Emergence of novel circoviruses in humans and pigs and their possible importance for xenotransplantation and blood transfusions

As sequencing is becoming more broadly available, virus discovery continues. Small DNA viruses contribute to up to 60% of the overall virus load in pigs. Porcine circoviruses (PCVs) are small DNA viruses with a single‐stranded circular genome. They are common in pig breeds and have not been properly addressed for their potential risk in xenotransplantation. Whereas PCV1 is non‐pathogenic in pigs, PCV2 has been associated with various disease manifestations. Recently two new circoviruses have been described, PCV3 and PCV4. While PCV4 is currently present mainly in Asia, PCV3 is widely distributed, and has been identified in commercial pigs, wild boars, and pigs generated for xenotransplantation. In one case PCV3 was transmitted by pigs to baboons via heart transplantation. PCV3 pathogenicity in pigs was controversial initially, however, the virus was found to be associated with porcine dermatitis and nephropathy syndrome (PDNS), reproductive failure, and multisystemic inflammation. Inoculation studies with PCV3 infectious clones confirmed that PCV3 is pathogenic. Most importantly, recently discovered human circoviruses (CV) are closely related to PCV3.


INTRODUCTION
Circoviruses-single stranded, small, circular arranged DNA viruseshave been emerging in recent years in many animal species.The Circoviridae family includes the smallest (10-20 nm) autonomously replicating animal viral pathogens 1 and has two genera: Circovirus and Cyclovirus. 2 Within each genus, the species demarcation threshold is 80% genome-wide nucleotide sequence identity. 2Previously, cycloviruses have been found in 7%-17% of human stools and 3%-55% of meat samples tested, in both humans and animals. 3e family Circoviridae belongs to the order Cirlivirales and the phylum Cressdnaviricota. 4 The term CRESS DNA virus was selected in 2012 to refer to the group of ssDNA viruses encoding a replicationassociated protein (Rep) that appears to be descended from a common ancestor."CRESS DNA" stands for circular, rep-encoding ssDNA and encompasses both prokaryotic and eukaryotic viruses, although the Reps of each of these groups have distinct characteristics.5 CRESS DNA viruses are ubiquitous viruses with small genomes, present in all environments, with a diverse host range. Tere appears to be an increasing number of newly discovered CRESS viruses which highlights their diversity and helps to improve the classification of this virus group.The phylum Cressdnaviricota includes several orders, among them the order Cirlivirales with the family Circoviridae.Other families in the phylum are Bacilladnaviridae, Geminiviridae, Nanoviridae, Genomoviridae, and Smacoviridae.4 Little is currently known about these viruses.In a study published in 2020 6 it has been indicated that millions that can infect the different members of all three domains-archaea, bacteria, and eukarya.These viruses can be linear (minority), or circular arranged (majority).6 Initially, circoviruses were associated with infection of various bird species including canary circovirus, 7 gull circovirus, 8,9 penguin circovirus, 10 raven circovirus, 11 starling circovirus, 12 swan circovirus, 13 zebra finch circovirus, 14 and others.The first mammalian host known to harbor a circovirus was the pig, in 1974, when researchers noticed an unknown virus as a contaminant of a permanent porcine kidney cell line PK-15 15 and hence the corresponding virus was called porcine circovirus or PCV.16 Studies performed in the 1980s and 1990s indicated wide spread of the virus in pig farms 17 but association of the virus with disease in pigs was not demonstrated.18,19 However, in 1997 a novel PCV was identified [20][21][22] which quickly was associated with widespread disease outbreaks in pigs.Genomic analysis revealed differences in the two viruses and hence they were named PCV1 and PCV2.As of today, two additional pig associated circoviruses have been identified and are termed PCV3, discovered in 2016 23,24 and PCV4, discovered in 2019.25 PCV4 is present mainly in Asia, but meanwhile the virus was also detected in Europe in wild boars and domestic pigs.26 Circoviruses have also been identified in many other mammalian species, some of which develop disease; including dogs, 27 foxes, 28 cats, 29 and others.In addition, circoviruses have also been detected in fish, 30 shellfish, 31,32 amphibia, 33 reptiles, 34 and insects.35 Circoviruses in bats 36 can be divided into two major clades: Bat-clade-1 and Batclade 2. 37 It has been shown that PCV1 and PCV2 are similar to Batclade-2 whereas PCV3 is more similar to Bat clade 1. Tmost recently discovered circovirus in pigs, PCV4, clusters with mink circovirus.37 PCV3 can be found in most pigs on a global basis including wild boars and domestic pigs with or without clinical signs and are often associated with various other coinfections.38 Interestingly, it has also been Greek pig 39 and dippity pig syndrome in Göttingen minipigs.40 It should be mentioned that PCV3 can be found in invertebrates, in ticks and mosquitoes 41,42 which could indicate that these species may serve as a potential transmission vector in the life-cycle of PCV3.
