用于异种移植的DPF猪病原微生物检测与质量控制

Detection and quality control of pathogenic microorganisms in DPF pigs for xenotransplantation

摘要

异种器官移植是解决当前器官供体短缺的重要方式, 而猪作为器官供体在异种移植中具有重要的作用。然而, 猪的器官移植需要考虑生物安全问题, 特别是猪携带的人畜共患病原体, 一直是异种器官移植研究的焦点之一。异种移植受体感染人畜共患病的风险, 是由动物体内微生物种类和数量决定的, 并与移植受体的免疫抑制性质和强度有关。本文重点介绍了在异种移植手术中显著影响移植受体存活时间, 且可能通过垂直传播的猪巨细胞病毒、猪噬淋巴疱疹病毒、猪圆环病毒以及猪内源性逆转录病毒, 并综述了上述病毒的基本特征和流行病学特性, 讨论了这些病毒的检测和控制策略, 同时介绍了用于异种移植临床的无指定病原体猪的生物净化技术要领和设施管控方法, 期望为异种器官移植提供符合生物安全规范的医用供体猪。

Abstract

Xenotransplantation is considered an important solution for the current shortage of organ donors, and pigs as organ donors play an indispensable role in xenotransplantation. However, the biosafety of pig organ transplantation, especially the zoonotic viruses carried by pigs, has been one of the focuses of xenotransplantation research. The risk of zoonotic infection in xenograft recipients is determined by the type and quantity of microorganisms in the animal, and is related to the nature and intensity of immune suppression. This paper focuses on porcine cytomegalovirus, porcine lymphotropic herpesviruses, porcine circoviruses, and porcine endogenous retroviruses, which have been shown to significantly affect the survival time of transplant recipients in previous xenotransplantation surgeries and may be transmitted vertically, reviews the basic feature and epidemiological characteristics of these viruses, and discusses the detection and control strategies of these viruses. At the same time, this paper introduces the technical essentials of biological purification and facility control methods of designated pathogen-free pigs for clinical xenotransplantation, in order to provide medical donor pigs that meet biosafety standards for xenotransplantation.

关键词

异种移植 / 人畜共患病 / 无指定病原体猪 / 病原控制

Key words

xenotransplantation / zoonosis / designated pathogen-free pig / pathogen control

参考文献

[1] COOPER D K C, GASTON R, ECKHOFF D, et al. Xenotransplantation-the current status and prospects[J]. Br Med Bull, 2018, 125(1): 5-14. DOI: 10.1093/bmb/ldx043.

[2] FISHMAN J A. Risks of infectious disease in xenotransplantation[J]. N Engl J Med, 2022, 387(24): 2258-2267. DOI: 10.1056/NEJMra2207462.

[3] KIM S J, MOON J. Narrative review of the safety of using pigs for xenotransplantation: characteristics and diagnostic methods of vertical transmissible viruses[J]. Biomedicines, 2024, 12(6): 1181. DOI: 10.3390/biomedicines12061181.

[4] ROBBINS J R, BAKARDJIEV A I. Pathogens and the placental fortress[J]. Curr Opin Microbiol, 2012, 15(1): 36-43. DOI: 10.1016/j.mib.2011.11.006.

[5] DONE J T. An inclusion body rhinitis of pigs[J]. Vet Rec, 1955, 67: 525-527.

[6] GU W W, ZENG N F, ZHOU L, et al. Genomic organization and molecular characterization of porcine cytomegalovirus[J]. Virology, 2014, 460/461: 165-172. DOI: 10.1016/j.virol.2014.05.014.

[7] GUEDES M I M C, RISDAHL J M, WISEMAN B, et al. Reactivation of porcine cytomegalovirus through allogeneic stimulation[J]. J Clin Microbiol, 2004, 42(4): 1756-1758. DOI: 10.1128/JCM.42.4.1756-1758.2004.

[8] DENNER J, LÄNGIN M, REICHART B, et al. Impact of porcine cytomegalovirus on long-term orthotopic cardiac xenotransplant survival[J]. Sci Rep, 2020, 10(1): 17531. DOI: 10.1038/s41598-020-73150-9.

[9] CAVICCHIOLI L, ZANETTI R, FERRARESSO S, et al. Expression of recipient cytomegalovirus in immunosuppressed and xenotransplanted Macaca fascicularis may be related to more severe gastrointestinal lesions[J]. Xenotransplantation, 2015, 22(2): 135-143. DOI: 10.1111/xen.12153.

