- Email: [email protected]
Genetic Prevalence of Porcine Endogenous Retrovirus in Chinese Experimental Miniature Pigs
Genetic Prevalence of Porcine Endogenous Retrovirus in Chinese Experimental Miniature Pigs G. Liu, Z. Li, M. Pan, M. Ge, Y. Wang, and Y. Gao ABSTRACT Pig-to-human xenotransplantation poses the potential risk of interspecies transmission of porcine endogenous retrovirus (PERV). The Chinese experimental miniature pig may be used as a pig-to-human xenograft donor. However, data for the distribution of PERV provirus in genomic DNA and PERV expression at the RNA level for the Chinese experimental miniature pig population are lacking. In this study, PERV was investigated in this regard using polymerase chain reaction (PCR), real-time quantitative PCR, and real-time quantitative reverse transcription PCR. The results showed that the genotype distribution was PERV-A subtype 100%, PERV-B subtype 100%, and PERV-C subtype 30% among 20 pig genomic DNA samples. Both PERV copy number in genomic DNA and PERV expression at the RNA level varied significantly among individuals, ranging from 3.95 ⫾ 0.14 to 95.52 ⫾ 2.20 and 3.66 ⫾ 0.13 to 43.03 ⫾ 2.50, respectively. For some individuals, the PERV copy number (eg, 3.95 ⫾ 0.14) in genomic DNA and PERV expression (eg, 3.66 ⫾ 0.13) at the RNA level were low. These results suggested that the Chinese experimental miniature pig is a possible donor for xenotransplantation. Our results provide reference information for selective breeding, which will benefit the application of these animals for the study of xenotransplantation. IG-to-human xenotransplantation is one possible solution to alleviate the shortage of human allograft donors.1 However, the problem of human recipient infection with zoonosis caused by porcine pathogens remains in xenotransplantation. Porcine endogenous retrovirus (PERV) is one major concern.2,3 PERV belongs to the ␥-retrovirus family, with high nucleic and amino acid homology to monkey leukemia virus, murine leukemia virus, and other ␥-retroviruses.4 Proviral DNA of PERV integrated into the pig genome can not be eliminated under specific pathogenfree conditions.5 Of the 3 open reading frames (gag, pol, and env genes), the gag and pol genes are highly conserved, whereas the env gene is more variable.6 Based on differences in the receptor binding domain of env, PERV are divided into 3 subtypes: PERV-A, PERV-B, and PERV-C.7 In vitro the PERV-A and PERV-B subtypes infect a variety of human cells, and PERV-C subtypes infect only 2 kinds of pig cells.8 In the process of in vitro culture, a recombinant PERV-A/C has been demonstrated to be derived from PERV-A and PERV-C. Its infectivity for human cells is approximately 500 times stronger than that of PERV-A.9 With the advantages of appropriate organ size, strong disease resistance, clear genetic background, stable physio-
P
logical and biochemical indices, and economic affordability,10 the Chinese experimental miniature pig has become a promising donor source for pig-to-human xenotransplantation. According to a recent clinical report, PERV reverse transcriptase activity was not observed in sera from 3 patients treated with a porcine liver cell-based bioartificial liver from a Chinese experimental miniature pig.11 The PERV copy number carried in pig breeds serves as an important reference value for potential viral infectivity and the species’ suitability as a future donor. It is known that From the Institute of Regenerative Medicine (G.L., Z.L., Y.W., Y.G.), Zhujiang Hospital, Southern Medical University, and Department of Hepatobiliary Surgery (G.L., Z.L., M.P., M.G., Y.W., Y.G.), Zhujiang Hospital, Southern Medical University, Guangzhou, China. This work was supported by National High Technology Research and Development Program Project of China (2006AA02A141). Address reprint requests to Yan Wang, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou 510280, China. E-mail: [email protected]; and Yi Gao, Department of Hepatobiliary Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou 510280, China. E-mail: [email protected]
0041-1345/11/$–see front matter doi:10.1016/j.transproceed.2011.06.061
© 2011 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
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Table 1. PCR Primer Sequences for PERV and TFRC Gene Amplification Gene
Sequence of Primer
env-A6
F:5=-TGGAAAGATTGGCAACAGCG-3= R: 5=-AGTGATGTTAGGCTCAGTGG-3= F: 5=-TTCTCCTTTGTCAATTCCGG-3= R:5=-TACTTTATCGGGTCCCACTG-3= F:5=-CTGACCTGGATTAGAACTGG-3= R:5=-ATGTTAGAGGATGGTCCTGG-3= F: 5=-AGCTCCGGGAGGCCTACTC-3= R:5=-ACAGCCGTTGGTGTGGTCA-3= F:5=-GAGACAGAAACTTTCGAAGC-3= R:5=-GAAGTCTGTGGTATCCAATCC-3=
env-B6 env-C8 Pol6 15
TFRC
Fragment Size (bp)
359 263 281 150 80
Abbreviations: TFRC, transferrin receptor; bp, base pair.
there are multiple PERV copies in most pig breeds; the copy number differs among breeds and individuals of the same breed.12,13 After testing 67 Chinese experimental miniature pigs, Lu et al found that 2 individuals were negative for PERV.14 However, data for the distribution of
PERV provirus in genomic DNA and PERV expression at the RNA level among the population of Chinese experimental miniature swine are lacking. In this study, we identified PERV genotypes in the genomes of Chinese experimental miniature swine detecting PERV copy number in genomic DNA and PERV expression at the RNA level. These data may lay the foundation for selective breeding to benefit the application of these donors to the study of xenotransplantation. MATERIALS AND METHODS Sample Collection Twenty Chinese experimental miniature pigs were randomly selected from Changping experimental farm at China Agricultural University (Beijing, China). Tissue samples (1 cm3) obtained from the ears were stored in 70% ethanol.
