- Email: [email protected]
Porcine Endogenous Retrovirus Transmission Characteristics From a Designated Pathogen-Free Herd
Porcine Endogenous Retrovirus Transmission Characteristics From a Designated Pathogen-Free Herd O. Garkavenko, S. Wynyard, D. Nathu, M. Muzina, Z. Muzina, L. Scobie, R.D. Hector, M.C. Croxson, P. Tan, and B.R. Elliott ABSTRACT Previously, a strategy for monitoring pigs intended for cell transplantation was developed and successfully applied to several representative herds in New Zealand. A better understanding of porcine viruses’ epidemiology in New Zealand has been achieved, and, as a result, a designated pathogen-free (DPF) herd has been chosen as a good candidate for xenotransplantation. This herd is free of all infectious agents relevant to xenotransplantation. The presented study of pig endogenous retrovirus (PERV) transmission with cocultures in vitro has shown no evidence of PERV transmission from DPF pig tissue. Additionally, in PERV-C–positive DPF donor pigs tested, a specific locus for PERV-C present in miniature swine possibly associated with the transmission of PERV was absent. The data on PERV transmission allowed classifying the DPF potential donors as “null” or noninfectious pigs.
I
T IS ARGUED that some of the infectious risk associated with xenotransplantation can be assessed before the broad application of the emerging technology. A specific program for choosing and maintaining a source herd should be developed. Such a program should be designed for a specific geographical area (PHS Guidelines on Infectious Disease Issues in Xenotransplantation). A strategy to select and maintain the source pig herd intended for islet cell xenotransplantation has been developed for New Zealand representative herds.1 Following this strategy, several representative herds have been screened. We now have a much better understanding of the pig virus epidemiology in New Zealand. We have described the prevalence of pig hepatitis E virus,2 pig circovirus type 2,3 pig cytomegalovirus, encephalomyocarditis virus, and pig lymphotropic herpesvirus,1 and a designated pathogen-free (DPF) herd has been identified.1 However, one potential pathogen, the porcine endogenous retrovirus (PERV), cannot be eliminated with stringent microbiological control. This study is focused on the infectious characteristics of the PERV from the DPF herd.
MATERIALS AND METHODS Animals Five 7-day-old pig donors of LCT DPF herd were used for the coculture infectivity test. The same animals were used in a test for the locus associated with PERV class C. 0041-1345/08/$–see front matter doi:10.1016/j.transproceed.2008.01.051 590
Infectivity of PERV In Vitro Cell Cultures. Coculture infectivity tests used the following cell cultures: ● ● ●
●
Human embryonic kidney epithelial cells (HEK293) [ATCC CRL-1573]; Porcine embryo kidney epithelial cells (PK15) (obtained from MAF, New Zealand); Porcine primary cells: peripheral blood mononuclear cells (PBMCs), choroid plexus, and islets cell clusters (ICC) from the DPF herd; and Foetal swine testis (St-Iowa) ATCC CRL-1746 (kindly provided by Dr Linda Scobie, University of Glasgow).
Three flasks were used for each condition. Cells were maintained in standard media: ●
Dulbecco modified medium (DMEM), supplemented with 10% fetal bovine serum (FBS) for HEK293;
From Living Cell Technologies Ltd, Otara, Manukau, Auckland, New Zealand; the Institute of Comparative Medicine, University of Glasgow, Bearsden, Glasgow, United Kingdom; and the Virology Immunology Department, Auckland Hospital, Auckland, New Zealand. Supported by the European Commission funded project LSHB-CT-2006-037377 (LS/RDH). Address reprint requests to O. Garkavenko, Living Cell Technologies Ltd, 19 Laureston Avenue, Otara, Manukau 2025, Auckland, New Zealand. E-mail: [email protected] © 2008 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 40, 590 –593 (2008)
PERV TRANSMISSION CHARACTERISTICS ● ● ●
Minimal Eagle’s medium (MEM), supplemented with 3% FBS for PK15; RPMI 1640 Medium, supplemented with 10% FBS for PBMCs, ICC, and choroid plexus cells; and MEM supplemented with 10% FBS for St-Iowa.
