Investigation of Porcine Endogenous Retrovirus in the Conservation Population of Ningxiang Pig

Investigation of Porcine Endogenous Retrovirus in the Conservation Population of Ningxiang Pig X.W. Xing, W.J. Hawthorne, S. Yi, D.M. Simond, Q. Dong, B. Ye, Q.J. Tong, Z. Ye, and W. Wang ABSTRACT Porcine endogenous retrovirus (PERV) varies between pig breeds. Screening and analysis of PERV in putative pig breeds may provide basic parameters to evaluate the biological safety of xenotransplantation from pigs to humans. In this study, PERV was investigated among the conservation population of the Ningxiang pig. The result revealed that the genotype of PERV distribution was subtype A, 100%; subtype B, 100%; and subtype C, 100%. The env sequences of PERV-A and -B showed 11 clones detected by KpnI and MboI digestion, indicating that there existed multiple variants of PERV-A and -B in the Ningxiang pig. Reverse transcriptase polymerase chain reaction results showed that PERV had transcriptional activity in these individuals. In addition, PERV A/C recombinant was detected in most individuals of Ningxiang pig. Because PERV A/C recombinants increase the potential infectious risk, the breed may not be a proper donor for xenotransplantation. ENOTRANSPLANTATION using porcine organs or cells may provide an unlimited resource to alleviate the shortage of human donor organs. The pig is regarded as the most suitable donor in xenotransplantation. However, porcine endogenous retrovirus (PERV) is still a concern.1,2 PERVs belong to the genus Gammaretrovirus. Because multiple copies of PERV genomes are present in pig genomes, PERV may be difficult to eliminate from porcine tissues.1 According to the sequences of their envelope proteins, PERVs have been classed into PERV-A, -B and -C. PERV-A and -B could infect human cells in vitro (human-tropic virus), but PERV-C infects only pig cells (ecotropic virus).1–3 The in vitro expression of PERV varies among pig breeds and tissues. PERV-A and -B are present in the genome of all pigs, whereas PERV-C is not ubiquitous.4,5 The recombination of PERV-A and PERV-C has been reported in some pig breeds. Recombinant PERVA/C is able to infect human cells, increasing the potential infectious risk in therapeutic xenotransplantation.5 China has at least a 7000-year history of domesticating pigs and ⬎100 pig breeds. About one third of the world’s pig breeds exist in China.6 Ningxiang pig (also called Caochong pig or Liusha River pig) is a famous native pig breeds in the Hunan region, which possess some unique hereditary properties including an high reproduction rate and better adaptability. However, from the biological safety aspect, it is still unclear whether this breed is proper putative donor for xenotransplantation. In this study, we investigated PERV in the conservation population of

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Ningxiang pig to provide basic information on the biological safety of xenotransplantation from pig to human. MATERIALS AND METHODS Genomic DNA and total RNA Isolation Peripheral blood samples from 21 individual Ningxiang pigs were randomly collected of in heparin. Genomic DNA and total RNA were extracted according to the manufacture’s instruction (Gentra); the concentrations of DNA and RNA were measured with ultraviolet (UV) spectrophotometry.

Amplification of PERV Proviral DNA by Polymerase Chain Reaction Polymerase chain reaction (PCR) was performed to amplify PERV env-A, env-B, env-C and porcine mitochondrial (mt) DNA cytochrome oxidase subunit II (Coll) using specific primers (Table 1). The PCR reaction volume (20 ␮L) contained: nuclease-free water From the Cell Transplantation and Gene Therapy Institute of Central South University (X.W.X., Q.D., B.Y., Q.J.T., Z.Y., W.W.), Changsha, P. R. China; and the Centre for Transplant & Renal Research (W.J.H., S.Y., D.M.S.), Westmead Millennium Institute, University of Sydney, Westmead, NSW, Australia. Supported by grant of Human Nature Science Foundation of China (Grant No. 07JJ3066) and Program from Public Health of Human in China (Grant No. B2006-080). Address reprint requests to Wei Wang, Professor, Cell Transplantation & Gene Therapy Center, Third Hospital of Central South University, Changsha, 410013, China. E-mail: wawe01cn@ yahoo.com.cn