Extraction of Genomic DNA and Total RNA Genomic DNA was extracted from samples using the genomic DNA Extraction kit protocol (QIAGEN, Hamburg, Germany). Sample total RNA was extracted using an RNA Extraction kit
Fig 1. PCR analysis of the detection specificity for PERV DNA. DNA sample from PK-15 cell line was used as the positive control: lane’s 2, 4, and 6 were env-C, env-B, and env-A, respectively. Sample no. 5 was randomly selected as the testing sample: lane’s 1, 3, and 5 were env-C, env-B, and env-A, respectively. Distilled water was used as the negative crontrol in lane N. Lane M was the DNA marker.
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(Promega, Wisconsin, United States). The OD260nm value was determined for each DNA and RNA sample using a spectrophotometer (Eppendorf, Hamburg, Germany). The average of 3 measurements performed for each sample allowed samples to be adjusted to 200 ng/L.
Polymerase Chain Reaction Detection of PERV Genotypes Using the reported sequences for PERV,6,8 3 primer pairs were designed and synthesized to individually detect PERV genotypes (PERV-A, PERV-B, and PERV-C; (Sangon Biotech Co., Ltd., Shanghai, China; Table 1). To control for false-positive polymerase chain reaction (PCR) reactions we selected PK-15 cell genomic DNA as a positive control; a negative control (blank) confirmed the absence of nonspecific amplification. Using the primers described above, we amplified the corresponding genes in a PCR assay with a total volume of 25 L (Bio-Rad, United States), including 1 ⫻ PCR buffer (TaKaRa, Dalian, China), 200 mol/L dNTPs (TaKaRa), 2 U Taq DNA polymerase (TaKaRa), both primers, and 15 pmol of each DNA template, and distilled water added to bring the final total volume to 25 L. PCR reaction conditions for env-A, env-B, and env-C were as follows: 95°C for 5 minutes to initially denature, 95°C for 30 seconds 55°C for 45 seconds, 72°C for 1 minute repeated for a total of 30 cycles, with final extension at 72°C for 7 minutes followed by 2% agarose gel electrophoresis.
Real-Time Quantitative PCR Assay Because the pol segment is highly conserved among PERV gene sequences, it was selected as a target gene to design specific primers (Sangon Biotech Co., Ltd.).6 Meanwhile, the house-keeping gene porcine transferrin receptor (TFRC) was selected as an internal reference gene and primers were also designed to amplify its sequence (Sangon Biotech Co., Ltd.; Table 1).15 After purification and gel recovery, the pol and TFRC gene PCR amplification products were independently ligated into the PMD-18T
vector (TaKaRa) for transformation in DH5a Escherichia coli (Tiangen Biotech Co., Ltd., Beijing, China). Using blue-white screening, we selected positive clones. EcoRI/Hind III enzyme (New England Biolabs, Mass, USA) digestions were performed to identify and extract plasmids containing pol and TFRC, respectively. After plasmid concentrations were determined with an ultra violet (UV) spectrophotometer (Eppendorf, Hamburg, Germany), the 2 standard plasmids were diluted with a 10⫻ gradient. Plasmids of 5 continuous gradient concentrations were selected to detect the copy number and cycle threshold values for the TFRC and pol genes. An absolute quantitative standard curve was constructed accordingly. SYBR Green, a commonly used fluorescent DNA binding dye, based real-time qPCR assays were performed with a 7500 Real Time PCR System (Applied Biosystems, Calif, USA) using the SYBR Premix Ex Taq Kit (TaKaRa) in a total volume of 20 L, including 10 L of 2 ⫻ SYBR Premix Ex Ta (TaKaRa), 0.8 L each of forward and reverse primers, 0.4 L of ROX Reference Dye (Haoran Biological Technology Co., Ltd., Shanghai, China), and 50 ng plasmid DNA or sample DNA, and finally nuclease-free water was added for a total volume of 20.0 L. The qPCR reaction conditions were 95°C for 5 minutes to initially denature, 95°C for 30 seconds, 55°C for 45 seconds, 72°C for 1 minute for a total of 30 cycles, with final extension at 72°C for 7 minutes. The temperature was then slowly increased to 95°C at a rate of 0.1°C/s. The first complementary DNA (cDNA) strand was synthesized according to the previously described method.16 Real-time quantitative reverse transcription PCR assays were performed using primers for the pol and TFRC genes as described above. For each reaction we used 50 ng total RNA. The qPCR reactions were performed with the temperature conditions as described above. Melting curves were constructed for the amplification products from the plasmid DNA, sample DNA, and RNA for verification. Reaction data were analyzed using Sequence Detection System software (Applied Biosystems).