Cell Isolation Pig PBMCs were isolated using HISTOPAQUE-1077 (SigmaAldrich, St Louis, MO). ICC was extracted as described by Elliott et al4 and choroid plexus cells as described by Borlongan et al.5
Mitogenic Stimulation of Cells Cells (106) were mitogenically stimulated with phytohemagglutin (PHA, 2 g/mL) and phorbol 12-myristate-13-acetate (PMA) 10 ng/mL in RPMI supplemented with FBS (10%), 1.5 mL of ciprofloxacin (Ciproxin IV 200, Bayer AG) and 2 mL of Fungizone (Gibco, Grand Isle, NY) per 500 mL. PBMCs were maintained at 37°C in 5% CO2 for at least 3 days.
Cell Counts Cells were counted using a hemacytometer. Cell viability was determined by Trypan blue staining.
Reverse Transcriptase PERV Assay Reverse transcriptase (RT) activity to determine PERV production in the cell culture supernatant and blood plasma was determined using a commercial assay (Cavidi Tech, Uppsala, Sweden), according to the manufacturers protocol.
Infectivity Assay After 6 days, 105 stimulated porcine primary cells (PBMCs, ICC, or choroid plexus) or PK15 cells were lethally irradiated (2000 rads from 137Cs source, GammaCell), mixed with 105 HEK 293 target cells, and plated with appropriate medium in 25-cm2 flasks. Alternatively 1 mL of supernatant from stimulated cells was added to St Iowa target cells. Cocultures were maintained for up to 20 weeks. Cell aliquots were taken weekly.
Nucleic Acid Isolation Aliquots of cell suspension were obtained at routine passage of cells and stored at ⫺80°C. Subsequently, samples were thawed and pelleted by centrifugation at 14,000 rpm for 1 minute, the supernatant removed and the cellular pellet divided for DNA and RNA isolation. All DNA purifications were carried out using the GENERATION Capture Column Kit (Gentra Systems, Minneapolis, Minn), following the manufacturer’s instructions. Total RNA was isolated from cocultured pig PBMC, PK15, and HEK293 cells using TRI Reagent LS (Molecular Research Center, Inc, Cincinnati, Ohio), following the manufacturer’s instructions. DNA and RNA were quantified by UV spectrophotometry using an Eppendorf BioPhotometer (Eppendorf, North Ryde, Australia).
591
Polymerase Chain Reaction Amplification Specific primers have been used for COII and PERV pol region4,6 and env region7 amplification. To check the integrity of isolated nucleic acids from HEK293 cocultures, the following primers for human endogenous retrovirus HERV-K (Y10392) were used: forward 5=-ATGGGGCCTCTCCAACCCGG-3=; reverse 5=-CGTTTCT GCAGCACATAAAATATCA-3= for the first round and 5=GCCCTCTCCGGCCATGATCC-3= and 5=-TCAATATAATGAA TAATATAACAGT CTG-3= for the second round. First-round polymerase chain reaction (PCR) was performed for each primer pair in 25-L reactions containing 2.5 L of 10⫻ HotMaster Taq Buffer with 25 mmol/L Mg⫹⫹ (Eppendorf), 0.2 mmol/L dNTPs, 0.1 mol/L of each primer pair, 1 U Eppendorf Hotmaster Taq, and 1 L cDNA or approximately 100 ng genomic DNA. A Bio-Rad iCycler (Bio-Rad, Hercules, Calif) was used for all PCRs with thermocycling conditions as follows: 1 cycle of 94°C for 2 minutes; 45 cycles of 94°C for 20 seconds, 55°C for 10 seconds, 72°C for 30 seconds, and 10 minutes at 72°C. For PERV-C, the PCR conditions were 92°C for 2 minutes, 30 cycles of 94°C for 30 seconds, 58°C for 45 seconds, 72°C for 30 seconds, and 1 cycle at 72°C for 5 minutes. Second-round (nested) PCR was conducted under the same conditions as described with specific primers, with 2 L of PCR product from the first round amplification. Products were visualised by electrophoresis in 1.5% ethidium bromide-stained agarose gel (Probiogen, Murrarrie, Queensland, Australia).
