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

Abstract 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.


TA B L E 1 A list of ssDNA viruses prevalent in pigs
Developments in genome editing technology, such as the wide availability of the CRISPR-Cas9 system, have opened avenues to engineer the pig genome and create immunologically safe organ donors for humans in the near future. 8 Apart from the compatibility and immunological rejection of the xenotransplant, microbial safety is also a primary concern in xenotransplantation. Pigs are a relatively distant species to humans compared to nonhuman primates, and overall, there is a perceived lower risk of cross-species pathogen transmission. However, pigs are known to be a reservoir of viruses that are pathogenic to humans such as Japanese encephalitis virus, Nipah virus, swine influenza A virus, Menangle virus, and Hepatitis E virus. [9][10][11][12][13][14] In addition to these viruses, porcine cytomegalovirus and vesicular stomatitis virus are also known to have zoonotic potential. 15,16 Cross-species transmissions of viruses between pigs and humans can be broadly described to occur under three scenarios. The first "classical scenario" is facilitated by shared ecosystems of humans and pigs and a certain degree of susceptibility of humans to these viruses. 9,12 This scenario includes exposure of humans to pig viruses via the food chain, farming, and veterinary activities. In the second scenario, humans are exposed to pig viruses without being in the proximity of pigs, through products of pig origin used in pharmaceutical products for human patients. These products may include anticoagulants, respiratory agents, and digestive supplements that contain or are based on pork by-products.

| PI G S ELEC TI ON FOR XENOTR ANS PL ANTATION
There is a wide availability of diagnostic reagents and wellestablished monitoring programs for selected pathogens important to commercial pig production. In contrast, pigs bred and maintained for xenotransplantation under high biosecurity procedures require a different approach to ensure inadvertent transmission of porcine viruses to the recipient. A real concern in xenotransplanation is the presence of asymptomatic viral swine infections, which are not part of routine pig veterinary screening. Viruses of pigs which are of potential risk in xenotransplantation have been widely researched and reviewed. 15  approaching that of RNA viruses. 22 The three major families of ssDNA viruses of potential importance in xenotransplantation using pig-derived organs are Anelloviridae, Circoviridae, and

| S ING LE-S TR ANDED DNA VIRUS E S IN PI G S
Parvoviridae (Table 1).

| Epidemiology in humans and pigs
The members of the Anelloviridae have closed circular genomes and typically encode four genes, including a replicase gene which is essential for virus replication. 24 Anelloviruses are estimated to have a mutation rate of 10 -4 nucleotide substitutions per site per year. 25 The first member of this family, torque teno virus (TTV), was isolated in a human case of post-transfusion hepatitis. 26 This virus is now known to be ubiquitous in the human population worldwide, and in one instance has been identified in 94% of analyzed healthy individuals. 27 Five major phylogenetic clusters of TTV differing mainly in the ORF1 (replicase gene) are observed. 28 Although the TTV has not been definitively associated with any disease, it is known to suppress the host interferon response. 28  from healthy pig serum in Japan. 34 It is now known that TTSuVs are prevalent worldwide and two distinct genera, TTSuV1 and TTSuV2 with 40-50% sequence identity, have been identified (Table 1).
TTSuV1 and TTSuV2 are both further subclassified into two genotypes. [35][36][37] Antigenic cross-reactivity is observed between the two genotypes TTSuV1a and TTSuV1b but not between the two species TTSuV1a/b and TTSuV2. 35 Although varying levels of prevalence of TTSuVs in farmed pigs are reported, prevalence rates generally increase with age and may reach up to 100% in finisher and breeding pigs. [37][38][39] In addition, natural mixed infections of TTSuV1 and TTSuV2 have been observed. 38 TTSuVs are transmitted by the oralfecal and vertical routes, with the former considered the main route of transmission. 38,40 TTSuV is also found in boar semen used for artificial insemination, but the importance of this virus transmission route is unknown. 41