Crown Copyright © 2009 Published by Elseiver Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 41, 4389 – 4393 (2009)

0041-1345/09/$–see front matter doi:10.1016/j.transproceed.2009.09.051 4389

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Table 1. Primer Pairs for PCR Amplification of PERV and mtDNA Gene CoII Genes

Sequences of Primers

env-A

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= -CCACAGGGCAACGGCAGTATCC-3= R:5= -TTGGAGGGTCAACACAGTGATGG-3= F:5= -CGGCAAGAGAAGAATTTGACT-3= R:5= -CAGTTCCTTGCCCAGTGTCC-3= F:5= -TCACCCATCATAGAAGAACTCCTACA-3= R:5= -TTTTACGGTTAAGGCTGGGTTATTAAT-3=

env-B env-C pol gag Co II

Fragments (bp)

360 264 281 212 188 281

12 ␮L, 10 ⫻ PCR reaction buffer 2 ␮L, 25 mmol/L MgCl2 1.6 ␮L, 2.5 mmol/L dNTP 2 ␮L, Taq DNA polymerase 0.4 ␮L, primer mix 0.4 ␮L, and DNA 1.6 ␮L. The cycling reaction was performed in a programmable thermal cycler, (PE9700) using denaturation at 95°C for 2 minutes 30 seconds, and then 94°C for 40 seconds, 58 – 60°C for 40 seconds, 72°C for 40 seconds, for 35 cycles. The extension step in the last cycle step was 5 minutes. The PCR products were then analyzed on 2% agarose gel. The sensitivities of PCR for PERV and Coll have described been elsewhere.7,8

Detection of the Expression of PERV in the Peripheral Blood by Reverse Transcriptase PCR The first cDNA strand was synthesized according to the previously described method.9 RT-PCR was performed to detect the expression of PERV pol, gag according to the previous method.8

accession no. Y12238) and -B (GenBank accession no. Y12239) envelope genes, were employed to search for novel variants in the less conserved region and to amplify as many envelope fragments as possible from PERV proviruses in the pig genome.10 After preliminary analysis with electrophoresis, these fragments were treated with agarose gel DNA purification kit (TaKaRa) and mixed together. Then, we inserted these fragments into pUCm-T vectors. PCR was used to identify these inserts; products from each clone were analyzed by restriction enzymes KpnI and MboI. Finally, distinct restriction digestion patterns of PERV clones were sequenced for analysis using the BLAST software in The National Center for Biotechnology Information available: (http://blast. ncbi.nlm.nih.gov/Blast.cgi).

Screen of PERV A/C Recombinants in PERV-C Positive Individuals The tropism of PERV was determined by 2 sequences in the receptor binding domain (RBD) in the surface envelope protein and variable regions (VR)-A and VR-B. PERV A/C recombinants in PERV-C–positive individuals were amplified using 2 sets of primer pairs: (1) PERV A-VRAF (5=-ATGTCTGCCTTCGATCAGTAATCCC-3=) and PERV C-TMR (5=-CTCAAACCACCCTTGAGTAGTTTCC-3=); (2) PERV A-VRBF(5=-CCTACCAGTTATAATCAATTT AATTATGGC-3=) and PERV C-TMR (5=-CTCAAACCACCCTTGAGT AGTTTCC-3=).11,12 Easy-A high-fidelity PCR cloning enzyme was used to amplify PERV A/C recombinant by PCR. Each PCR was repeated ⱖ3 times. Finally, a PERV-A/C recombinant cloned into pUCm-T vector was sequenced and analyzed using BLAST software in National Center for Biotechnology Information available: (http://blast.ncbi.nlm. nih.gov/Blast.cgi) and CLUSTALW in the ExPASY Proteomics Server available: (http://www.ebi.ac.uk/Tools/clustalw2/index.html).