Assay Sensitivity Sensitivity of nested PCR was estimated for the pol region of PERV using genomic DNA extracted from the PK15 cell line. The sensitivity of the PERV PCR was determined by limiting dilution. The sensitivity was 10⫺7 g DNA per reaction for pol PERV and 10⫺7 g DNA per reaction for COII.
Sequence Analysis Amplified products were purified using the High Pure PCR Product Purification Kit (Boehringer Mannheim GMBH, Germany) following the manufacturer’s protocol. Sequencing was carried out on an ABI 373A Sequencer (Centre for Gene Technology, Auckland University).
PERV-C and Recombinant PERV Transmission Analysis of PERV-C in miniature swine of known transmission status revealed that some of these “null” animals lacked a specific full-length PERV-C locus that had been identified as present in animals capable of infecting human and/or pig cells.8 To determine if the DPF donor pigs that were PERV-C positive retained this locus, DNA from those animals was tested using the primer sequences and cycling conditions for the PERV-C 2213 locus as described.8 To confirm that the DNA was suitable for amplification, PCR for the porcine -globin gene was carried out.9
Reverse Transcription
RESULTS Cell Counts
Total RNA was treated with DNase I, Amplification Grade (Invitrogen) before RT according to the manufacturer’s instructions. Total RNA (1 g) was reverse transcribed by M-MLV RT (Invitrogen) with 2 g of Random Primers (Invitrogen) at 42°C for 1 hour.
After mitogenic stimulation, all primary cells and cell cultures showed an increase in cell count of at least 2 to 10 times compared with the nonstimulated control.
592
Reverse Transcriptase Activity
All cell supernatant samples from the cocultures were assessed for PERV C-type RT activity. PBMC, choroid plexus cells, and pig islet cells were stimulated with PHA/ PMA. No C-type retroviral-RT activity was shown at week 0 before coculture, nor was C-type retroviral-RT activity found in the supernatant from these cells after 16 weeks of coculture in either pig or human cells. The increase of PK15 retroviral-RT activity was detected after stimulation (6.8 mU/mL before stimulation and 9.8 mU/mL after stimulation) and throughout the 16 weeks. Amplification of PERV pol-Specific Sequences, Cytochrome Oxidase II for Evidence of Microchimerism, and HERV-K Assay for Integrity of Isolated Nucleic Acids
None of the PBMC/HEK293, choroid plexus/HEK293, or ICC/HEK293 cocultures had detectable PERV DNA or mRNA at 10 weeks after establishment. Cytochrome oxidase II (CoII) DNA persisted in several cocultures of PBMC/HEK293 for a period of up to 14 weeks. PERV infection and expression, in the absence of microchimerism, was shown in the HEK293 cells cocultured with the porcine cell line PK15. All cocultures negative for PERV and COII DNA and RNA were positive for HERV DNA and mRNA, indicating template integrity.
GARKAVENKO, WYNYARD, NATHU ET AL
RT activity detected in St Iowa cell line after coculturing with stimulated porcine primary cells. Recently, it was determined that screening pigs for PERV sequences is no longer sufficient. It is now necessary to identify the ability of PERV-A and PERV-C classes to recombine because recombinants show higher titers when cultured with human cells in vitro.14 –17 Using the coculture infectivity test with human HEK293 or porcine St Iowa target cell lines, we have established that the primary cells of the Auckland Islands donor pigs do not release either eco- or xenotropic infective viruses. We have also shown that although some of the donors have PERV-C, these pigs lack a putative marker that may be associated with the transmission of PERV identified previously in miniature swine.8 This finding is entirely consistent with our infectivity data, indicating that the Auckland Islands pigs have an extremely low potential risk of transmitting PERV infection and can therefore be qualified as “null” pigs.15 In conclusion, after extensive microbiological screening, we have selected a DPF herd as donors for cell xenotransplantation. This herd is free of all tested conventional and xenotransplantation related pathogens. Negative transmission of PERV in the infectivity test in vitro with both human and pig cell lines, as well as absence of a locus associated with transmission in miniature swine, allow us to classify the DPF pigs as nontransmitters of both xeno- and ecotropic PERV or “null” pigs.