| Infection in pigs and tropism
Experimental infections of TTSuV1 and TTSuV2 of gnotobiotic pigs suggest a pathogenic potential of these viruses; however, conclusive evidence of the pathogenicity of TTSuVs in pigs is not established to date. 36,42 Although a positive correlation between the TTSuV DNA levels and the major pig pathogen porcine circovirus type 2 (PCV2) has been observed in clinically affected pigs, this is interpreted to be an effect of immunosuppression caused by high levels of PCV2. 43 TTSuV1 and TTSuV2 are distributed in many organs of naturally infected pigs, with highest virus concentration in bone marrow.
Moreover, T lymphocytes seem to carry a high TTSuV genome load. [43][44][45][46] In farmed pigs, organs of potential value in transplantation such as kidney or liver are found to harbor TTSuV1 and TTSuV2 with a prevalence of more than 50%. 43,45,46 Islet cells in the pancreas could contain TTSuV acquired through circulation; however, this has not been examined. In addition, TTSuV DNA was identified in porcine biologicals such as Mycoplasma hyopneumoniae bacterins, porcine products such as trypsin, and in cell cultures derived from different species. 47,48

| Zoonotic potential of TTSuV
Interestingly, TTV DNA is found in pigs and TTSuV DNA is found in humans. 49,50 Preliminary evidence indicates TTSuV1 replication in human peripheral blood monocytes (PBMCs) and the presence of antibodies against TTSuV1 ORF2 (a nonstructural protein) in human serum samples suggests a zoonotic potential. 50 Of note, TTSuV1infected human PBMCs are shown to be impaired in their mitogenic response. 50 In addition to humans, TTSuV1 DNA and antibodies against TTSuV1 ORF2 antibodies are found in many mammalian hosts such as horses, cattle, sheep, and dogs, indicative of promiscuity in the host range of TTSuV1. 49 At present, cell culture propagation of TTSuV is not established. The potential of this virus to infect humanorigin cell lines needs to be further explored, as this may offer clues to its zoonotic potential. Although the pathogenicity of neither TTV nor TTSuVs has been clearly established, the potential of TTSuV to cross the species barrier and suppress the mitogenic response of human PBMCs raises a concern in xenotransplantation. 49,50

| Negative pig sources
Infections with TTSuV are thought to be widely prevalent and persistent; however, fetuses can be negative for TTSuV and it appears feasible to derive TTSuV1 and TTSuV2 negative herds by a combination of cesarean delivery and high biosecurity conditions of rearing. 41 However, previous contamination of the pig housing facility with Anelloviruses may be an issue and needs to be resolved if existing facilities are being re-utilized rather than using new buildings for donor pig housing. Currently, there is no commercial vaccine available against TTSuV.

| Epidemiology in humans and pigs
The  [66][67][68] However, it has also been associated with enteric disease and skin infections in growing pigs. 69 In pigs, PPVs and PBoVs, in line with the corresponding viruses infecting mammals, have a high rate of mutation. 70

| Infection in pigs and tropism
Other than for PPV1, pathogenicity of any of the newly identified PPVs or PBoVs has not yet been conclusively demonstrated.
Parvovirus DNA can be detected, even in healthy pigs, in a wide range of pig tissues including tissues of possible interest for xenotransplantation such as liver and kidney. 71

| Zoonotic potential of PPV
Porcine parvovirus 1 does not infect cell lines of human or primate origin. 74 Hemophilia patients treated with porcine clotting factor VIII, in which PPV1 DNA was detected, did not develop any antibodies to PPV1. 76 There is no information on the ability of other PPVs to replicate in human cells. However, the general ability of parvoviruses to cross-species barrier has been well noted among virologists. 77,78 The history of emergence of canine parvovirus (CPV) from feline panleukopenia virus (FPV) and its further evolution into novel genotypes with varying host range (CPV2a, b and c) is a paradigm in virology. 77,79 The evolution of CPV has been recapitulated in vitro and a single mutation is sufficient to allow replication in a novel host cell. 77 Similarly, the rodent H1 parvovirus is well studied for its oncolytic potential in human cancer cells. 80,81 The ability of the H1 virus to selectively infect human cancer cells is thought to be determined by the capsid protein and enabled by the increased rate of proliferation of the cancerous cells and their subdued antiviral mechanisms. 81,82 In the case of oncolytic rodent H1 parvovirus, its ability to replicate in human cells is proof of having sufficient factors for the virus to replicate and produce an infective progeny H1 virus in cancerous cells. Given the rapid mutation rate of PPVs and the overall ability of parvoviruses to cross-species barriers, the risk of widely prevalent PPVs in a xenotransplantation setting, where the recipient will undergo prolonged immunosuppressive regimen, is worth closer scrutiny.

