A newly cloned pig dolichyl‐phosphate mannosyl‐transferase for preventing the transmission of porcine endogenous retrovirus to human cells

Porcine endogenous retrovirus (PERV) is a major problem associated with successful clinical xenotransplantation. In our previous study, reducing the high mannose type of N‐glycan content proved to be very effective in downregulating PERV infectivity. In this study, dolichyl‐phosphate mannosyltransferase (D‐P‐M), an enzyme related to the early stages of N‐linked sugar synthesis was studied. The pig cDNA of the encoding D‐P‐M was newly isolated. The RNA interference (siRNA) for the D‐P‐M was applied and transfected to PEC(Z)/PB cells, a pig endothelial cell line with the Lac Z gene and PERV‐B, to reduce the levels of high mannose type N‐glycans. Compared with the mock line, the temporary PEC(Z)/PB lines showed a decreased mRNA expression for pig D‐P‐M, and each line then showed a clear destruction of PERV infectivity to human cells in the Lac Z pseudotype assay. The PEC(Z)/PB was next transfected with pSXGH‐siRNA, H1‐RNA gene promoter. The established PEC(Z)/PB clones with pSXGH‐siRNA clearly led to the downregulation of PERV infectivity, as evidenced by the decreased levels of the mRNA for pig D‐P‐M. Reducing D‐P‐M enzyme activity represents a potentially useful approach to address the problem of PERV infections in clinical xenotransplantations.     

Differential human serum‐mediated neutralization of PERV released from pig cells transfected with variants of hDAF

Abstract: Background: Expression of complement regulatory proteins (CRP) on pig endothelial cells (PEC) is an effective means of avoiding induction of hyperacute rejection by human sera. However, pig endogenous retrovirus (PERV) from PEC transfected with CRP may acquire resistance to human sera. This study investigated a form of transfected CRP that is easily expressed on PERV particles. Methods: The PEC line was transfected with the Lac Z gene and PERV‐B to investigate PERV infectivity using a Lac Z pseudo‐type assay. The cDNAs of several modified DAF (CD55) were then transfected into the PEC(Lac Z)/P‐B lines using lipofection. DAF expression was verified by FACS analysis. Complement‐dependent PEC lysis was tested to verify the complement regulatory function of the expressed DAF. HEK293 cells were incubated with PEC culture supernatants with or without human sera. The inoculated 293 cells were histochemically stained and Lac Z‐positive blue foci were counted. The rate of reduction in Lac Z‐positive cells resulting from the addition of human serum was then calculated. In addition, to assess the localization of the expressed DAF, flotation sucrose density analysis was performed. Results: While PERV released from PEC expressing delta‐short consensus repeat 2 (delta‐SCR2) DAF (lacking CRP function) showed no change in resistance to human serum compared to control cells, PERV from cells expressing delta‐SCR1 DAF (with CRP function) showed a significant increase in resistance. The DAF‐blocking antibody assay indicated that PERV from the DAF transfectants expressed DAF molecules on the surface of the retrovirus. While delta‐SCR1 DAF (PI‐anchor form) significantly inhibited the reduction of Lac Z‐positive cells by human serum, the reduction of Lac Z‐positive cells by human serum was less inhibited in the case of transmembrane (TM)‐types of DAF–HLA‐G, modified influenza hemagglutinin (HA) and MCP (delta‐CYT form). However, the reduction in each TM‐type DAF was slightly less than that observed in naive and mock cells. The flotation sucrose density analysis of these transfectants indicated that the PI‐anchor form of DAF is a raft‐associated protein, and most TM‐types of DAF are non‐raft proteins. Conclusion: Induction of resistance to human serum in PERV, depends on the form of the CRP tail. The CRP/TM hybrid that does not associate with lipid rafts, is a suitable form of CRP for gene transduction.     