PERV-C and Transmission of Recombinant PERV
DNA from the animals was tested for both the presence of PERV-C and for the previously characterized PERV-C locus 2213.9 All five samples from the DPF herd were positive for PERV-C proviral sequences as determined by amplification of the env gene. None were positive for the PERV-C locus 2213. DISCUSSION
A standard infectivity method6,10 has been applied to check the transmission of PERV from primary cells—PBMC, ICC, and choroid plexus— derived from DPF pigs. To ensure the release of infectious human tropic PERV particles (if any), cells were PHA/PMA stimulated11–13 before coculture with the standard cell lines HEK 293 and St Iowa. Positive stimulation was confirmed with a cell count as well as by a standard RT assay (for PK15 control cell line). After stimulation, the cell number increased in all of the stimulated cell cultures. However, there was no evidence of RT activity in supernatant of primary cells before or after stimulation. PK15 control cells showed increased RT activity after stimulation. Although PHA/PMA stimulation enhanced proliferation of all tested cells, no evidence of PERV transmission was detected in the infectivity tests: There was no detectable PERV DNA or RNA in cocultures of primary porcine cells with the target cell line HEK 293. Thus, it was concluded that the tested DPF pigs can be classified as nontransmitters for human tropic recombinant PERV. There was also no
REFERENCES 1. Garkavenko O, Muzina M, Muzina Z, et al: Monitoring for potentially xenozoonotic viruses in New Zealand pigs. J Med Virol 72:338, 2004 2. Garkavenko O, Obriadina A, Meng J, et al: Detection and characterisation of swine hepatitis E virus in New Zealand. J Med Virol 65:525, 2001 3. Garkavenko O, Elliott RB, Croxson MC: Identification of pig circovirus type 2 in New Zealand pigs. Transplant Proc 37:506, 2005 4. Elliott RB, Escobar L, Garkavenko O, et al: No evidence of infection with porcine endogenous retrovirus in recipients of encapsulated porcine islet xenografts. Cell Transplant 9:895, 2000 5. Borlongan CV, Skinner SJM, Geany M, et al: CNF grafts of rat choroid plexus against cerebral ischemia in adult rats. Neuroreport 15:1543, 2004 6. Patience C, Takeuchi Y, Weiss RA: Infection of human cells by an endogenous retrovirus of pigs. Nat Med 3:282, 1997 7. Takeuchi Y, Patience C, Magre S, et al: Host range and interference studies of three classes of pig endogenous retrovirus. J Virol 72:9986, 1998 8. Hector RD, Meikle S, Grant L, et al: Pre-screening of miniature swine may reduce the risk of transmitting human tropic recombinant porcine endogenous retroviruses. Xenotransplantation 14:222, 2007 9. Herring C, Quinn G, Bower R, et al: Mapping full-length porcine endogenous retroviruses in a large white pig. J Virol 75:12252, 2001 10. Wilson CA, Wong S, Muller J, et al: Type C retrovirus released from porcine primary peripheral blood mononuclear cells infects human cells. J Virol 72:3082, 1998 11. Patience C, Switzer WM, Takeuchi Y, et al: Multiple groups of novel retroviral genomes in pigs and related species. J Virol 75:2771, 2001
PERV TRANSMISSION CHARACTERISTICS 12. Cunningham DA, Dos Santos Cruz GJ, Fernandez-Suarez XM, et al: Activation of primary porcine endothelial cells induces release of porcine endogenous retroviruses. Transplantation 77:1071, 2004 13. Tacke SJ, Specke V, Denner J: Differences in release and determination of subtype of porcine endogenous retroviruses produced by stimulated normal pig blood cells. Intervirology 46:17, 2003 14. Bartosch B, Stefanidis D, Myers R, et al: Evidence and consequence of porcine endogenous retrovirus recombination. J Virol 78:13880, 2004