[10] MUELLER N J, LIVINGSTON C, KNOSALLA C, et al. Activation of porcine cytomegalovirus, but not porcine lymphotropic herpesvirus, in pig-to-baboon xenotransplantation[J]. J Infect Dis, 2004, 189(9): 1628-1633. DOI: 10.1086/383351.

[11] MOHIUDDIN M M, SINGH A K, SCOBIE L, et al. Graft dysfunction in compassionate use of genetically engineered pig-to-human cardiac xenotransplantation: a case report[J]. Lancet, 2023, 402(10399): 397-410. DOI: 10.1016/S0140-6736(23)00775-4.

[12] YAMADA K, TASAKI M, SEKIJIMA M, et al. Porcine cytomegalovirus infection is associated with early rejection of kidney grafts in a pig to baboon xenotransplantation model[J]. Transplantation, 2014, 98(4): 411-418. DOI: 10.1097/TP.0000000000000232.

[13] SEKIJIMA M, WAKI S, SAHARA H, et al. Results of life-supporting galactosyltransferase knockout kidneys in cynomolgus monkeys using two different sources of galactosyltransferase knockout Swine[J]. Transplantation, 2014, 98(4): 419-426. DOI: 10.1097/TP.0000000000000314.

[14] PLOWRIGHT W, EDINGTON N, WATT R G. The behaviour of porcine cytomegalovirus in commercial pig herds[J]. J Hyg, 1976, 76(1): 125-135. DOI: 10.1017/s0022172400055017.

[15] EDINGTON N, WATT R G, PLOWRIGHT W. Experimental transplacental transmission of porcine cytomegalovirus[J]. J Hyg, 1977, 78(2): 243-251. DOI: 10.1017/s0022172400056138.

[16] EDINGTON N, BROAD S, WRATHALL A E, et al. Superinfection with porcine cytomegalovirus initiating transplacental infection[J]. Vet Microbiol, 1988, 16(2): 189-193. DOI: 10.1016/0378-1135(88)90043-0.

[17] CLARK D A, FRYER J F L, TUCKER A W, et al. Porcine cytomegalovirus in pigs being bred for xenograft organs: progress towards control[J]. Xenotransplantation, 2003, 10(2): 142-148. DOI: 10.1034/j.1399-3089.2003.01128.x.

[18] EGERER S, FIEBIG U, KESSLER B, et al. Early weaning completely eliminates porcine cytomegalovirus from a newly established pig donor facility for xenotransplantation[J]. Xenotransplantation, 2018, 25(4): e12449. DOI: 10.1111/xen.12449.

[19] DEGRÉ M, RANNEBERG-NILSEN T, BECK S, et al. Human cytomegalovirus productively infects porcine endothelial cells in vitro[J]. Transplantation, 2001, 72(7): 1334-1337. DOI: 10.1097/00007890-200110150-00028.

[20] WHITTEKER J L, DUDANI A K, TACKABERRY E S. Human fibroblasts are permissive for porcine cytomegalovirus in vitro[J]. Transplantation, 2008, 86(1): 155-162. DOI: 10.1097/TP.0b013e31817d4823.

[21] EHLERS B, ULRICH S, GOLTZ M. Detection of two novel porcine herpesviruses with high similarity to gammaherpesviruses[J]. J Gen Virol, 1999, 80(Pt 4): 971-978. DOI: 10.1099/0022-1317-80-4-971.

[22] CHMIELEWICZ B, GOLTZ M, FRANZ T, et al. A novel porcine gammaherpesvirus[J]. Virology, 2003, 308(2): 317-329. DOI: 10.1016/S0042-6822(03)00006-0.

[23] DENNER J. Porcine lymphotropic herpesviruses (PLHVs) and xenotranplantation[J]. Viruses, 2021, 13(6): 1072. DOI: 10.3390/v13061072.

[24] DOUCETTE K, DOR F J M F, WILKINSON R A, et al. Gene expression of porcine lymphotrophic herpesvirus-1 in miniature Swine with posttransplant lymphoproliferative disorder[J]. Transplantation, 2007, 83(1): 87-90. DOI: 10.1097/01.tp.0000228237.32549.16.