No in vivo infection of triple immunosuppressed non‐human primates after inoculation with high titers of porcine endogenous retroviruses*

Abstract: Background: Porcine endogenous retroviruses (PERVs) released from pig tissue can infect selected human cells in vitro and therefore represent a safety risk for xenotransplantation using pig cells, tissues, or organs. Although PERVs infect cells of numerous species in vitro, attempts to establish reliable animal models failed until now. Absence of PERV transmission has been shown in first experimental and clinical xenotransplantations; however, these trials suffered from the absence of long‐term exposure (transplant survival) and profound immunosuppression. Methods: We conducted infectivity studies in rhesus monkeys, pig‐tailed monkeys, and baboons under chronic immunosuppression with cyclosporine A, methylprednisolone, and the rapamycin derivative. These species were selected because they are close to the human species and PERVs can be transmitted in vitro to cells of these species. In addition, the animals received twice, a C1 esterase inhibitor to block complement activation before inoculation of PERV. In order to overcome the complications of microchimerism, animals were inoculated with high titers of cell‐free PERV. In addition, to enable transmission via cell–cell contact, some animals also received virus‐producing cells. For inoculation the primate cell‐adapted strain PERV/5° was used which is characterized by a high infectious titer. Produced on human cells, this virus does not express alpha 1,3 Gal epitopes, does not contain porcine antigens on the viral surface and is therefore less immunogenic in non‐human primates compared with pig cell‐derived virus. Finally, we present evidence that PERV/5° productively infects cells from baboons and rhesus monkeys. Results: In a follow‐up period of 11 months, no antibody production against PERV and no integration of proviral DNA in blood cells was observed. Furthermore, no PERV sequences were detected in the DNA of different organs taken after necropsy. Conclusion: These results indicate that in a primate model, in the presence of chronic immunosuppression, neither the inoculation of cell‐free nor cell‐associated PERV using a virus already adapted to primate cells results in an infection; this is despite the fact that peripheral blood mononuclear cells of the same animals are infectible in vitro.     

Knockdown of porcine endogenous retrovirus (PERV) expression by PERV-specific shRNA in transgenic pigs

Abstract: Background: Xenotransplantation using porcine cells, tissues or organs may be associated with the transmission of porcine endogenous retroviruses (PERVs). More than 50 viral copies have been identified in the pig genome and three different subtypes of PERV were released from pig cells, two of them were able to infect human cells in vitro. RNA interference is a promising option to inhibit PERV transmission. Methods: We recently selected an efficient si (small interfering) RNA corresponding to a highly conserved region in the PERV DNA, which is able to inhibit expression of all PERV subtypes in PERV‐infected human cells as well as in primary pig cells. Pig fibroblasts were transfected using a lentiviral vector expressing a corresponding sh (short hairpin) RNA and transgenic pigs were produced by somatic nuclear transfer cloning. Integration of the vector was proven by PCR, expression of shRNA and PERV was studied by in‐solution hybridization analysis and real‐time RT PCR, respectively. Results: All seven born piglets had integrated the transgene. Expression of the shRNA was found in all tissues investigated and PERV expression was significantly inhibited when compared with wild‐type control animals. Conclusion: This strategy may lead to animals compatible with PERV safe xenotransplantation.     

Long-term effects of PERV-specific RNA interference in transgenic pigs

Semaan M, Kaulitz D, Petersen B, Niemann H, Denner J. Long‐term effects of PERV‐specific RNA interference in transgenic pigs. Xenotransplantation 2012; 19: 112–121. © 2012 John Wiley & Sons A/S. Abstract: Background: Porcine endogenous retroviruses (PERVs) represent a risk of xenotransplantation using porcine cells, tissues, or organs, as they are integrated in the porcine genome and have been shown to be able to infect human cells in vitro. To increase viral safety by RNA interference, transgenic pigs expressing a PERV‐specific small hairpin (sh)RNA targeted to a highly conserved sequence in the pol gene (pol2) were generated in which expression of PERVs was reduced (Xenotransplantation, 15, 2008, 38). However, it remains to be shown how long expression of the shRNA and the RNA interference is effective in reducing PERV expression. Methods: To analyze the long‐term duration of RNA interference, expression of the PERV‐specific pol2 shRNA and inhibition of PERV expression was studied repeatedly in fibroblasts and peripheral blood mononuclear cells (PBMCs) of transgenic pigs over a period of 3 yr, when animals were sacrificed and expression was studied in different organs. Expression of the PERV‐specific shRNA was measured using a newly developed real‐time PCR, and expression of PERV was measured using a PERV‐specific real‐time PCR. Results: Over a period of 3 yr, PERV‐specific shRNA and green fluorescent protein (GFP) as reporter of the vector system were consistently expressed in transgenic animals. PERV expression was significantly reduced during the entire period. Levels of PERV and shRNA expression were different in the various organs. PERV expression was highest in the spleen and the lungs and lowest in liver and heart. However, in all organs of the transgenic pigs, PERV expression was inhibited compared with the vector control animals. Conclusions: Transgenic pigs expressing PERV‐specific shRNA maintained their specific RNA interference long term, suggesting that PERV expression in the xenotransplants will be suppressed over extended periods of time.     