593 15. Wood JC, Quinn G, Suling KM, et al: Identification of exogenous forms of human-tropic porcine endogenous retrovirus in miniature swine. J Virol 78:2494, 2004 16. Harrison I, Takeuchi Y, Bartosch B, et al: Determinants of high titer in recombinant porcine endogenous retroviruses. J Virol 78:13871, 2004 17. Scobie L, Taylor S, Wood JC, et al: Absence of replicationcompetent human-tropic porcine endogenous retroviruses in the germ line DNA of inbred miniature Swine. J Virol 78:2502, 2004
I
T IS ARGUED that some of the infectious risk associated with xenotransplantation can be assessed before the broad application of the emerging technology. A specific program for choosing and maintaining a source herd should be developed. Such a program should be designed for a specific geographical area (PHS Guidelines on Infectious Disease Issues in Xenotransplantation). A strategy to select and maintain the source pig herd intended for islet cell xenotransplantation has been developed for New Zealand representative herds.1 Following this strategy, several representative herds have been screened. We now have a much better understanding of the pig virus epidemiology in New Zealand. We have described the prevalence of pig hepatitis E virus,2 pig circovirus type 2,3 pig cytomegalovirus, encephalomyocarditis virus, and pig lymphotropic herpesvirus,1 and a designated pathogen-free (DPF) herd has been identified.1 However, one potential pathogen, the porcine endogenous retrovirus (PERV), cannot be eliminated with stringent microbiological control. This study is focused on the infectious characteristics of the PERV from the DPF herd.
MATERIALS AND METHODS Animals Five 7-day-old pig donors of LCT DPF herd were used for the coculture infectivity test. The same animals were used in a test for the locus associated with PERV class C. 0041-1345/08/$–see front matter doi:10.1016/j.transproceed.2008.01.051 590
Infectivity of PERV In Vitro Cell Cultures. Coculture infectivity tests used the following cell cultures: ● ● ●
●
Human embryonic kidney epithelial cells (HEK293) [ATCC CRL-1573]; Porcine embryo kidney epithelial cells (PK15) (obtained from MAF, New Zealand); Porcine primary cells: peripheral blood mononuclear cells (PBMCs), choroid plexus, and islets cell clusters (ICC) from the DPF herd; and Foetal swine testis (St-Iowa) ATCC CRL-1746 (kindly provided by Dr Linda Scobie, University of Glasgow).
Three flasks were used for each condition. Cells were maintained in standard media: ●
Dulbecco modified medium (DMEM), supplemented with 10% fetal bovine serum (FBS) for HEK293;
From Living Cell Technologies Ltd, Otara, Manukau, Auckland, New Zealand; the Institute of Comparative Medicine, University of Glasgow, Bearsden, Glasgow, United Kingdom; and the Virology Immunology Department, Auckland Hospital, Auckland, New Zealand. Supported by the European Commission funded project LSHB-CT-2006-037377 (LS/RDH). Address reprint requests to O. Garkavenko, Living Cell Technologies Ltd, 19 Laureston Avenue, Otara, Manukau 2025, Auckland, New Zealand. E-mail: [email protected] © 2008 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 40, 590 –593 (2008)
PERV TRANSMISSION CHARACTERISTICS ● ● ●
Minimal Eagle’s medium (MEM), supplemented with 3% FBS for PK15; RPMI 1640 Medium, supplemented with 10% FBS for PBMCs, ICC, and choroid plexus cells; and MEM supplemented with 10% FBS for St-Iowa.
Cell Isolation Pig PBMCs were isolated using HISTOPAQUE-1077 (SigmaAldrich, St Louis, MO). ICC was extracted as described by Elliott et al4 and choroid plexus cells as described by Borlongan et al.5
Mitogenic Stimulation of Cells Cells (106) were mitogenically stimulated with phytohemagglutin (PHA, 2 g/mL) and phorbol 12-myristate-13-acetate (PMA) 10 ng/mL in RPMI supplemented with FBS (10%), 1.5 mL of ciprofloxacin (Ciproxin IV 200, Bayer AG) and 2 mL of Fungizone (Gibco, Grand Isle, NY) per 500 mL. PBMCs were maintained at 37°C in 5% CO2 for at least 3 days.
