How, where and when to screen for porcine cytomegalovirus (PCMV) in donor pigs for xenotransplantation

Abstract

Porcine cytomegalovirus (PCMV), that is actually a porcine roseolovirus (PRV), is a common herpesvirus in domestic pigs and wild boars. In xenotransplantation, PCMV/PRV has been shown to significantly reduce the survival time of pig kidneys and hearts in preclinical trials with different non-human primates. Furthermore, PCMV/PRV has been transmitted in the first pig to human heart xenotransplantation and contributed to the death of the patient. Although transmitted to the recipient, there is no evidence that PCMV/PRV can infect primate cells including human cells. PCMV/PRV is closely related to the human herpesviruses 6 and 7, and only distantly related to the human CMV (HCMV). Antiviral drugs used for the treatment of HCMV are less effective against PCMV/PRV. However, there are well described strategies to eliminate the virus from pig facilities. In order to detect the virus and to eliminate it, highly sensitive detection methods and the knowledge of how, where and when to screen the donor pigs is required. Here, a comparative testing of organs from pigs of different ages using polymerase chain reaction (PCR)-based and immunological methods was performed. Testing young piglets, PCMV/PRV was detected effectively by PCR in blood, bronchoalveolar lavage fluid, tonsils and heart. In adult animals, detection by PCR was not successful in most cases, because the virus load was below the detection limit or the virus was in its latent stage. Therefore, detection of antibodies against selected recombinant proteins corresponding to epitopes detected by nearly all infected animals in a Western blot assay is advantageous. By contrast, immunological testing is not beneficial in young animals as piglets might have PCMV/PRV-specific antibodies obtained from their infected mother via the colostrum. Using a thoughtful combination of PCR-based and immunological methods, detection of PCMV/PRV in donor pigs for xenotransplantation is feasible and a controlled elimination of the virus by early weaning or other methods is possible.

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

“…The detection of PCMV/PRV was performed using a real-time PCR assay with specific primers and a probe developed by Mueller et al [13] (Table 1). All assays were performed as duplex real-time PCR using as reference gene porcine glyceraldehyde-3-phosphate-dehydrogenase (pGAPDH) with a specific primer-probe mixture (Table 1) [14] running 45 cycles as described previously [15][16][17]. All experiments were performed with the Sen-siFAST Probe No-ROX kit (Meridian Bioscience, Cincinnati, OH, USA) and the qTOWER3 G qPCR cycler (Analytik Jena, Jena, Germany).…”

“…Western blot analysis was performed as previously described [16,18]. Briefly, for the detection of antibodies against PCMV/PRV, the Western Blot assay designed by Plotzki et al [18] was re-established, but only the C-terminal fragment R2 of the gB protein of PCRV/PRV was used as antigen because the R2 protein was shown to be immunodominant [18].…”

“…Briefly, for the detection of antibodies against PCMV/PRV, the Western Blot assay designed by Plotzki et al [18] was re-established, but only the C-terminal fragment R2 of the gB protein of PCRV/PRV was used as antigen because the R2 protein was shown to be immunodominant [18]. The R2 fragment of the gB of PCMV/PRV was produced in E. coli BL21 cells using the pET16b expression vector encoding PCMV-R2 as described in detail [16,18]. Cell were induced with 1 mM isopropyl-β-D-thiogalactopyranosid (Roth, Karlsruhe, Germany), harvested, and dissolved in 10 mL 8 M urea, 0.5 M NaCl, 15 mM imidazole, 20 mM Tris pH 7.5.…”

Porcine cytomegalovirus detection by nanopore-based metagenomic sequencing in a Hungarian pig farm

“…3 However, since xenotransplantation from PCMV-infected pigs affects the recovery and survival of human patients, screening these donor animals has become an important issue. 4–6…”

“…3 However, since xenotransplantation from PCMV-infected pigs affects the recovery and survival of human patients, screening these donor animals has become an important issue. [4][5][6] Although the virus has already been identified by PCR in Hungary 7 , a phylogenetic comparison of its sequence has not yet been performed. In addition to ONT-based viral detection, the similarity of its genome sequence to the available genomes is studied in this work.…”