Similar frequency of Porcine circovirus 3 (PCV‐3) detection in serum samples of pigs affected by digestive or respiratory disorders and age‐matched clinically healthy pigs

Porcine circovirus 3 (PCV‐3) has been identified in pigs affected by different disease conditions, although its pathogenicity remains unclear. The objective of the present study was to assess the frequency of PCV‐3 infection in serum samples from animals suffering from post‐weaning respiratory or digestive disorders as well as in healthy animals. A total of 315 swine serum samples were analysed for PCV‐3 DNA detection by conventional PCR; positive samples were further assayed with a quantitative PCR and partially sequenced. Sera were obtained from 4 week‐ to 4 month‐old pigs clinically diagnosed with respiratory (n = 129) or digestive (n = 126) disorders. Serum samples of age‐matched healthy animals (n = 60) served as negative control. Pigs with clinical respiratory signs had a wide variety of pulmonary lesions including suppurative bronchopneumonia, interstitial pneumonia, fibrinous‐necrotizing pneumonia and/or pleuritis. Animals with enteric signs displayed histopathological findings like villus atrophy and fusion, catarrhal enteritis and/or catarrhal colitis. Overall, PCV‐3 DNA was detected in 19 out of 315 analysed samples (6.0%). Among the diseased animals, PCV‐3 was found in 6.2% (8 out of 129) and 5.6% (7 out of 126) of pigs with respiratory and digestive disorders, respectively. The frequency of PCV‐3 PCR positive samples among healthy pigs was 6.7% (4 out of 60). No apparent association was observed between PCR positive cases and any type of histopathological lesion. The phylogenetic analysis of the partial genome sequences obtained showed high identity among viruses from the three groups of animals studied. In conclusion, PCV‐3 was present in the serum of diseased and healthy pigs to similar percentages, suggesting that this virus does not seem to be causally associated with respiratory or enteric disorders.     

Review on porcine endogenous retrovirus detection assays—impact on quality and safety of xenotransplants

Xenotransplantation of porcine organs, tissues, and cells inherits a risk for xenozoonotic infections. Viable tissues and cells intended for transplantation have to be considered as potentially contaminated non‐sterile products. The demands on microbial testing, based on the regulatory requirements, are often challenging due to a restricted shelf life or the complexity of the product itself. In Europe, the regulatory framework for xenogeneic cell therapy is based on the advanced therapy medicinal products (ATMP) regulation (2007), the EMA CHMP Guideline on xenogeneic cell‐based medicinal products (2009), as well as the WHO and Council of Europe recommendations. In the USA, FDA guidance for industry (2003) regulates the use of xenotransplants. To comply with the regulations, validated test methods need to be established that reveal the microbial status of a transplant within its given shelf life, complemented by strictly defined action alert limits and supported by breeding in specific pathogen‐free (SPF) facilities. In this review, we focus on assays for the detection of the porcine endogenous retroviruses PERV‐A/‐B/‐C, which exhibit highly polymorphic proviral loci in pig genomes. PERVs are transmitted vertically and cannot be completely eliminated by breeding or gene knock out technology. PERVs entail a public health concern that will persist even if no evidence of PERV infection of xenotransplant recipients in vivo has been revealed yet. Nevertheless, infectious risks must be minimized by full assessment of pigs as donors by combining different molecular screening assays for sensitive and specific detection as well as a functional analysis of the infectivity of PERV including an adequate monitoring of recipients.     