Cell Counts Cells were counted using a hemacytometer. Cell viability was determined by Trypan blue staining.
Reverse Transcriptase PERV Assay Reverse transcriptase (RT) activity to determine PERV production in the cell culture supernatant and blood plasma was determined using a commercial assay (Cavidi Tech, Uppsala, Sweden), according to the manufacturers protocol.
Infectivity Assay After 6 days, 105 stimulated porcine primary cells (PBMCs, ICC, or choroid plexus) or PK15 cells were lethally irradiated (2000 rads from 137Cs source, GammaCell), mixed with 105 HEK 293 target cells, and plated with appropriate medium in 25-cm2 flasks. Alternatively 1 mL of supernatant from stimulated cells was added to St Iowa target cells. Cocultures were maintained for up to 20 weeks. Cell aliquots were taken weekly.
Nucleic Acid Isolation Aliquots of cell suspension were obtained at routine passage of cells and stored at ⫺80°C. Subsequently, samples were thawed and pelleted by centrifugation at 14,000 rpm for 1 minute, the supernatant removed and the cellular pellet divided for DNA and RNA isolation. All DNA purifications were carried out using the GENERATION Capture Column Kit (Gentra Systems, Minneapolis, Minn), following the manufacturer’s instructions. Total RNA was isolated from cocultured pig PBMC, PK15, and HEK293 cells using TRI Reagent LS (Molecular Research Center, Inc, Cincinnati, Ohio), following the manufacturer’s instructions. DNA and RNA were quantified by UV spectrophotometry using an Eppendorf BioPhotometer (Eppendorf, North Ryde, Australia).
591
Polymerase Chain Reaction Amplification Specific primers have been used for COII and PERV pol region4,6 and env region7 amplification. To check the integrity of isolated nucleic acids from HEK293 cocultures, the following primers for human endogenous retrovirus HERV-K (Y10392) were used: forward 5=-ATGGGGCCTCTCCAACCCGG-3=; reverse 5=-CGTTTCT GCAGCACATAAAATATCA-3= for the first round and 5=GCCCTCTCCGGCCATGATCC-3= and 5=-TCAATATAATGAA TAATATAACAGT CTG-3= for the second round. First-round polymerase chain reaction (PCR) was performed for each primer pair in 25-L reactions containing 2.5 L of 10⫻ HotMaster Taq Buffer with 25 mmol/L Mg⫹⫹ (Eppendorf), 0.2 mmol/L dNTPs, 0.1 mol/L of each primer pair, 1 U Eppendorf Hotmaster Taq, and 1 L cDNA or approximately 100 ng genomic DNA. A Bio-Rad iCycler (Bio-Rad, Hercules, Calif) was used for all PCRs with thermocycling conditions as follows: 1 cycle of 94°C for 2 minutes; 45 cycles of 94°C for 20 seconds, 55°C for 10 seconds, 72°C for 30 seconds, and 10 minutes at 72°C. For PERV-C, the PCR conditions were 92°C for 2 minutes, 30 cycles of 94°C for 30 seconds, 58°C for 45 seconds, 72°C for 30 seconds, and 1 cycle at 72°C for 5 minutes. Second-round (nested) PCR was conducted under the same conditions as described with specific primers, with 2 L of PCR product from the first round amplification. Products were visualised by electrophoresis in 1.5% ethidium bromide-stained agarose gel (Probiogen, Murrarrie, Queensland, Australia).
Assay Sensitivity Sensitivity of nested PCR was estimated for the pol region of PERV using genomic DNA extracted from the PK15 cell line. The sensitivity of the PERV PCR was determined by limiting dilution. The sensitivity was 10⫺7 g DNA per reaction for pol PERV and 10⫺7 g DNA per reaction for COII.