Table 2. Distribution of PERV Subtypes in 20 Porcine Genomic DNA Samples PERV Subtype Sample No.
Gender
Age (mo)
BW (kg)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Male Male Female Male Female Female Male Male Male Female Female Male Male Male Male Female Female Female Male Female
21 8 6 12 10 12 7 7 15 22 13 13 8 18 10 6 9 17 12 11
47.5 30 26 38.5 34 39.5 28 28.5 41.5 48 39 28.5 29 45 34.5 26 32.5 42.5 39 35.5
Original Place
Guizhou, Guizhou, Guizhou, Guizhou, Guizhou, Guizhou, Guizhou, Guizhou, Guizhou, Guizhou, Guizhou, Guizhou, Guizhou, Guizhou, Guizhou, Guizhou, Guizhou, Guizhou, Guizhou, Guizhou,
China China China China China China China China China China China China China China China China China China China China
PERV-A
PERV-B
PERV-C
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
⫺ ⫺ ⫹ ⫺ ⫹ ⫺ ⫹ ⫺ ⫺ ⫹ ⫺ ⫹ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺
Abbreviations: BW, body weight; ⫹, a PERV subtype was present in the sample genome; ⫺, a PERV subtype was absent in the sample genome.
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Fig 2. A standard curve for the internal reference gene TFRC (R ⫽ .9807). The standard plasmid for TFRC was diluted into 5 gradient concentrations of 10⫻ dilution: 106, 105, 104, 103, and 102 (copies/L). The qPCR was run individually and corresponding Ct values were measured. The standard curve for the TFRC gene was generated by plotting the corresponding Ct values (ordinate) against the logarithm values of the copy number of TFRC in the standard plasmid (abscissa). Abbreviations: Ct, cycle threshold, the number of cycles when the fluorescent signal reaches the set threshold in each reaction tube; N, copy number of the TFRC gene.
Statistical Analysis Experimental data were expressed as the mean values ⫾ standard deviations (x ⫾ SD). A P value less than .05 was considered significant. Statistical analysis was performed using SPSS13.0 statistical software (SPSS Co., Ill, USA). A completely randomized design analysis of variance (ANOVA; one-factor ANOVA) was used to evaluate test data.
RESULTS Detection of 3 PERV Genotypes: PERV-A, PERV-B, and PERV-C
To detect the distribution of PERV genotypes among the genomes of Chinese experimental miniature pigs, we evaluated genomic DNA by PCR using specific primers for env-A, env-B, and env-C. Nonspecific amplification products did not appear among the negative controls, and the positive controls confirmed no false-positive results (Fig 1). PERV proviral DNA were found in all 20 samples of pig genomic DNA. The distribution of the 3 PERV genotypes was 20/20 (100%) samples with PERV-A and PERV-B
subtypes (Table 2), whereas PERV-C subtypes were detected in 6/20 (30%) samples (Table 2). Quantitation of PERV Copy Number in Porcine Genomic DNA
To confirm the specificity of the PERV-pol sequence detected using SYBR Green real-time qPCR, the same amount of DNA isolated from HEK-293 cells was used as a negative control. There was no amplification signal for the pol sequences observed. The pol amplification signals were detected for the plasmid and sample DNA. According to the absolute quantitative standard curves for pol and TFRC based on the 10⫻ plasmid dilution gradient (Fig 2 and Fig 3) and the internal reference copy number of the TFRC gene for each sample, the copy number of PERV was evaluated in genomic DNA. The experiment was repeated 3 times for each genomic DNA sample. The presence of PERV proviral DNA was positively detected in all 20 tested samples of Chinese experimental miniature pigs. The copy number of PERV in
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Fig 3. A standard curve for target gene pol (R ⫽ .9936). The standard plasmid for pol was diluted into 5 gradient concentrations of 10⫻ dilution: 106, 105, 104, 103, and 102 (copies/L). The qPCR was run individually and corresponding Ct values were measured. The standard curve for pol gene was generated by plotting the corresponding Ct values (ordinate) against the logarithm values of the copy number of pol in the standard plasmid (abscissa). Abbreviations: Ct, cycle threshold; N, copy number of the pol gene.
genomic DNA varied significantly among individual Chinese experimental miniature pigs (P ⬍ .05; one-factor ANOVA), ranging from 3.95 ⫾ 0.14 to 95.52 ⫾ 2.20 (Fig 4).
specificity of the PERV-pol sequence detected using SYBR Green qPCR assay was analyzed followed by negative amplification signal in a negative control (HEK-293 cells). The pol amplification signals were detected for the sample RNA.
