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. 2023 Jan 27;20(1):15.
doi: 10.1186/s12985-023-01975-7.

Detection of porcine cytomegalovirus, a roseolovirus, in pig ovaries and follicular fluid: implications for somatic cells nuclear transfer, cloning and xenotransplantation

Sabrina Hansen  1 , Konrad Fischer  2 , Ludwig Krabben  1 , Alexander Rinke Carrapeiro  2 , Bernhard Klinger  2 , Angelika Schnieke  2 , Benedikt Kaufer  1 , Joachim Denner  3
Affiliations

Detection of porcine cytomegalovirus, a roseolovirus, in pig ovaries and follicular fluid: implications for somatic cells nuclear transfer, cloning and xenotransplantation

Sabrina Hansen et al. Virol J. .

Abstract

Background: Porcine cytomegalovirus (PCMV) is a porcine roseolovirus (PCMV/PRV) which is widely distributed in pigs. Transmission of PCMV/PRV in preclinical xenotransplantations was shown to significantly reduce the survival time of the pig transplants in non-human primates. PCMV/PRV was also transmitted in the first transplantation of a pig heart into a human patient. To analyze how PCMV/PRV could be introduced into pig breeds, especially considering cloned transgenic pigs, and subsequently spread in breeding facilities, we screened ovaries and derived materials which are used to perform somatic cell nuclear transfer (SCNT).

Methods: DNA was isolated from ovarian tissues, follicular fluids, oocytes with cumulus cells, denuded oocytes and parthenotes. A real-time PCR with PCMV/PRV-specific primers and a probe was performed to detect PCMV/PRV. Furthermore, a Western blot assay using a recombinant fragment of the gB protein of PCMV/PRV was performed to screen for virus-specific antibodies in the follicular fluids.

Results: PCMV/PRV was found by real-time PCR in ovarian tissues, in the follicular fluid and in oocytes. In parthenotes the virus could not be detected, most-likely due to the low amount of DNA used. By Western blot assay specific antibodies against PCMV/PRV were found in 19 of 20 analyzed follicular fluids.

Conclusion: PCMV/PRV was found in ovarian tissues, in the follicular fluids and also in denuded oocytes, indicating that the virus is present in the animals of which the oocytes were taken from. Despite several washing steps of the denuded oocytes, which are subsequently used for microinjection or SCNT, the virus could still be detected. Therefore, the virus could infect oocytes during genetic modifications or stay attached to the surface of the oocytes, potentially infecting SCNT recipient animals.

Keywords: Cloning; Oocytes; Porcine cytomegalovirus; Porcine roseolovirus; Somatic cell nuclear transfer; Xenotransplantation.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Western blot analysis of 18 follicular fluids (1–18). A a 12% gel was used for SDS-PAGE, B, C a 17% gel was used. The molecular weight marker (M), a serum from a positive control animal (P) and the location of the recombinant R2 protein of the gB protein of PCMV/PRV are indicated. The serum dilution was 1:150
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