Analysis of PERV env-A and env-B Sequences

RESULTS Three Subtypes of PERV Were Detected

Platinum Taq DNA polymerase high fidelity was used to amplify 1.8-kb envelope gene fragments from 3 individuals using consensus primers for the type A and type B viruses. The consensus primers (sence primer: 5=-CATGCATCCCACGTTAAGC-3=, antisense primer: 5=-ACCATCCTTCAAACCACCC-3=), chosen from the highly conserved regions at either end of the PERV-A (GenBank

According to the PERV env sequences, we amplified PERV proviral DNA using type-specific primers. We detected PERV env-A, env-B, env-C and Coll gene in all 21 genomic DNA samples: subtype A, 100%; subtype B, 100%; and subtype C, 100% (Fig 1).

Fig 1. PERV env-A, env-B, and env-C were detected in Ningxiang pigs by PCR. M, pUC mix 8 marker; P, positive control; N, H2O, lanes 1– 8 indicate DNAs from different individuals.

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Fig 2. PERV pol and gag could be detected in all individuals. M, pUC mix 8 marker; P, positive control; N, H2O, lanes 1–9 indicated RNAs from different individuals.

PERV Has Transcriptional Activity in Each Individual

To clarify whether PERV has transcriptional activity in the peripheral blood, RT-PCR was performed to amplify PERV pol, gag in all 21 samples. The specific bands of pol and gag gene were detected in all samples, suggesting that PERV had transcriptional activity (Fig 2). Multiple Variants of PERV env-A and env-B Were Found

Using the consensus primers, we amplified 1.8-kb envelope gene fragments from 3 individuals (Fig 3A). These env PCR products were gel-purified for insertion into pUCm-T vectors. We identified the 42 positive clones by PCR (Fig 3B). Each clone was amplified by PCR using the consensus primers and the PCR products analyzed by restriction enzymes Kpn I and Mbo I. Six pattern of KpnI and 8 pattern of Mbo I digestion were identified in Ningxiang pigs (Fig 3C). The combined results for the 2 restriction enzymes, showed 11 clones, which were amplified by platinum Taq DNA polymerase high fidelity and shown to be distinct from other pig breeds

Fig 3. Identification of PERV env-A and env-B sequences. (A) Envelope gene fragments (1.8 kb) were amplified from 3 individuals (lanes 1–3) using consensus primers. (B) The inserts were identified by PCR using the consensus primer pairs. M, 100-bp ladder; lane 1– 8 PCR products were amplified from different clones. (C) Restriction digestion patterns of PERV clones. Six patterns (A–F) of KpnI digestion and 8 patterns (a– g) of MboI digestion were identified in the PCR product amplified by platinum Taq DNA polymerase high fidelity.

as previously published (Table 2). In 42 clones, predominant types were Bb type (n ⫽ 22) and Aa type (n ⫽ 9) (Table 2). KpnI restriction digestion pattern A is characteristic of PERV-A and pattern B is characteristic of PERV-B, according to the published PERV-A and PERV-B sequences. About 30% of the envelope sequences are classified as PERV-A. Further, we sequenced 4 clones from type Aa, Bb, Ac, and Bh. The sequences were initially aligned with the published PERV-A (GenBank accession no. Y12238) and PERV-B (GenBank accession no. Y12239). The result showed that two sequences (from Aa, Ac) were similar to PERV-A and the other 2 sequences (from Bb, Bh) to PERV-B. There existed variants both in PERV-A and PERV-B sequences but there was no PERV-A/B recombinant. Prevalence of PERV-A/C Recombinant Virus in Ningxiang Pigs

Using VRAF and TMR PCR primers, we amplified PERVA/C recombinant virus from genomic DNA of Ningxiang

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Table 2. KpnI and MboI Restriction Digestion Patterns of PERV Clones KpnI Digestion Pattern

MboI Digestion Pattern

Clone Numbers

A A A A B B B C D E F

a c d g b f h d e b b

9 2 1 1 22 1 1 2 1 1 1

pigs. The PERV-A/C recombinants were detected in 90.5% (19/21) of the individuals. However, using VRBF and TMR PCR primers, we did not PERV-A/C recombinant virus. This result indicated that there existed PERV-A/C recombinants virus in most Ningxiang pigs, mainly in the VRA region of PERV-A and the TM region of PERV-C (Fig 4). The PERV-A/C recombinant from individual 8 was sequenced to be aligned with the published PERV-A/C sequence (GenBank accession no. AY534306). The homologies of nucleic acid and amino acid were 86% (1032/1193) and 78% (396/504), respectively. This result indicated that PERV A/C recombinants were variable between pig breeds. DISCUSSION