Quantitation of PERV Expression at Messenger RNA Level
PERV expression was evaluated in the RNA samples using the constructed absolute quantitative standard curves for pol and TFRC as described above. The expression of PERV at the RNA level was positively detected in all 20 tested samples of Chinese experimental miniature pigs. The PERV expression at the RNA level also varied significantly in different individuals (P ⬍ .05; one-factor ANOVA), ranging from 3.66 ⫾ 0.13 to 43.03 ⫾ 2.50 (Fig 5). To prevent detection errors caused by DNA contamination when quantifying PERV expression at the RNA level, DNase I endonuclease digestion was performed during the total RNA extraction. In addition, a control without reverse transcriptase was used during reverse transcription of RNA into cDNA and then tested using PCR with pol primers. There was no amplification product indicating that the extracted RNA samples were not affected by DNA contamination. The
DISCUSSION
Due to their physiological similarity to humans, low production costs, and adequate supply, pigs have become the preferred donors for human xenotransplantation.17,18 However, whether humans can be infected with PERV after proviral DNA integration into the pig genome to cause disease has become a focus of debate. Although no evidence of in vivo PERV infection in other animals or humans has been found,19 –21 it has been shown that in vitro PERV infection occurs in several types of human cells.22,23 The possibility of human PERV infection during xenotransplantation still cannot be ruled out. Human immunodeficiency virus (HIV), which has similar properties to PERV, can cause acquired immuno deficiency syndrome (AIDS), which clearly confirms the hazard of retroviruses in humans.24
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Fig 4. PERV copy numbers in genomic DNA for Chinese experimental miniature pigs. 1–20: number of tissue sample from Chinese experimental miniature pigs.
Chinese miniature pigs are mainly used in experiments. Based on the advantages of clear genetic background, subtle individual differences, and a similar organ size to humans, they are considered to be suitable transplant donors.25 The Chinese experimental miniature pig is one model in this regard. A previous study reported 2 PERVfree cases of Chinese experimental miniature pigs.14 It has been acknowledged that PERV genomic copy number, especially the one at RNA expression level, show a positive correlation with infectivity.26 Only the intact PERV proviral DNA copies integrated into porcine genome can be transcripted into PERV retrovirus RNA, generating infectious retrovirus particles.27–29 Thus information on genomic expression can be valuable to select the porcine strain/ individual with lower PERV infectivity. For the Chinese experimental miniature pig, experimental data regarding the PERV proviral distribution of its genomic DNA and RNA expression are still limited. In this regard, we surveyed the population of Chinese experimental miniature pigs to examine these characteristics. Although PERV-C does not infect human cells, it is still a threat to humans. PERV-C and PERV-A can be recombined into PERV-A/C, which has stronger in vitro infectiv-
ity in human cells.30 By PCR detection, we observed that PERV-A and PERV-B were common among genomes Chinese of experimental miniature pigs (100%), but PERV-C has not, occurring in only 30% of samples. Therefore, we can reduce the PERV infection risk by screening individuals not containing PERV-C. SYBR Green qPCR provides simple, sensitive, and specific detection of PERV copy number in the porcine genome.13,31 Our results with this assay confirmed the previous report that the copy number of PERV differs among individuals of the same breed.32 The PERV copy number in the Chinese experimental miniature pig genome ranged from 3.95 ⫾ 0.14 to 95.52 ⫾ 2.20. We also observed that the amount of PERV expression at the RNA level ranged from 3.66 ⫾ 0.13 to 43.03 ⫾ 2.50 by SYBR Green real-time qPCR. Although the PERV copy number was great in some individual’s genomes, there were still some pigs with low copy numbers for PERV. In addition, for some individuals, the PERV expression at the RNA level was low. For instance, sample no. 17 (1/20) not only had fewer copy number of PERV in DNA and lower PERV expression at the RNA level, but also no PERV-C subtype. This member is considered to be ideal for further selective
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Fig 5. Amount of PERV expression at RNA level for Chinese experimental miniature pigs. 1–20: number of tissue sample from Chinese experimental miniature pigs.
breeding. Through selective breeding technology or gene knockouts, we hope to obtain individuals with no risk of PERV infection, which are ideally suitable for xenotransplantation is future studies. In summary, due to its advantages, the Chinese experimental miniature pig is a promising donor for human xenotransplantation. By investigating the PERV genotypes in the genome of Chinese experimental miniature pigs, PERV copy number, and PERV expression at the RNA level, our results may provide a foundation for selective breeding, to benefit the application of these pigs in the study of xenotransplantation.