Safety of xenotransplantation: screening the donor pig and the recipient

Introduction: Xenotransplantation using pig cells and tissues may be associated with the transmission of porcine microorganisms including bacteria, parasites, fungi and viruses to the human recipient and may result in zoonones. Porcine endogenous retroviruses (PERVs) represent a special risk since PERV‐A and PERV‐B are present in the genome of all pigs and infect human cells. PERV‐C is not present in all pigs and does not infect human cells. However, recombinants between PERV‐A and PERV‐C have been observed in normal pigs characterised by higher replication rates compared with PERV‐A, and they are also able to infect human cells (1). Methods: In the past years numerous assays based on the PCR technology have been developed to screen for the prevalence and expression of PERV and other porcine microorganisms in the donor pig (2). Whereas most microorganisms may be eliminated by designated pathogen‐free breeding, PERVs cannot be removed this way. In addition, assays have been developed to analyse the recipient for the transmission of PERV and other microorganisms, either using PCR methods or immunological assays to detect an antibody production as a result of infection (3). Results: Using these assays, no transmission of PERV as well as of other porcine microorganisms has been observed in first preclinical and clinical xenotransplantations or animal infection experiments. This was especially true for the first clinical transplantation of pig islet cells approved by the New Zealand government (4). Until now there is no susceptible animal model to study PERV transmission and transplantations of porcine cells or organs to non‐human primates as they are associated with limitations concerning the safety aspect, which do not allow transmitting the negative findings to humans (5). Different experimental approaches are under development to reduce the probability of PERV transmission, e.g. the generation of transgenic pigs expressing PERV‐specific siRNA inhibiting PERV expression by RNA interference (6), genotypic selection of pigs with a low prevalence and expression of PERV and neutralising antibodies against the envelope proteins inhibiting PERV infection (7). Conclusion: Investigations of the last years resulted in highly sensitive and specific methods to study PERV and other microorganisms in donor pigs and human recipients of xenotransplants. These methods showed absence of PERV transmission in all investigated cases, both in more than 200 human xenotransplant recipients, mostly recipients of cellular xenotransplants, as well as in non‐human primates and small animals. New technologies under development may further decrease the probability of transmission. References: 1. Denner J. Recombinant porcine endogenous retroviruses (PERV‐A/C): A new risk for xenotransplantation? Arch Virol 2008; 153: 1421–1426. 2. Kaulitz D, Mihica D, Dorna J, Costa MR, Petersen B, Niemann H, TÖnjes RR, Denner J. Development of sensitive methods for detection of porcine endogenous retrovirus‐C (PERV‐C) in the genome of pigs J Virol Methods 2011; 175(1): 60–65. 3. Denner, J. Infectious risk in xenotransplantation – what post‐transplant screening for the human recipient? Xenotransplantation 2011; 18(3): 151–157. 4. Wynyard S, Garkavenko O, Nathu D, Denner J, Elliott R. Microbiological safety of the first clinical pig islet xenotransplantation trial in New Zealand, submitted. 5. Mattiuzzo G, Takeuchi Y. Suboptimal porcine endogenous retrovirus infection in non‐human primate cells: implication for preclinical xenotransplantation. PLoS One 2010; 5(10): e13203. 6. Semaan M, Kaulitz D, Petersen B, Niemann H, Denner J. Long‐term effects of PERV‐specific RNA interference in transgenic pigs. Xenotransplantation 2012; 19(2): 112–21. 7. Kaulitz D, Fiebig U, Eschricht M, Wurzbacher C, Kurth R, Denner J. Generation of neutralising antibodies against porcine endogenous retroviruses (PERVs). Virology 2011; 411(1): 78–86.     

Emerging infectious diseases and xenotransplantation

A total of 335 infectious diseases was reported in the global human population between 1940 and 2004, the majority of which were caused by zoonotic pathogens [ 1 ]. Although viral pathogens constitute only 25%, some have spread worldwide with most starting from Central Africa. These include human immunodeficiency virus (HIV) causing acquired immunodeficiency syndromes (AIDS), chikungunya virus and West Nile virus, which also cause severe diseases in humans. HIV‐1 and HIV‐2, for example, are the result of trans‐species transmission from non‐human primates [ 2 ] to humans sometime in the last century. The spread of two henipaviruses causing fatal diseases in horses, pigs and humans has been observed in Asia and Australia, and although these viruses represent transspecies transmissions from bats, secondary transmissions from pigs to humans have also occurred. These and many other examples of emerging infectious diseases call for strong safety considerations in the field of xenotransplantation. Whereas known viruses can easily be eliminated from donor pigs, strategies should be developed to detect new zoonotic pathogens. In addition, all pigs carry porcine endogenous retroviruses (PERVs) in their genome. Two of these, PERV‐A and PERV‐B, as wells as recombinant PERV‐A/C are able to infect human cells. The greatest threat appears to come from the recombinant PERV‐A/C viruses as they appear to have an increased infectivity [ 3 , 4 ]. An increase in PERV expression was not observed in multitransgenic pigs expressing DAF, TRAIL and HLAE, generated to prevent immune rejection [ 5 ]. Our laboratory has developed a variety of strategies to prevent PERV transmission following xenotransplantation: (i) selection of animals that do not harbour PERV‐C genomes in order to prevent recombination, (ii) selection of PERV‐A and PERV‐B low‐producers [ 6 ], (iii) development of an antiviral vaccine to protect xenotransplant recipients [ 7 ] and (iv) generation of transgenic pigs in which PERV expression is inhibited via RNA interference. Inhibition of PERV expression using either synthetic small interfering (si) RNA or short hairpin (sh) RNA was demonstrated in PERV infected human cells [ 8 ], in primary pig cells [ 9 ] and in all transgenic piglets born [ 10 ]. A second generation of pigs expressing PERV‐specific siRNA is now under study and experiments have been started to introduce multiple shRNA. Supported by Deutsche Forschungsgemeinschaft, DFG, DE729/4.     