[25] MUELLER N J, KUWAKI K, KNOSALLA C, et al. Early weaning of piglets fails to exclude porcine lymphotropic herpesvirus[J]. Xenotransplantation, 2005, 12(1): 59-62. DOI: 10.1111/j.1399-3089.2004.00196.x.

[26] GOLTZ M, ERICSSON T, PATIENCE C, et al. Sequence analysis of the genome of porcine lymphotropic herpesvirus 1 and gene expression during posttransplant lymphoproliferative disease of pigs[J]. Virology, 2002, 294(2): 383-393. DOI: 10.1006/viro.2002.1390.

[27] TUCKER A W, MCNEILLY F, MEEHAN B, et al. Methods for the exclusion of circoviruses and gammaherpesviruses from pigs[J]. Xenotransplantation, 2003, 10(4): 343-348. DOI: 10.1034/j.1399-3089.2003.02048.x.

[28] CHMIELEWICZ B, GOLTZ M, LAHRMANN K H, et al. Approaching virus safety in xenotransplantation: a search for unrecognized herpesviruses in pigs[J]. Xenotransplantation, 2003, 10(4): 349-356. DOI: 10.1034/j.1399-3089.2003.02074.x.

[29] WYNYARD S, NATHU D, GARKAVENKO O, et al. Microbiological safety of the first clinical pig islet xenotransplantation trial in New Zealand[J]. Xenotransplantation, 2014, 21(4): 309-323. DOI: 10.1111/xen.12102.

[30] TISCHER I, RASCH R, TOCHTERMANN G. Characterization of papovavirus-and picornavirus-like particles in permanent pig kidney cell lines[J]. Zentralbl Bakteriol Orig A, 1974, 226(2): 153-167.

[31] NAYAR G P, HAMEL A, LIN L. Detection and characterization of porcine circovirus associated with postweaning multisystemic wasting syndrome in pigs[J]. Can Vet J, 1997, 38(6): 385-386.

[32] PALINSKI R, PIÑEYRO P, SHANG P C, et al. A novel porcine circovirus distantly related to known circoviruses is associated with porcine dermatitis and nephropathy syndrome and reproductive failure[J]. J Virol, 2016, 91(1): e01879-e01816. DOI: 10.1128/JVI.01879-16.

[33] ZHANG H H, HU W Q, LI J Y, et al. Novel circovirus species identified in farmed pigs designated as Porcine circovirus 4, Hunan Province, China[J]. Transbound Emerg Dis, 2020, 67(3): 1057-1061. DOI: 10.1111/tbed.13446.

[34] OPRIESSNIG T, KARUPPANNAN A K, CASTRO A M M G, et al. Porcine circoviruses: current status, knowledge gaps and challenges[J]. Virus Res, 2020, 286: 198044. DOI: 10.1016/j.virusres.2020.198044.

[35] ALLAN G M, MCNEILLY F, CASSIDY J P, et al. Pathogenesis of porcine circovirus; experimental infections of colostrum deprived piglets and examination of pig foetal material[J]. Vet Microbiol, 1995, 44(1): 49-64. DOI: 10.1016/0378-1135(94)00136-k.

[36] CHAE C. A review of porcine circovirus 2-associated syndromes and diseases[J]. Vet J, 2005, 169(3): 326-336. DOI: 10.1016/j.tvjl.2004.01.012.

[37] MADSON D M, OPRIESSNIG T. Effect of porcine circovirus type 2 (PCV2) infection on reproduction: disease, vertical transmission, diagnostics and vaccination[J]. Anim Health Res Rev, 2011, 12(1): 47-65. DOI: 10.1017/S1466252311000053.

[38] OH T, CHAE C. First isolation and genetic characterization of porcine circovirus type 3 using primary porcine kidney cells[J]. Vet Microbiol, 2020, 241: 108576. DOI: 10.1016/j.vetmic.2020.108576.

[39] FRANZO G, GRASSI L, TUCCIARONE C M, et al. A wild circulation: high presence of Porcine circovirus 3 in different mammalian wild hosts and ticks[J]. Transbound Emerg Dis, 2019, 66(4): 1548-1557. DOI: 10.1111/tbed.13180.