REFERENCES 1. Boneva RS, Folks TM, Chapman LE: Infectious disease issues in xenotransplantation. Clin Microbiol Rev 14:1, 2001 2. Lee D, Lee J, Park N, et al: Analysis of natural recombination in porcine endogenous retrovirus envelope genes. J Microbiol Biotechnol 18:585, 2008 3. Chapman LE, Folks TM, Salomon DR, et al: Xenotransplantation and xenogeneic infections. N Engl J Med 333:1498, 1995 4. Li Z, Ping Y, Shengfu L, et al: Phylogenetic relationship of porcine endogenous retrovirus (PERV) in Chinese pigs with some type C retroviruses. Virus Res 105:167, 2004
5. Switzer WM, Shanmugam V, Chapman L, et al: Polymerase chain reaction assays for the diagnosis of infection with the porcine endogenous retrovirus and the detection of pig cells in human and nonhuman recipients of pig xenografts. Transplantation 68:183, 1999 6. Le Tissier P, Stoye JP, Takeuchi Y, et al: Two sets of human-tropic pig retrovirus. Nature 389:681, 1997 7. Magre S, Takeuchi Y, Bartosch B: Xenotransplantation and pig endogenous retroviruses. Rev Med Virol 13:311, 2003 8. 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 9. Harrison I, Takeuchi Y, Bartosch B, et al: Determinants of high titer in recombinant porcine endogenous retroviruses. J Virol 78:13871, 2004 10. Yao SK, Zhang Q, Sun FZ, et al: Genetic diversity of seven miniature pig breeds (strains) analyzed by using microsatellite markers. Yi Chuan 28:407, 2006 11. Liu Q, Liu Z, Dalakas E: Prevalence of porcine endogenous retrovirus in Chinese pig breeds and in patients treated with a porcine liver cell-based bioreactor. World J Gastroenterol 11:4727, 2005 12. Louz D, Bergmans HE, Loos BP, et al: Reappraisal of biosafety risks posed by PERVs in xenotransplantation. Rev Med Virol 18:53, 2008 13. Ma Y, Yang Y, Lv M, et al: Real-time quantitative polymerase chain reaction with SYBR green indetection for estimating
PORCINE ENDOGENOUS RETROVIRUS copy numbers of porcine endogenous retrovirus from Chinese miniature pigs. Transplant Proc 42:1949, 2010 14. Lu Q, Han H, Lian Z, et al: The screening and identification of endogenous retrovirus free CEMPs. Sci China C Life Sci 47:562, 2004 15. Wang Y, Ren J, Lan L, et al: Characterization of polymorphisms of transferrin receptor and their association with susceptibility to ETEC F4ab/ac in pigs. J Anim Breed Genet 124:225, 2007 16. Xing XW, Hawthorne WJ, Yi S, et al: Investigation of porcine endogenous retrovirus in the conservation population of Ningxiang pig. Transplant Proc 41:4389, 2009 17. Platt JL: New directions for organ transplantation. Nature 392:11, 1998 18. Cooper DK, Gollackner B, Knosalla C, et al: Xenotransplantation-how far have we come? Transpl Immunol 9:251, 2002 19. Patience C, Patton GS, Takeuchi Y, et al: No evidence of pig DNA or retroviral infection in patients with short-term extracorporeal connection to pig kidneys. Lancet 352:699, 1998 20. Winkler ME, Winkler M, Burian R, et al: Analysis of pig-to-human porcine endogenous retrovirus transmission in a triple-species kidney xenotransplantation model. Transpl Int 17: 848, 2005 21. Issa NC, Wilkinson RA, Griesemer A, et al: Absence of replication of porcine endogenous retrovirus and porcine lymphotropic herpesvirus type 1 with prolonged pig cell microchimerism after pig-to-baboon xenotransplantation. J Virol 82:12441, 2008 22. Martin U, Kiessig V, Blusch JH, et al: Expression of pig endogenous retrovirus by primary porcine endothelial cells and infection of human cells. Lancet 352:692, 1998 23. Wilson CA, Wong S, VanBrocklin M, et al: Extended analysis of the in vitro tropism of porcine endogenous retrovirus. J Virol 74:49, 2000
2769 24. Denner J: Porcine endogenous retroviruses (PERVs) and xenotransplantation: screening for transmission in several clinical trials and in experimental models using non-human primates. Ann Transplant 8:39, 2003 25. Wu J, Ma Y, Lv M, et al: Large-scale survey of porcine endogenous retrovirus in Chinese miniature pigs. Comp Immunol Microbiol Infect Dis 31:367, 2008 26. Moon HJ, Kim HK, Park SJ, et al: Comparison of the age-related porcine endogenous retrovirus (PERV) expression using duplex RT-PCR. J Vet Sci 10:317, 2009 27. Niebert M, Rogel-Gaillard C, Chardon P, et al: Characterization of chromosomally assigned replication-competent gamma porcine endogenous retroviruses derived from a large white pig and expression in human cells. J Virol 76:2714, 2002 28. Patience C, Switzer WM, Takeuchi Y, et al: Multiple groups of novel retroviral genomes in pigs and related species. J Virol 75:2771, 2001 29. 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 30. Denner J: Recombinant porcine endogenous retroviruses (PERV-A/C): a new risk for xenotransplantation? Arch Virol 153:1421, 2008 31. Zhang P, Yu P, Wang W, et al: An effective method for the quantitative detection of porcine endogenous retrovirus in pig tissues. In Vitro Cell Dev Biol Anim 46:408, 2010 32. Herring C, Quinn G, Bower R, et al: Mapping full-length porcine endogenous retroviruses in a large white pig. J Virol 75:12252, 2001
P
logical and biochemical indices, and economic affordability,10 the Chinese experimental miniature pig has become a promising donor source for pig-to-human xenotransplantation. According to a recent clinical report, PERV reverse transcriptase activity was not observed in sera from 3 patients treated with a porcine liver cell-based bioartificial liver from a Chinese experimental miniature pig.11 The PERV copy number carried in pig breeds serves as an important reference value for potential viral infectivity and the species’ suitability as a future donor. It is known that From the Institute of Regenerative Medicine (G.L., Z.L., Y.W., Y.G.), Zhujiang Hospital, Southern Medical University, and Department of Hepatobiliary Surgery (G.L., Z.L., M.P., M.G., Y.W., Y.G.), Zhujiang Hospital, Southern Medical University, Guangzhou, China. This work was supported by National High Technology Research and Development Program Project of China (2006AA02A141). Address reprint requests to Yan Wang, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou 510280, China. E-mail: [email protected]; and Yi Gao, Department of Hepatobiliary Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou 510280, China. E-mail: [email protected]
0041-1345/11/$–see front matter doi:10.1016/j.transproceed.2011.06.061
© 2011 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
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Transplantation Proceedings, 43, 2762–2769 (2011)
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Table 1. PCR Primer Sequences for PERV and TFRC Gene Amplification Gene
Sequence of Primer
env-A6
F:5=-TGGAAAGATTGGCAACAGCG-3= R: 5=-AGTGATGTTAGGCTCAGTGG-3= F: 5=-TTCTCCTTTGTCAATTCCGG-3= R:5=-TACTTTATCGGGTCCCACTG-3= F:5=-CTGACCTGGATTAGAACTGG-3= R:5=-ATGTTAGAGGATGGTCCTGG-3= F: 5=-AGCTCCGGGAGGCCTACTC-3= R:5=-ACAGCCGTTGGTGTGGTCA-3= F:5=-GAGACAGAAACTTTCGAAGC-3= R:5=-GAAGTCTGTGGTATCCAATCC-3=
env-B6 env-C8 Pol6 15
TFRC
Fragment Size (bp)
359 263 281 150 80
Abbreviations: TFRC, transferrin receptor; bp, base pair.