First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes—Chapter 2a: source pigs—preventing xenozoonoses

Chapter 2 of the original consensus statement published in 2009 by IXA represents an excellent basis for the production of safe donor pigs and pig‐derived materials for porcine islet xenotransplantation. It was intended that the consensus statement was to be reviewed at interval to remain relevant. Indeed, many of the original salient points remain relevant today, especially when porcine islet xenotransplantation is performed in conjunction with immunosuppressants. However, progress in the field including demonstrated safe clinical porcine xenograft studies, increased understanding of risks including those posed by PERV, and advancement of diagnostic capabilities now allow for further consideration. Agents of known and unknown pathogenic significance continue to be identified and should be considered on a geographic, risk‐based, dynamic, and product‐specific basis, where appropriate using validated, advanced diagnostic techniques. PERV risk can be sufficiently reduced via multicomponent profiling including subtype expression levels in combination with infectivity assays. Barrier facilities built and operated against the AAALAC Ag Guide or suitable alternative criteria should be considered for source animal production as long as cGMPs and SOPs are followed. Bovine material‐free feed for source animals should be considered appropriate instead of mammalian free materials to sufficiently reduce TSE risks. Finally, the sponsor retention period for archival samples of donor materials was deemed sufficient until the death of the recipient if conclusively determined to be of unrelated and non‐infectious cause or for a reasonable period, that is, five to 10 yrs. In summary, the safe and economical production of suitable pigs and porcine islet xenograft materials, under appropriate guidance and regulatory control, is believed to be a viable means of addressing the unmet need for clinical islet replacement materials.     

Islet isolation from adult designated pathogen-free pigs: use of the newer bovine nervous tissue-free enzymes and a revised donor selection strategy would improve the islet graft function

Jin S‐M, Shin JS, Kim KS, Gong C‐H, Park SK, Kim J‐S, Yeom S‐C, Hwang ES, Lee CT, Kim S‐J, Park C‐G. Islet isolation from adult designated pathogen‐free pigs: use of the newer bovine nervous tissue–free enzymes and a revised donor selection strategy would improve the islet graft function. Xenotransplantation 2011; 18: 369–379. © 2011 John Wiley & Sons A/S. Abstract: Background: In clinical trials using adult porcine islet products, islets should be isolated from the designated pathogen‐free (DPF) pigs under the current good manufacturing practice (GMP) regulations. Our previous studies suggested that male DPF pigs are better donors than retired breeder pigs and histomorphometrical parameters of donor pancreas predict the porcine islet quality. We aimed to investigate whether the use of the newer bovine nervous tissue–free enzymes and a revised donor selection strategy could improve the islet graft function in the context of islet isolation with DPF pigs. Methods: Using 30 DPF pigs within a closed herd, we compared the islet yield of porcine islets isolated with Liberase PI (n = 11, as a historical control group), Liberase MTF C/T, which is a GMP‐grade enzyme (n = 12), and CIzyme collagenase MA/BP protease (n = 7). We analyzed the relationship between the diabetes reversal rate of recipient NOD/SCID mice (n = 75) and histomorphometric parameters of each donor pancreas as well as donor characteristics. Results: Proportion of islets larger than 200 μm from the biopsied donor pancreas (P = 0.006) better predicted islet yield than age (P = 0.760) or body weight (P = 0.371) of donor. The proportion of islets larger than 200 μm from the biopsied donor pancreas was not related to the sex of the donor miniature pig (P = 0.358). The islet yield obtained with the three enzymes did not differ, even after stratification of the donor with the histomorphometric parameters of the biopsied donor pancreas and the sex of donor. The use of the newer bovine nervous tissue–free enzymes (P < 0.001), a higher proportion of large islets in donor pancreas (P = 0.006), and a male sex of the donor (P = 0.025) were independent predictors of earlier diabetes reversal. Conclusions: Use of the newer bovine nervous tissue–free enzymes including a GMP‐grade enzyme resulted in better islet quality than that of islet isolated using Liberase PI. To obtain high‐quality islet from DPF pigs, the donor should be male pig and histomorphometrical parameters from donor pancreas should be considered.     