[40] HA Z, LI J F, XIE C Z, et al. First detection and genomic characterization of porcine circovirus 3 in mosquitoes from pig farms in China[J]. Vet Microbiol, 2020, 240: 108522. DOI: 10.1016/j.vetmic.2019.108522.

[41] KRÜGER L, LÄNGIN M, REICHART B, et al. Transmission of porcine circovirus 3 (PCV3) by xenotransplantation of pig hearts into baboons[J]. Viruses, 2019, 11(7): 650. DOI: 10.3390/v11070650.

[42] PHAN T G, GIANNITTI F, ROSSOW S, et al. Detection of a novel circovirus PCV3 in pigs with cardiac and multi-systemic inflammation[J]. Virol J, 2016, 13(1): 184. DOI: 10.1186/s12985-016-0642-z.

[43] 窦科峰, 张玄. 临床异种器官移植十大问题的思考[J]. 器官移植, 2022, 13(4): 411-416. DOI: 10.3969/j.issn.1674-7445.2022.04.001.

[44] DENNER J. Microchimerism, PERV and xenotransplantation[J]. Viruses, 2023, 15(1): 190. DOI: 10.3390/v15010190.

[45] AKIYOSHI D E, DENARO M, ZHU H, et al. Identification of a full-length cDNA for an endogenous retrovirus of miniature swine[J]. J Virol, 1998, 72(5): 4503-4507. DOI: 10.1128/JVI.72.5.4503-4507.1998.

[46] PATIENCE C, TAKEUCHI Y, WEISS R A. Infection of human cells by an endogenous retrovirus of pigs[J]. Nat Med, 1997, 3(3): 282-286. DOI: 10.1038/nm0397-282.

[47] WILSON C A, WONG S, MULLER J, et al. Type C retrovirus released from porcine primary peripheral blood mononuclear cells infects human cells[J]. J Virol, 1998, 72(4): 3082-3087. DOI: 10.1128/JVI.72.4.3082-3087.1998.

[48] MARTIN U, KIESSIG V, BLUSCH J H, et al. Expression of pig endogenous retrovirus by primary porcine endothelial cells and infection of human cells[J]. Lancet, 1998, 352(9129): 692-694. DOI: 10.1016/S0140-6736(98)07144-X.

[49] PORRETT P M, ORANDI B J, KUMAR V, et al. First clinical-grade porcine kidney xenotransplant using a human decedent model[J]. Am J Transplant, 2022, 22(4): 1037-1053. DOI: 10.1111/ajt.16930.

[50] NIU D, WEI H J, LIN L, et al. Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9[J]. Science, 2017, 357(6357): 1303-1307. DOI: 10.1126/science.aan4187.

[51] HALECKER S, KRABBEN L, KRISTIANSEN Y, et al. Rare isolation of human-tropic recombinant porcine endogenous retroviruses PERV-A/C from Göttingen minipigs[J]. Virol J, 2022, 19(1): 30. DOI: 10.1186/s12985-022-01742-0.

[52] MA Y Y, JIA J T, FAN R, et al. Screening and identification of the first non-CRISPR/Cas9-treated Chinese miniature pig with defective porcine endogenous retrovirus pol genes[J]. Front Immunol, 2022, 12: 797608. DOI: 10.3389/fimmu.2021.797608.

[53] MONTGOMERY R A, MEHTA S A, PARENT B, et al. Next steps for the xenotransplantation of pig organs into humans[J]. Nat Med, 2022, 28(8): 1533-1536. DOI: 10.1038/s41591-022-01896-y.

[54] WILSON C A. Endogenous retroviruses: porcine endogenous retroviruses and xenotransplantation[J]. Cell Mol Life Sci, 2008, 65(21): 3399-3412. DOI: 10.1007/s00018-008-8498-z.

[55] HALECKER S, HANSEN S, KRABBEN L, et al. How, where and when to screen for porcine cytomegalovirus (PCMV) in donor pigs for xenotransplantation[J]. Sci Rep, 2022, 12(1): 21545. DOI: 10.1038/s41598-022-25624-1.

[56] GRIFFITH B P, GOERLICH C E, SINGH A K, et al. Genetically modified porcine-to-human cardiac xenotransplantation[J]. N Engl J Med, 2022, 387(1): 35-44. DOI: 10.1056/NEJMoa2201422.