there are multiple PERV copies in most pig breeds; the copy number differs among breeds and individuals of the same breed.12,13 After testing 67 Chinese experimental miniature pigs, Lu et al found that 2 individuals were negative for PERV.14 However, data for the distribution of
PERV provirus in genomic DNA and PERV expression at the RNA level among the population of Chinese experimental miniature swine are lacking. In this study, we identified PERV genotypes in the genomes of Chinese experimental miniature swine detecting PERV copy number in genomic DNA and PERV expression at the RNA level. These data may lay the foundation for selective breeding to benefit the application of these donors to the study of xenotransplantation. MATERIALS AND METHODS Sample Collection Twenty Chinese experimental miniature pigs were randomly selected from Changping experimental farm at China Agricultural University (Beijing, China). Tissue samples (1 cm3) obtained from the ears were stored in 70% ethanol.
Extraction of Genomic DNA and Total RNA Genomic DNA was extracted from samples using the genomic DNA Extraction kit protocol (QIAGEN, Hamburg, Germany). Sample total RNA was extracted using an RNA Extraction kit
Fig 1. PCR analysis of the detection specificity for PERV DNA. DNA sample from PK-15 cell line was used as the positive control: lane’s 2, 4, and 6 were env-C, env-B, and env-A, respectively. Sample no. 5 was randomly selected as the testing sample: lane’s 1, 3, and 5 were env-C, env-B, and env-A, respectively. Distilled water was used as the negative crontrol in lane N. Lane M was the DNA marker.
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(Promega, Wisconsin, United States). The OD260nm value was determined for each DNA and RNA sample using a spectrophotometer (Eppendorf, Hamburg, Germany). The average of 3 measurements performed for each sample allowed samples to be adjusted to 200 ng/L.
Polymerase Chain Reaction Detection of PERV Genotypes Using the reported sequences for PERV,6,8 3 primer pairs were designed and synthesized to individually detect PERV genotypes (PERV-A, PERV-B, and PERV-C; (Sangon Biotech Co., Ltd., Shanghai, China; Table 1). To control for false-positive polymerase chain reaction (PCR) reactions we selected PK-15 cell genomic DNA as a positive control; a negative control (blank) confirmed the absence of nonspecific amplification. Using the primers described above, we amplified the corresponding genes in a PCR assay with a total volume of 25 L (Bio-Rad, United States), including 1 ⫻ PCR buffer (TaKaRa, Dalian, China), 200 mol/L dNTPs (TaKaRa), 2 U Taq DNA polymerase (TaKaRa), both primers, and 15 pmol of each DNA template, and distilled water added to bring the final total volume to 25 L. PCR reaction conditions for env-A, env-B, and env-C were as follows: 95°C for 5 minutes to initially denature, 95°C for 30 seconds 55°C for 45 seconds, 72°C for 1 minute repeated for a total of 30 cycles, with final extension at 72°C for 7 minutes followed by 2% agarose gel electrophoresis.