Long-term absence of porcine endogenous retrovirus infection in chronically immunosuppressed patients after treatment with the porcine cell-based Academic Medical Center bioartificial liver

Di Nicuolo G, D’Alessandro A, Andria B, Scuderi V, Scognamiglio M, Tammaro A, Mancini A, Cozzolino S, Di Florio E, Bracco A, Calise F, Chamuleau RAFM. Long‐term absence of porcine endogenous retrovirus infection in chronically immunosuppressed patients after treatment with the porcine cell–based Academic Medical Center bioartificial liver. Xenotransplantation 2010; 17: 431–439. © 2010 John Wiley & Sons A/S. Abstract: Background: Clinical use of porcine cell–based bioartificial liver (BAL) support in acute liver failure as bridging therapy for liver transplantation exposes the patient to the risk of transmission of porcine endogenous retroviruses (PERVs) to human. This risk may be enhanced when patients receive liver transplant and are subsequently immunosuppressed. As further follow‐up of previously reported patients (Di Nicuolo et al. 2005), an assessment of PERV infection was made in the same patient population pharmacologically immunosuppressed for several years after BAL treatment and in healthcare workers (HCWs) involved in the clinical trial at that time. Methods: Plasma and peripheral blood mononuclear cells (PBMCs) from eight patients treated with the Academic Medical Center‐BAL (AMC‐BAL), who survived to transplant, and 13 HCWs, who were involved in the trial, were assessed to detect PERV infection. A novel quantitative real‐time polymerase chain reaction assay has been used. Results: Eight patients who received a liver transplant after AMC‐BAL treatment are still alive under long‐term pharmacological immunosuppression. The current clinical follow‐up ranges from 5.6 to 8.7 yr after BAL treatment. A new q‐real‐time PCR assay has been developed and validated to detect PERV infection. The limit of quantification of PERV DNA was ≥5 copies per 1 × 10⁵ PBMCs. The linear dynamic range was from 5 × 10⁰ to 5 × 10⁶ copies. In both patients and HCWs, neither PERV DNA in PBMCs nor PERV RNA in plasma and PBMC samples have been found. Conclusion: Up to 8.7 yr after exposure to treatment with porcine liver cell–based BAL, no PERV infection has been found in long‐term immunosuppressed patients and in HCWs by a new highly sensitive and specific q‐real‐time PCR assay.     

Possible risks posed by single‐stranded DNA viruses of pigs associated with xenotransplantation

Routine large‐scale xenotransplantation from pigs to humans is getting closer to clinical reality owing to several state‐of‐the‐art technologies, especially the ability to rapidly engineer genetically defined pigs. However, using pig organs in humans poses risks including unwanted cross‐species transfer of viruses and adaption of these pig viruses to the human organ recipient. Recent developments in the field of virology, including the advent of metagenomic techniques to characterize entire viromes, have led to the identification of a plethora of viruses in many niches. Single‐stranded DNA (ssDNA) viruses are the largest group prevalent in virome studies in mammals. Specifically, the ssDNA viral genomes are characterized by a high rate of nucleotide substitution, which confers a proclivity to adapt to new hosts and cross‐species barriers. Pig‐associated ssDNA viruses include torque teno sus viruses (TTSuV) in the Anelloviridae family, porcine parvoviruses (PPV), and porcine bocaviruses (PBoV) both in the family of Parvoviridae, and porcine circoviruses (PCV) in the Circoviridae family, some of which have been confirmed to be pathogenic to pigs. The risks of these viruses for the human recipient during xenotransplantation procedures are relatively unknown. Based on the scant knowledge available on the prevalence, predilection, and pathogenicity of pig‐associated ssDNA viruses, careful screening and monitoring are required. In the case of positive identification, risk assessments and strategies to eliminate these viruses in xenotransplantation pig stock may be needed.