To better understand the pathogenicity of PCV3, inoculation studies involving PCV3 infectious clones have been conducted.Rescued virus from cells transfected with an infectious DNA clone of a PCV3 derived in China was able to induce PDNS with anorexia, coughing, sneezing, diarrhea, enlarged lymph nodes, hepatitis, and splenitis in specific pathogen-free piglets. 43The levels of proinflammatory cytokines and chemokines in the PCV3 infected pigs were significantly upregulated. 43Another infectious PCV3 DNA clone induced changes in alveolar and cardiac tissues of infected mice 44,45 Recently, experimental inoculation of a PCV3 positive tissue homogenate resulted in vascular lesions and localization of PCV3 in lymph nodes in addition to PCV3 viremia approximately from week 1 to 4. 46 Isolation of PCV3 is generally considered difficult but was achieved from different tissues from weak piglets, stillborn and mummified fetuses into PK15 cells which were free of PCV1 and PCV2. 47Today is commonly accepted that PCV3 can be associated with reproductive disease including late abortions, malformations, mummified, stillborn and weak piglets, cardiac and multisystemic inflammation, in addition to PCV3 systemic disease characterized by wasting, weight loss, ill thrift, poor doers and possible also neurological signs. 23,24,48V3 has importance for xenotransplantation 49 as the virus was found in genetically modified pigs generated for xenotransplantation and PCV3 was, in a few cases, transmitted to baboons after pig heart transplantation.Interestingly the baboons to which PCV3 was transmitted had the longest survival times following the xenotransplant. 50It is well known that PCV2 causes immunosuppression, leading to secondary infections with other pathogens 51,52 ; however, whether PCV3 can cause immunosuppression needs further clarification.53 Nevertheless, PCV3 is commonly found in healthy animals indicating that it could attribute to a seemingly undetected and uncontrolled circulation of PCV3.54 It may be speculated that some pig breeds have viral restriction factors preventing disease or that pathogenicity depends on the virus load.
Recently, human circoviruses have been identified in two instances and in two geographic locations France and China.The report from France described circovirus infection in a 61-year-old woman who had undergone heart and lung transplantation 18 years previously.In March 2023 she was hospitalized due to acute hepatitis. 55Numerous coinfections including cytomegalovirus colitis, parvovirus B19 bicytopenia, and aspergillus bronchitis were identified.A shotgun metagenomics sequencing approach identified a novel human circovirus which initially was called Circovirus parisii or HCirV-1. 55There was a temporal association between hepatitis and a high load of human circovirus 56 in the liver and blood of the patient.Furthermore, a novel human circovirus was also identified in intravenous drug users in Yunnan, China. 57That virus was named HuCV2. 57circovirus in humans was initially discovered in 2010, using human stools samples from Nigeria, 3 that virus was discovered without association with any disease.The authors suggested the virus could have been present due to contamination of the food. 3With the detection of the first human circovirus associated with increased virus load and clinical signs it is likely that additional human circoviruses will follow soon.
Based on extensive tests in pigs, we know that circoviruses are very thermostable. 58For instance, it has been shown that a virus stock maintained at 120 • C for 30 min only resulted in 1 log reduction in virus titre. 58Circoviruses are also resistant to many disinfectants. 59In a source animals barrier facility for generating designated pathogenfree pigs to serve as donor animals in xenotransplantation two outbreaks with PCV2 were recorded and the eradication of the virus from the facility was very difficult. 60The first outbreak was due to The Neighbor-Joining tree constructed based on the genomes of human circoviruses and other represent circoviruses, using the p-distance model.The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches (only >70 are shown).Evolutionary analyses were conducted in MEGA7. 65Circoviruses in pigs were shown in blue font and transmissible encephalopathy (TSE)-associated circular DNAs were used as an outgroup.The classification of bat circoviruses (clade 1 and clade 2) was reported previously. 68wly installed steel penning, the second by employee activities.The room and penning were foamed with potassium peroxymonosulfate, sterilized with chlorine dioxide fog, and disinfected with potassium peroxymonosulfate fog and glutaraldehyde ammonium foam. 60In a similar case in the U.S., PCV2 was eliminated by using VirkonS and a protocol of exposing equipment to ultraviolet light via natural sun light exposure. 61e fact that PCV3 is pathogenic in some pigs, but non-pathogenic in others is of great interest.Clinically healthy pigs generated for xeno-transplantation transmitted PCV3 to baboons. 50Most interestingly, two of the baboons receiving the virus had the longest survival time (195 and 182 days) ever observed in this set of experiments. 62The virus was detected in all analyzed organs of the baboon (spleen, liver, lung, kidney, skin, and muscle) as well as in the explanted pig heart.
The virus load was correlated with survival time of the recipient, indicating that the virus was replicating.However, it is unclear whether PCV3 infected baboon cells or whether the virus was replicating in the pig transplant and disseminated pig cells in the body of the baboons as seen in the case of porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV). 63 2004 the idea of searching for a human circoviruses was first introduced. 64However, the authors found no evidence of such a virus despite screening 1101 samples and concluded that the results rendered the existence of a human circovirus unlikely. 64Nevertheless, in 2023 two independent reports were published almost simultaneously and both reported a novel human circovirus 55 which, based on our analysis, appear to belong to different species.Three different species including human CV1, human CV2, and human CV3 have been identified to date.This probably implies that there may be additional human circoviruses in circulation and waiting for discovery.
The objective of this work was to summarize and combine findings of human circoviruses and cycloviruses in different geographic areas and to contrast the human circoviruses to their counterparts in pigs, as pigs are commonly used for xenotransplantation.