Real-Time Quantitative PCR Assay Because the pol segment is highly conserved among PERV gene sequences, it was selected as a target gene to design specific primers (Sangon Biotech Co., Ltd.).6 Meanwhile, the house-keeping gene porcine transferrin receptor (TFRC) was selected as an internal reference gene and primers were also designed to amplify its sequence (Sangon Biotech Co., Ltd.; Table 1).15 After purification and gel recovery, the pol and TFRC gene PCR amplification products were independently ligated into the PMD-18T
vector (TaKaRa) for transformation in DH5a Escherichia coli (Tiangen Biotech Co., Ltd., Beijing, China). Using blue-white screening, we selected positive clones. EcoRI/Hind III enzyme (New England Biolabs, Mass, USA) digestions were performed to identify and extract plasmids containing pol and TFRC, respectively. After plasmid concentrations were determined with an ultra violet (UV) spectrophotometer (Eppendorf, Hamburg, Germany), the 2 standard plasmids were diluted with a 10⫻ gradient. Plasmids of 5 continuous gradient concentrations were selected to detect the copy number and cycle threshold values for the TFRC and pol genes. An absolute quantitative standard curve was constructed accordingly. SYBR Green, a commonly used fluorescent DNA binding dye, based real-time qPCR assays were performed with a 7500 Real Time PCR System (Applied Biosystems, Calif, USA) using the SYBR Premix Ex Taq Kit (TaKaRa) in a total volume of 20 L, including 10 L of 2 ⫻ SYBR Premix Ex Ta (TaKaRa), 0.8 L each of forward and reverse primers, 0.4 L of ROX Reference Dye (Haoran Biological Technology Co., Ltd., Shanghai, China), and 50 ng plasmid DNA or sample DNA, and finally nuclease-free water was added for a total volume of 20.0 L. The qPCR reaction conditions were 95°C for 5 minutes to initially denature, 95°C for 30 seconds, 55°C for 45 seconds, 72°C for 1 minute for a total of 30 cycles, with final extension at 72°C for 7 minutes. The temperature was then slowly increased to 95°C at a rate of 0.1°C/s. The first complementary DNA (cDNA) strand was synthesized according to the previously described method.16 Real-time quantitative reverse transcription PCR assays were performed using primers for the pol and TFRC genes as described above. For each reaction we used 50 ng total RNA. The qPCR reactions were performed with the temperature conditions as described above. Melting curves were constructed for the amplification products from the plasmid DNA, sample DNA, and RNA for verification. Reaction data were analyzed using Sequence Detection System software (Applied Biosystems).
Table 2. Distribution of PERV Subtypes in 20 Porcine Genomic DNA Samples PERV Subtype Sample No.
Gender
Age (mo)
BW (kg)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Male Male Female Male Female Female Male Male Male Female Female Male Male Male Male Female Female Female Male Female
21 8 6 12 10 12 7 7 15 22 13 13 8 18 10 6 9 17 12 11
47.5 30 26 38.5 34 39.5 28 28.5 41.5 48 39 28.5 29 45 34.5 26 32.5 42.5 39 35.5
Original Place
Guizhou, Guizhou, Guizhou, Guizhou, Guizhou, Guizhou, Guizhou, Guizhou, Guizhou, Guizhou, Guizhou, Guizhou, Guizhou, Guizhou, Guizhou, Guizhou, Guizhou, Guizhou, Guizhou, Guizhou,
China China China China China China China China China China China China China China China China China China China China
PERV-A
PERV-B
PERV-C
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
⫺ ⫺ ⫹ ⫺ ⫹ ⫺ ⫹ ⫺ ⫺ ⫹ ⫺ ⫹ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺
Abbreviations: BW, body weight; ⫹, a PERV subtype was present in the sample genome; ⫺, a PERV subtype was absent in the sample genome.
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Fig 2. A standard curve for the internal reference gene TFRC (R ⫽ .9807). The standard plasmid for TFRC was diluted into 5 gradient concentrations of 10⫻ dilution: 106, 105, 104, 103, and 102 (copies/L). The qPCR was run individually and corresponding Ct values were measured. The standard curve for the TFRC gene was generated by plotting the corresponding Ct values (ordinate) against the logarithm values of the copy number of TFRC in the standard plasmid (abscissa). Abbreviations: Ct, cycle threshold, the number of cycles when the fluorescent signal reaches the set threshold in each reaction tube; N, copy number of the TFRC gene.
Statistical Analysis Experimental data were expressed as the mean values ⫾ standard deviations (x ⫾ SD). A P value less than .05 was considered significant. Statistical analysis was performed using SPSS13.0 statistical software (SPSS Co., Ill, USA). A completely randomized design analysis of variance (ANOVA; one-factor ANOVA) was used to evaluate test data.
RESULTS Detection of 3 PERV Genotypes: PERV-A, PERV-B, and PERV-C
To detect the distribution of PERV genotypes among the genomes of Chinese experimental miniature pigs, we evaluated genomic DNA by PCR using specific primers for env-A, env-B, and env-C. Nonspecific amplification products did not appear among the negative controls, and the positive controls confirmed no false-positive results (Fig 1). PERV proviral DNA were found in all 20 samples of pig genomic DNA. The distribution of the 3 PERV genotypes was 20/20 (100%) samples with PERV-A and PERV-B
subtypes (Table 2), whereas PERV-C subtypes were detected in 6/20 (30%) samples (Table 2). Quantitation of PERV Copy Number in Porcine Genomic DNA
To confirm the specificity of the PERV-pol sequence detected using SYBR Green real-time qPCR, the same amount of DNA isolated from HEK-293 cells was used as a negative control. There was no amplification signal for the pol sequences observed. The pol amplification signals were detected for the plasmid and sample DNA. According to the absolute quantitative standard curves for pol and TFRC based on the 10⫻ plasmid dilution gradient (Fig 2 and Fig 3) and the internal reference copy number of the TFRC gene for each sample, the copy number of PERV was evaluated in genomic DNA. The experiment was repeated 3 times for each genomic DNA sample. The presence of PERV proviral DNA was positively detected in all 20 tested samples of Chinese experimental miniature pigs. The copy number of PERV in
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Fig 3. A standard curve for target gene pol (R ⫽ .9936). The standard plasmid for pol was diluted into 5 gradient concentrations of 10⫻ dilution: 106, 105, 104, 103, and 102 (copies/L). The qPCR was run individually and corresponding Ct values were measured. The standard curve for pol gene was generated by plotting the corresponding Ct values (ordinate) against the logarithm values of the copy number of pol in the standard plasmid (abscissa). Abbreviations: Ct, cycle threshold; N, copy number of the pol gene.
genomic DNA varied significantly among individual Chinese experimental miniature pigs (P ⬍ .05; one-factor ANOVA), ranging from 3.95 ⫾ 0.14 to 95.52 ⫾ 2.20 (Fig 4).
specificity of the PERV-pol sequence detected using SYBR Green qPCR assay was analyzed followed by negative amplification signal in a negative control (HEK-293 cells). The pol amplification signals were detected for the sample RNA.
