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

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.

Screening for Viruses in Indigenous Greek Black Pigs

“…Since the symptoms in baboons with PCMV/PRV-positive transplants are similar to the symptoms in the Baltimore patient, the virus obviously contributed to the death of the patient. The real-time PCR developed by Mueller et al [ 17 ] was modified and performed as a duplex real-time PCR detecting simultaneously porcine GAPDH [ 30 ]. Furthermore, gene blocks comprising the virus-specific oligosequences corresponding to the primers and probes were used as positive control and for the standard curves ( Supplementary Figure S1 ).…”

“…The R2 fragment was shown immunodominant in the gB protein [ 28 ] and gave similar results when compared with an ELISA using synthetic peptides corresponding to the R2 sequence [ 35 , 36 ]. Our Western blot assay was used repeatedly to determine the antibody response in different pig breeds [ 29 , 30 ].…”

“…Western blotting was performed as described previously in detail using the recombinant R2 fragment of the gB protein of PCMV/PRV [ 28 , 29 , 30 ]. The sera were tested at a dilution of 1:150.…”

Xenotransplantation literature update March 2021–March 2023

“…However, the inherent latency of the virus presents a significant challenge. PCMV has even been found to transmit through somatic cells after washing steps 50,51 …”

“…PCMV has even been found to transmit through somatic cells after washing steps. 50,51 Although PCMV and porcine roseolovirus (PRV) are not known to infect human and non-human primate cells, it is a highly replicative virus with no standard treatment. 52 There has been limited effect of standard anti-viral medications, such as ganciclovir and valacyclovir, on PCMV after xenotransplantation.…”