Selected GenBank numbers
The Genbank numbers selected for this study are summarized in Table 1.

Analysis
The Neighbor-Joining tree method was used in addition to the p-distance model.Evolutionary analyses were conducted in MEGA7. 65

PubMed search outcomes
To get an idea of the level of interest in publishing on certain circovirus in pigs or humans we did a basic PubMed search.The results are presented in Table 1.It would appear most publications have been focused on PCV2, followed by PCV3, PCV4, and finally human circoviruses (See Table 2).

Phylogenetic and genetic distances of the human circoviruses and other representative circoviruses
A human circovirus, GQ404856, was discovered initially in a human stool sample in 2007 from Nigerian children with no further information provided. 3It is of interest that initially that virus was classified as cyclovirus 3 Only recently two studies focusing on taxonomy of the family Circoviridae were published and GQ404856 from 2010 was clearly classified as circovirus in 2017. 2,66Human CV1 has a similar genomic structure as other circoviruses with which it clusters.
The more recent human circoviruses from France, both from the same patient but in different publications, 55,56 are, as expected, almost identical except for a single nucleotide difference, resulting in a single amino acid difference in the rep gene.In contrast, the human circovirus from China is substantially different from the French strains with genome identities of ca.80% (Table 3, Figure 1) which appears sufficient to define a new circovirus species 2 hence we classified them into different species (human CV1, CV2, and CV3).It also would appear that human circoviruses are closely related to PCV3.In fact, there are few more similar circovirus species in comparisons between human CV2, human CV3 and PCV3 based on searching from GenBank sequences.The exception is a newly discovered circovirus recovered in four wolverine fecal samples collected during 2018-2019.In Figure 1 we included one of the sequences, MW686210, from a Wolverine (Gulo gulo) which belongs to the Mustelidae family. 67This sequence is closer in identity to PCV3 (63%) than to human circovirus 2 or 3 (approximately 56%).Hence it might be possible that human circoviruses emerged from pigs or vice versa.However, as the genome identity between them is less than 60%, there may be other circoviruses from other species (such as the Wolverine circovirus) which have not been discovered yet.

CONCLUSION
Human circoviruses have been recently discovered for the first time.It is likely that more findings will be occurring soon, and it is reasonable to believe that human CVs may be widely distributed, mainly resulting in subclinical infections.It seems imperative to quickly develop serological assays and PCR kits so that people undergoing blood transfusions but also organ transplantations can be tested beforehand.It also may be necessary to test pigs for presence of these viruses to ensure a safe xenotransplantation.

AUTHOR CONTRIBUTIONS
All authors contributed substantial to the data acquisition, analysis, and interpretation of the data.All authors also contributed to the drafting of the manuscript, and all approved the final version.We acknowledge participation in the Transplant Peer Review Network and complied with the journal's author guidelines and policies.
Results of a basic Pubmed search on circoviruses in pigs and humans.
TA B L E 1Selected Genbank numbers used to create a representative dendogram of circoviruses in pigs, selected other animals and humans.aSearch results using the abbreviatd name: For example: PCV1.b Search results uing the full name for example: Porcine circovirus type 1. c Not applicable.
Sequence identities in % between the three represent human circovirus (Human CV) species and porcine circovirus 3 (PCV3).
TA B L E 3a Indicates the identities of amino acids of Rep and Cap proteins as a percentage.b 99.95.associated with unusual syndromes including erythema multiform in a