Sequence Analysis Amplified products were purified using the High Pure PCR Product Purification Kit (Boehringer Mannheim GMBH, Germany) following the manufacturer’s protocol. Sequencing was carried out on an ABI 373A Sequencer (Centre for Gene Technology, Auckland University).
PERV-C and Recombinant PERV Transmission Analysis of PERV-C in miniature swine of known transmission status revealed that some of these “null” animals lacked a specific full-length PERV-C locus that had been identified as present in animals capable of infecting human and/or pig cells.8 To determine if the DPF donor pigs that were PERV-C positive retained this locus, DNA from those animals was tested using the primer sequences and cycling conditions for the PERV-C 2213 locus as described.8 To confirm that the DNA was suitable for amplification, PCR for the porcine -globin gene was carried out.9
Reverse Transcription
RESULTS Cell Counts
Total RNA was treated with DNase I, Amplification Grade (Invitrogen) before RT according to the manufacturer’s instructions. Total RNA (1 g) was reverse transcribed by M-MLV RT (Invitrogen) with 2 g of Random Primers (Invitrogen) at 42°C for 1 hour.
After mitogenic stimulation, all primary cells and cell cultures showed an increase in cell count of at least 2 to 10 times compared with the nonstimulated control.
592
Reverse Transcriptase Activity
All cell supernatant samples from the cocultures were assessed for PERV C-type RT activity. PBMC, choroid plexus cells, and pig islet cells were stimulated with PHA/ PMA. No C-type retroviral-RT activity was shown at week 0 before coculture, nor was C-type retroviral-RT activity found in the supernatant from these cells after 16 weeks of coculture in either pig or human cells. The increase of PK15 retroviral-RT activity was detected after stimulation (6.8 mU/mL before stimulation and 9.8 mU/mL after stimulation) and throughout the 16 weeks. Amplification of PERV pol-Specific Sequences, Cytochrome Oxidase II for Evidence of Microchimerism, and HERV-K Assay for Integrity of Isolated Nucleic Acids
None of the PBMC/HEK293, choroid plexus/HEK293, or ICC/HEK293 cocultures had detectable PERV DNA or mRNA at 10 weeks after establishment. Cytochrome oxidase II (CoII) DNA persisted in several cocultures of PBMC/HEK293 for a period of up to 14 weeks. PERV infection and expression, in the absence of microchimerism, was shown in the HEK293 cells cocultured with the porcine cell line PK15. All cocultures negative for PERV and COII DNA and RNA were positive for HERV DNA and mRNA, indicating template integrity.
GARKAVENKO, WYNYARD, NATHU ET AL
RT activity detected in St Iowa cell line after coculturing with stimulated porcine primary cells. Recently, it was determined that screening pigs for PERV sequences is no longer sufficient. It is now necessary to identify the ability of PERV-A and PERV-C classes to recombine because recombinants show higher titers when cultured with human cells in vitro.14 –17 Using the coculture infectivity test with human HEK293 or porcine St Iowa target cell lines, we have established that the primary cells of the Auckland Islands donor pigs do not release either eco- or xenotropic infective viruses. We have also shown that although some of the donors have PERV-C, these pigs lack a putative marker that may be associated with the transmission of PERV identified previously in miniature swine.8 This finding is entirely consistent with our infectivity data, indicating that the Auckland Islands pigs have an extremely low potential risk of transmitting PERV infection and can therefore be qualified as “null” pigs.15 In conclusion, after extensive microbiological screening, we have selected a DPF herd as donors for cell xenotransplantation. This herd is free of all tested conventional and xenotransplantation related pathogens. Negative transmission of PERV in the infectivity test in vitro with both human and pig cell lines, as well as absence of a locus associated with transmission in miniature swine, allow us to classify the DPF pigs as nontransmitters of both xeno- and ecotropic PERV or “null” pigs.
