Introduction: Porcine cytomegalovirus (PCMV) is an upper respiratory tract infection, widespread among swine herds, that can be prevented by early weaning piglets at birth into high-health herd barns. Although PCMV infection has not been documented in humans, there is a concern that it could be transmitted through xenotransplantation and potentially lead to xenograft failure. Recently we reported the first experimental case of xeno-heart transplant to a human. The pig (A328.1) had tested negative for PCMV in routine nasal swab DNA tests before being used as a heart donor. However, PCMV was subsequently detected in the recipient’s blood by cell free DNA sequencing. This suggests that latent PCMV was present in donor pig tissues and that nasal swab DNA testing was inadequate for detecting latent PCMV. Here, we retrospectively screened for PCMV in the donor pig PBMCs, spleen and explanted heart biopsy by nested PCR, RT-PCR and ddPCR.

Methods: DNA analysis for PCMV detection was performed by nested PCR, qRT-PCR and ddPCR, following standard protocols.  ddPCR was performed using 3 different PCMV probes (PCMV DNA pol gene) for robust and redundant analysis.

Results: In retrospective analyses, presence of PCMV was determined by qualitative, quantitative, and absolute DNA quantification analysis. PCMV was undetectable by nested PCR in the PBMC but was detected in spleen and postmortem explanted heart tissue. Low levels of PCMV DNA were detected by qRT-PCR in the pig’s PBMCs collected before heart procurement. Similar to qRT-PCR, ddPCR showed low copy number of PCMV in the pig’s PBMCs and spleen, but high copy number in the explanted heart. These results suggest that latent PCMV was present in the donor pig and that it subsequently replicated in the transplanted heart.

Conclusions: Collectively, our results revealed the inadequacy of PCR-based PCMV assays and showed the need for more reliable and robust methods to detect latent PCMV.  DNA tests on blood or nasal swab samples are more appropriate for viremic pigs with high viral loads. In pigs with latent or low levels of the virus, the PCR assays often produce hit-or-miss, ambiguous results, just above or below the threshold of detection, that could lead to false negatives, as in the case of this donor pig. Therefore, alternative approaches such as serological assays to screen donor serum for PCMV antibodies may be more appropriate before xenotransplantation.