; further surveillance is warranted because this spillover may threaten the health of swine herds.
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