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Title:   Author  item
                                            GAVA, D - Embrapa-Pigs And Poultryitem
                                            SOUZA, C - Federal University Of Rio Grande Do Sulitem
                                            Baker, Amyitem
                                            SCHAEFER, R - Embrapa-Pigs And Poultryitem
                                            CANTÃO, M - Embrapa-Pigs And Poultryitem
                                            COLDEBELLA, A - Embrapa-Pigs And Poultryitem
                                            CIACCI-ZANELLA, JANICE - Embrapa-Pigs And Poultry
  Submitted to: Journal of Virological Methods
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 3/13/2015
                                    Publication  Date: 7/1/2015
                                    Citation: Gava, D., Souza, C.K., Schaefer, R., Vincent, A.L., Cantão, M.E., Coldebella, A., Ciacci-Zanella, J.R. 2015. A TaqMan-based real-time PCR for detection and quantification of porcine parvovirus 4. Journal of Virological Methods. 219:14–17.Interpretive Summary: Porcine parvovirus 4 (PPV4) is a DNA virus recently detected in swine, but its epidemiology and pathology remain unclear. A TaqMan-based real-time polymerase chain reaction (qPCR) diagnostic testing assay to detect and quantify PPV4 was developed. The qPCR was highly sensitive, picking up very low amounts of virus. It was also highly specific, with no cross-reaction with porcine parvovirus, torque teno virus 1, torque teno virus 2, porcine circovirus type 1, porcine circovirus type 2, or pseudorabies virus. When compared with a conventional PCR (cPCR), the qPCR assay was 10 times more sensitive. Reproductive tissue samples of sows (ovarian follicular fluid, ovaries and uterus) were evaluated by qPCR. PPV4 was detected in 15 out of 83 (18.1%) ovaries and uterus pools, but it was not detected in ovarian follicular fluid. The viral DNA detected in the sow reproductive tissues was confirmed to be that of PPV4 by sequencing. The significance of finding PPV4 in sow reproductive tissue is unknown, but further study of this and other swine tissues are warranted and the test developed here will enable these types of future studies.
                                Technical Abstract: Porcine parvovirus 4 (PPV4) is a DNA virus, and a member of the Parvoviridae family within the Bocavirus genera. It was recently detected in swine, but its epidemiology and pathology remain unclear. A TaqMan-based real-time polymerase chain reaction (qPCR) assay targeting a conserved region of the ORF3 gene of PPV4 was developed. The qPCR detection limit was 9.5 X 10**1 DNA copies/μL. There was no cross-reaction with porcine parvovirus, torque teno virus 1, torque teno virus 2, porcine circovirus type 1, porcine circovirus type 2, nor with the pseudorabies virus. When compared with a conventional PCR (cPCR), the qPCR assay was 10 times more sensitive. Eighty-three reproductive tissue samples of sows (ovarian follicular fluid, ovaries and uterus) were evaluated by qPCR. PPV4 was detected in 15 out of 83 (18.1%) ovaries and uterus pools, but it was not detected in ovarian follicular fluid. The partial sequencing of the ORF3 gene showed a high nucleotide identity (98% and 99%) with a reference PPV4 sequence. The qPCR can be used as a fast and accurate assay for the detection and quantification of PPV4 and can provide a useful tool for clinical analysis and epidemiology of the virus in swine herds.


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