PERV-C and Transmission of Recombinant PERV
DNA from the animals was tested for both the presence of PERV-C and for the previously characterized PERV-C locus 2213.9 All five samples from the DPF herd were positive for PERV-C proviral sequences as determined by amplification of the env gene. None were positive for the PERV-C locus 2213. DISCUSSION
A standard infectivity method6,10 has been applied to check the transmission of PERV from primary cells—PBMC, ICC, and choroid plexus— derived from DPF pigs. To ensure the release of infectious human tropic PERV particles (if any), cells were PHA/PMA stimulated11–13 before coculture with the standard cell lines HEK 293 and St Iowa. Positive stimulation was confirmed with a cell count as well as by a standard RT assay (for PK15 control cell line). After stimulation, the cell number increased in all of the stimulated cell cultures. However, there was no evidence of RT activity in supernatant of primary cells before or after stimulation. PK15 control cells showed increased RT activity after stimulation. Although PHA/PMA stimulation enhanced proliferation of all tested cells, no evidence of PERV transmission was detected in the infectivity tests: There was no detectable PERV DNA or RNA in cocultures of primary porcine cells with the target cell line HEK 293. Thus, it was concluded that the tested DPF pigs can be classified as nontransmitters for human tropic recombinant PERV. There was also no
REFERENCES 1. Garkavenko O, Muzina M, Muzina Z, et al: Monitoring for potentially xenozoonotic viruses in New Zealand pigs. J Med Virol 72:338, 2004 2. Garkavenko O, Obriadina A, Meng J, et al: Detection and characterisation of swine hepatitis E virus in New Zealand. J Med Virol 65:525, 2001 3. Garkavenko O, Elliott RB, Croxson MC: Identification of pig circovirus type 2 in New Zealand pigs. Transplant Proc 37:506, 2005 4. Elliott RB, Escobar L, Garkavenko O, et al: No evidence of infection with porcine endogenous retrovirus in recipients of encapsulated porcine islet xenografts. Cell Transplant 9:895, 2000 5. Borlongan CV, Skinner SJM, Geany M, et al: CNF grafts of rat choroid plexus against cerebral ischemia in adult rats. Neuroreport 15:1543, 2004 6. Patience C, Takeuchi Y, Weiss RA: Infection of human cells by an endogenous retrovirus of pigs. Nat Med 3:282, 1997 7. Takeuchi Y, Patience C, Magre S, et al: Host range and interference studies of three classes of pig endogenous retrovirus. J Virol 72:9986, 1998 8. Hector RD, Meikle S, Grant L, et al: Pre-screening of miniature swine may reduce the risk of transmitting human tropic recombinant porcine endogenous retroviruses. Xenotransplantation 14:222, 2007 9. Herring C, Quinn G, Bower R, et al: Mapping full-length porcine endogenous retroviruses in a large white pig. J Virol 75:12252, 2001 10. Wilson CA, Wong S, Muller J, et al: Type C retrovirus released from porcine primary peripheral blood mononuclear cells infects human cells. J Virol 72:3082, 1998 11. Patience C, Switzer WM, Takeuchi Y, et al: Multiple groups of novel retroviral genomes in pigs and related species. J Virol 75:2771, 2001
PERV TRANSMISSION CHARACTERISTICS 12. Cunningham DA, Dos Santos Cruz GJ, Fernandez-Suarez XM, et al: Activation of primary porcine endothelial cells induces release of porcine endogenous retroviruses. Transplantation 77:1071, 2004 13. Tacke SJ, Specke V, Denner J: Differences in release and determination of subtype of porcine endogenous retroviruses produced by stimulated normal pig blood cells. Intervirology 46:17, 2003 14. Bartosch B, Stefanidis D, Myers R, et al: Evidence and consequence of porcine endogenous retrovirus recombination. J Virol 78:13880, 2004
593 15. Wood JC, Quinn G, Suling KM, et al: Identification of exogenous forms of human-tropic porcine endogenous retrovirus in miniature swine. J Virol 78:2494, 2004 16. Harrison I, Takeuchi Y, Bartosch B, et al: Determinants of high titer in recombinant porcine endogenous retroviruses. J Virol 78:13871, 2004 17. Scobie L, Taylor S, Wood JC, et al: Absence of replicationcompetent human-tropic porcine endogenous retroviruses in the germ line DNA of inbred miniature Swine. J Virol 78:2502, 2004