Quantitation of PERV Expression at Messenger RNA Level
PERV expression was evaluated in the RNA samples using the constructed absolute quantitative standard curves for pol and TFRC as described above. The expression of PERV at the RNA level was positively detected in all 20 tested samples of Chinese experimental miniature pigs. The PERV expression at the RNA level also varied significantly in different individuals (P ⬍ .05; one-factor ANOVA), ranging from 3.66 ⫾ 0.13 to 43.03 ⫾ 2.50 (Fig 5). To prevent detection errors caused by DNA contamination when quantifying PERV expression at the RNA level, DNase I endonuclease digestion was performed during the total RNA extraction. In addition, a control without reverse transcriptase was used during reverse transcription of RNA into cDNA and then tested using PCR with pol primers. There was no amplification product indicating that the extracted RNA samples were not affected by DNA contamination. The
DISCUSSION
Due to their physiological similarity to humans, low production costs, and adequate supply, pigs have become the preferred donors for human xenotransplantation.17,18 However, whether humans can be infected with PERV after proviral DNA integration into the pig genome to cause disease has become a focus of debate. Although no evidence of in vivo PERV infection in other animals or humans has been found,19 –21 it has been shown that in vitro PERV infection occurs in several types of human cells.22,23 The possibility of human PERV infection during xenotransplantation still cannot be ruled out. Human immunodeficiency virus (HIV), which has similar properties to PERV, can cause acquired immuno deficiency syndrome (AIDS), which clearly confirms the hazard of retroviruses in humans.24
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Fig 4. PERV copy numbers in genomic DNA for Chinese experimental miniature pigs. 1–20: number of tissue sample from Chinese experimental miniature pigs.
Chinese miniature pigs are mainly used in experiments. Based on the advantages of clear genetic background, subtle individual differences, and a similar organ size to humans, they are considered to be suitable transplant donors.25 The Chinese experimental miniature pig is one model in this regard. A previous study reported 2 PERVfree cases of Chinese experimental miniature pigs.14 It has been acknowledged that PERV genomic copy number, especially the one at RNA expression level, show a positive correlation with infectivity.26 Only the intact PERV proviral DNA copies integrated into porcine genome can be transcripted into PERV retrovirus RNA, generating infectious retrovirus particles.27–29 Thus information on genomic expression can be valuable to select the porcine strain/ individual with lower PERV infectivity. For the Chinese experimental miniature pig, experimental data regarding the PERV proviral distribution of its genomic DNA and RNA expression are still limited. In this regard, we surveyed the population of Chinese experimental miniature pigs to examine these characteristics. Although PERV-C does not infect human cells, it is still a threat to humans. PERV-C and PERV-A can be recombined into PERV-A/C, which has stronger in vitro infectiv-
ity in human cells.30 By PCR detection, we observed that PERV-A and PERV-B were common among genomes Chinese of experimental miniature pigs (100%), but PERV-C has not, occurring in only 30% of samples. Therefore, we can reduce the PERV infection risk by screening individuals not containing PERV-C. SYBR Green qPCR provides simple, sensitive, and specific detection of PERV copy number in the porcine genome.13,31 Our results with this assay confirmed the previous report that the copy number of PERV differs among individuals of the same breed.32 The PERV copy number in the Chinese experimental miniature pig genome ranged from 3.95 ⫾ 0.14 to 95.52 ⫾ 2.20. We also observed that the amount of PERV expression at the RNA level ranged from 3.66 ⫾ 0.13 to 43.03 ⫾ 2.50 by SYBR Green real-time qPCR. Although the PERV copy number was great in some individual’s genomes, there were still some pigs with low copy numbers for PERV. In addition, for some individuals, the PERV expression at the RNA level was low. For instance, sample no. 17 (1/20) not only had fewer copy number of PERV in DNA and lower PERV expression at the RNA level, but also no PERV-C subtype. This member is considered to be ideal for further selective
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Fig 5. Amount of PERV expression at RNA level for Chinese experimental miniature pigs. 1–20: number of tissue sample from Chinese experimental miniature pigs.
breeding. Through selective breeding technology or gene knockouts, we hope to obtain individuals with no risk of PERV infection, which are ideally suitable for xenotransplantation is future studies. In summary, due to its advantages, the Chinese experimental miniature pig is a promising donor for human xenotransplantation. By investigating the PERV genotypes in the genome of Chinese experimental miniature pigs, PERV copy number, and PERV expression at the RNA level, our results may provide a foundation for selective breeding, to benefit the application of these pigs in the study of xenotransplantation.
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