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Title: Evidence for porcine parvovirus type 4 (PPV4) in Brazilian swine herds

item GAVA, DANIELLE - Embrapa
item SA ROCHA, CAMILA - University Of Santa Catarina
item SIMON, NEIDE - Embrapa
item ZANELLA, ERALDO - Universidad De Passo Fundo
item CARON, LUIZINHO - Embrapa
item Cheung, Andrew

Submitted to: International Symposium on Emerging and Re-Emerging Pig Diseases
Publication Type: Proceedings
Publication Acceptance Date: 4/10/2011
Publication Date: 6/12/2011
Citation: Gava, D., Sa Rocha, C., Ritterbusch, G.A., Simon, N.L., Zanella, E.L., Caron, L., Cheung, A.K., Ciacci-Zanella, J.R. 2011. Evidence for porcine parvovirus type 4 (PPV4) in Brazilian swine herds. In: Proceedings of the 6th International Symposium on Emerging and Re-emerging Pig Diseases, June 12-15, 2011, Barcelona, Spain. p. 303.

Interpretive Summary:

Technical Abstract: Introduction Porcine bocaviruses were recently identified among swine co-infected with PCV2 (2,3) and suffering an acute-onset disease of high mortality in the United States, in pigs with PMWS in Sweden (1), and in pigs with reproductive and neurological disease in China (4). Parvoviruses are small and non-enveloped icosahedral viruses. According to the ICTV, the Parvovirinae subfamily is classified into five genera: Dependovirus, Bocavirus, Erythrovirus, Parvovirus and Amdovirus. Based on new virus discoveries, two new genera, Hokovirus and Cnvirus, have been proposed. In addition, there are two bovine isolates (BPV2 and BPV3) and one porcine isolate (PPV4, a bocavirus) that do not cluster with any member of the Parvovirinae subfamily (3,5,7). The PPV4 genome contains two major open reading frames (ORF1 and ORF2), coding for the non-structural protein, located at the 5'-end, and the capsid protein, located at the 3'-end (3,5). An additional ORF3 has been observed among viruses of the genus Bocavirus, including PPV4. ORF3 is located in the middle of the viral genome, between ORF1 and ORF2, and is essential for viral DNA replication (5,7). PPV, PCV2, TTV and other single stranded DNA viruses have been detected and associated with reproductive failure (6). The objective of this study was to detect and sequence the genome of PPV4 isolates from reproductive organs collected from culled sows, aiming to verify the presence of PPV4 at these sites. Materials and methods The ovaries and uteri from 83 sows were collected at a slaughterhouse in Santa Catarina state, Brazil, between July 2008 and July 2009. Viral DNA was extracted from a pool of uterus and ovary tissues of each sow with a standard phenol:chloroform:isoamyl alcohol extraction procedure. The PCR reaction was carried out using specific primers (F5'-TAT GTG GGC TGG GCA AGG AAT GTC-3') and (R5'-GTT GCG GAA TGC TAT CAG GCT CTT-3') designed for ORF3, targeting a 440 bp amplicon. The reaction mixture was heated at 95°C for 5 min, amplified for 35 cycles at 95°C for 45 s, 63°C for 1 min, and 72°C for 1 min, and then kept at 4°C. The PCR products were gel-purified using BigDye XTerminator Purification Kit (Qiagen) and nucleotide sequences were determined using an AB-3130xI Genetic Analyzer. Sequences obtained were analyzed using BLASTx and were aligned using Bioedit Sequence Alignment Editor and Clone Manager 7. Results Thirty-five out of 83 samples (42%) tested positive for PPV4, with a DNA amplicon of the expected size clearly detectable. Ten out of 35 positive samples were electrophoresed in an agarose gel, and three were purified and sequenced. Two out of three samples were aligned with results showing 99% and 100% identity with the other nine PPV4 submissions available in GenBank. A single synonymous nucleotide change in a conserved region was found (T to C) in one sample, thus encoding the same amino acid. Discussion Our findings represent the first identification of PPV4 in Brazilian herds. PPV4 has been described in USA (3) and in China (4), and appears to be ubiquitous. PPV4 ORF1 is 1,794 nt long and capable of encoding a protein of 588 residues, PPV4 ORF2 is 2,184 nt long and capable of encoding a protein of 728 residues, and ORF3 is 612 nt long and capable of encoding a protein of 204 residues. Whereas PPV4 is most closely related to BPV2, with respect to ORF1 and ORF2, the coding capacity and genome organization of PPV4 (ORF3), are more similar to those of viruses of the genus Bocavirus (3,4,7). In conclusion, the results of this study demonstrate that PPV4 exists in pig farms in Brazil, probably since 2008. Cloning of PPV4 followed by whole genome sequencing is being carried out. The prevalence, epidemiology and genetic diversity of PPV4 are currently being investigated to try to understand the significance of PPV4 within the swine population. Acknowledgments T