[57] LU T F, SUN B, YU T Y, et al. Porcine endogenous retroviruses: quantification of the viral copy number for the four miniature pig breeds in China[J]. Front Microbiol, 2022, 13: 840347. DOI: 10.3389/fmicb.2022.840347.

[58] PLOTZKI E, KELLER M, IVANUSIC D, et al. A new Western blot assay for the detection of porcine cytomegalovirus (PCMV)[J]. J Immunol Meth, 2016, 437: 37-42. DOI: 10.1016/j.jim.2016.08.001.

[59] KARUPPANNAN A K, OPRIESSNIG T. Porcine circovirus type 2 (PCV2) vaccines in the context of current molecular epidemiology[J]. Viruses, 2017, 9(5): 99. DOI: 10.3390/v9050099.

[60] KLAUMANN F, CORREA-FIZ F, FRANZO G, et al. Current knowledge on porcine circovirus 3 (PCV-3): a novel virus with a yet unknown impact on the swine industry[J]. Front Vet Sci, 2018, 5: 315. DOI: 10.3389/fvets.2018.00315.

[61] ZHANG C, FREISTAEDTER A, SCHMELAS C, et al. An RNA interference/adeno-associated virus vector-based combinatorial gene therapy approach against hepatitis E virus[J]. Hepatol Commun, 2022, 6(4): 878-888. DOI: 10.1002/hep4.1842.

[62] SEMAAN M, KAULITZ D, PETERSEN B, et al. Long-term effects of PERV-specific RNA interference in transgenic pigs[J]. Xenotransplantation, 2012, 19(2): 112-121. DOI: 10.1111/j.1399-3089.2012.00683.x.

[63] YUE Y N, XU W H, KAN Y N, et al. Extensive germline genome engineering in pigs[J]. Nat Biomed Eng, 2021, 5(2): 134-143. DOI: 10.1038/s41551-020-00613-9.

[64] LU C, PANG D X, LI M J, et al. CRISPR/Cas9-mediated hitchhike expression of functional shRNAs at the porcine mir-17-92 cluster[J]. Cells, 2019, 8(2): 113. DOI: 10.3390/cells8020113.

[65] NOORDERGRAAF J, SCHUCKER A, MARTIN M, et al. Pathogen elimination and prevention within a regulated, Designated Pathogen Free, closed pig herd for long-term breeding and production of xenotransplantation materials[J]. Xenotransplantation, 2018, 25(4): e12428. DOI: 10.1111/xen.12428.

[66] NANTEL-FORTIER N, LETELLIER A, LACHAPELLE V, et al. Detection and phylogenetic analysis of the hepatitis E virus in a Canadian swine production network[J]. Food Environ Virol, 2016, 8(4): 296-304. DOI: 10.1007/s12560-016-9252-6.

[67] DENNER J. Risk of pathogenic virus transmission by somatic cell nuclear transfer: implications for xenotransplantation[J]. Biol Reprod, 2022, 107(3): 717-722. DOI: 10.1093/biolre/ioac120.

[68] HANSEN S, FISCHER K, KRABBEN L, et al. Detection of porcine cytomegalovirus, a roseolovirus, in pig ovaries and follicular fluid: implications for somatic cells nuclear transfer, cloning and xenotransplantation[J]. Virol J, 2023, 20(1): 15. DOI: 10.1186/s12985-023-01975-7.

[69] MA W J. Swine influenza virus: current status and challenge[J]. Virus Res, 2020, 288: 198118. DOI: 10.1016/j.virusres.2020.198118.

[70] SUN H L, XIAO Y H, LIU J Y, et al. Prevalent Eurasian avian-like H1N1 swine influenza virus with 2009 pandemic viral genes facilitating human infection[J]. Proc Natl Acad Sci U S A, 2020, 117(29): 17204-17210. DOI: 10.1073/pnas.1921186117.

[71] HE W T, AUCLERT L Z, ZHAI X F, et al. Interspecies transmission, genetic diversity, and evolutionary dynamics of pseudorabies virus[J]. J Infect Dis, 2019, 219(11): 1705-1715. DOI: 10.1093/infdis/jiy731.

基金资助

陕西省创新能力支撑计划项目(2024CX-GXPT-36)

陕西省创新能力支撑计划项目(2024CX-GXPT-37)

四川省中央引导地方科技发展专项(2024ZYD0315)

空军军医大学“快速响应”科研课题(2022KXKT001)