| Negative pig sources
While vaccines are available for PPV1 and widely used in breeding herds, they do not prevent infection but rather protect against disease. 83 As the PPVs and PBoVs are not found in all sows and piglets, derivation of free piglets is feasible and the chances can be improved by cesarean section and colostrum deprivation. However, these viruses are widespread and very resistant to disinfection; thus, contamination of the pig housing facility may be difficult to resolve and could result in infection of piglets early in life. for replicase proteins and a single capsid protein in opposite orientations in addition to other putative genes. 24 These are referred to as circular rep-encoding ssDNA (CRESS-DNA) genomes. 24 The replicase protein consists of endonuclease and helicase motifs and is essential for the replication of the circoviral genome by rolling circle replication. 22  ples were collected from farms with 100 or more pigs and these sera were analyzed for PCV1 and PCV2 DNA. 94 While over 82% of sera from 185 farms were positive for PCV2 by PCR, only 2.4% were positive for PCV1. More than 80% of PCV2 DNA-positive pigs were also positive for anti-PCV2 antibodies. 94 Comprehensive data on prevalence of the novel PCV3 are yet being generated. However, similar to PCV1 and PCV2, it is considered to be prevalent worldwide. 92 replication is often limited to individual lymph nodes. Efficacious vaccines against PCV2 that prevent pathogenesis and reduce PCV2 viremia and shedding are available, but as with most vaccines, they

| Infection in pigs and tropism
do not prevent infection of pigs. 98

| Zoonotic potential of PCV
The threat posed by PCVs during xenotransplantation has been previously reviewed. 99 PCV1 and PCV2 are both propagated mainly in the porcine kidney cell line PK-15 but can be also cultured in other cells of porcine origin. [100][101][102] Under extreme in vitro conditions, it has been reported that PCV1 undergoes nonproductive replication in human cell lines (293, HeLa, and Chang liver cells), in which PCV1 replication and gene expression were detected but infectious virus particles are not produced. 102 Human blood leukocytes reportedly infected by PCV1-like particles were visualized by electron microscope and PCV1 DNA was detected in cells; however, infectivity was not determined. 103  In summary, PCVs have been widely prevalent in the global swine population for many decades. However, there is no conclusive evidence of human infections with PCV1 or PCV2 despite being constantly exposed to PCV by various routes and even in high-risk groups such as swine veterinarians. 112,113

| Negative pig sources
Breeding stock free of PCV1 and PCV2 has been derived by a combination of screening for PCV2 DNA, colostrum deprivation, and enhanced biosecurity in husbandry. 112,114,115

TTSuV 1a
TTSuV-F 5′-CGAATGGCTGAGTTTATGCC 38 Common primer for all TTSuVs and a specific probe for each species

TTSuV k2b
TTSuV2-Probe 5′-AACAGAGCTGAGTGTCTAACCGCCTG-3′  (Table 2). In addition, primer-free metagenomic sequencing, which is getting more affordable by the day, is a powerful technique to screen for the above viruses. Due to lack of in-depth knowledge on many aspects of ssDNA viruses, it may be beneficial to screen for them at various levels, such as in the source herd, in donor pigs, harvested organs or cells, and in the donor recipients. 124 It may also be beneficial to build dedicated facilities to rear such pigs. However, building and maintaining highlevel biosecurity pig units is usually associated with a high cost, and with the current state of knowledge perhaps not justifiable. This will need to be further discussed with regulatory agencies. The majority of the ssDNA viruses infecting pigs have not yet been cultured in vitro.
Therefore, the development of pig and human cell line repositories, perhaps genetically engineered to remove innate antiviral defenses, would help in understanding the biology and risk posed by these viruses in xenotransplantation. As ssDNA viruses are ubiquitous in pigs, they could be used as "indicators" to assess the level of "viral load" of the pigs intended for xenotransplantation to humans. 125

CO N FLI C T O F I NTE R E S T S
The authors declare no conflict of interests.

AUTH O R S' CO NTR I B UTI O N S
Anbu K. Karuppannan drafted the manuscript and both Anbu K.
Karuppannan and Tanja Opriessnig reviewed the literature and revised the final manuscript.