The Ningxiang pig, which possess many good biological and hereditary properties,13 is one unique native breeds in the Hunan region. In the early 1990s, the conservation population of Ningxiang pig was established to be supported by the government. Being an high-quality meat, it is welcome in the Hunan region. In a previous study, we cloned the SLA-DRA and SLA-DRB genes from Nningxiang pigs, observing that this breed showed good inherited immunity (un published result). However, it is still unclear whether this breed is better than others for xenotransplantation. Therefore, a series of screening experiments were performed to detect PERV in Ningxiang pigs. In this study, we randomly collected blood samples from the conservation population of Ningxiang pigs to investigate the prevalence of PERV. In all tested individuals we detected 3 subtype of PERV: PERV-A, PERV-B, and Fig 4. PERV A/C recombinants were found in ningxiang pigs by PCR. The sense primer specific for PERV-A was located in the VRA region, and the PERV-C specific antisense primer was located in the TM region. M, 100-bp ladder; 1–21 represented different individuals; P, positive control; N, H2O.

PERV-C. Further, we analyzed env sequences of PERV-A and -B using consensus primers chosen from the highly conserved regions at either end of the PERV-A and -B envelope genes. The fragments of PERV-A and -B were digested by restriction enzymes KpnI and MboI. The combined results for the 2 restriction enzymes showed, 11 clones with restriction enzyme digestion patterns different from those published for the Westran pig. According to the KpnI restriction digestion pattern, about 30% of the 42 envelope sequences described herein were classified as PERV-A, which is similar with that described in the Westran pig. Finally, based on the PERV-C screening experiment, we amplified PERV-A/C recombinants from PERV-A VR-A, VR-B region and the PERV-C TM region. A PERVA/C recombinant was detected in most individuals in the PERV-A VR-A region and the PERV-C TM region, but not in the PERV-A VR-B region or the PERV-C TM region. Comparing the PERV-A/C recombinant with the published PERV-A/C sequence (GenBank accession no. AY534306), we observed 86% (1032/1193) and 78% (396/ 504) homologies of nucleic acids and amino acids respectively, suggesting that PERV-A/C recombinants were variable among pig breeds. In early 1997, Patience et al14 demonstrated that PERV infected human kidney 293 cells in vitro. Yu et al15 reported that PERV may be transmitted from porcine to mouse cells in nude mice. As we knows, PERV-A and -B are polytropic viruses with wide host ranges in vitro, whereas PERV-C infects only pig cell lines.5 Recently, the existence of a recombinant PERV-A/C in pigs was identified; de novointegrated proviruses of these recombinants were observed in miniature pigs as well as in melanoma-bearing Munich miniature pigs.1,2,5 The recombinant PERV-A/C virus contains the PERV-A sequence between the middle of the SU and the end of pol region in env. The remaining sequence is derived from PERV-C. The PERV-A recombinant with PERV-C showed (approximately 500-fold) greater infectivity for human cells than PERV-A.12 Because the recombinant was able to infect human cells and its replication competence was increased when it was transmitted in human cells, the recombinant PERV-A/C represents a new risk for xenotransplantation.5,12 In this study, PERV-A/C recombinants were present in most Ningxiang pigs. Because PERV-A/C recombinants increase potential infectious risk, the biological safety point of view, demands that we not recommend this breed as a donor for xenotransplantation.

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4393 8. Xing XW, Xue LQ, Huang SQ, et al: Research of porcine endogenous retrovirus in Shaziling pigs. Yi Chuan 28:799, 2006 9. Xing XW, Li LY, Liu G, et al: Identification of a novel gene SRG4 expressed at specific stages of mouse spermatogenesis. Acta Biochim Biophys Sin (Shanghai) 36:351, 2004 10. Lee JH, Webb GC, Allen RD, et al: Characterizing and mapping porcine endogenous retroviruses in Westran pigs. J Virol 76:5548, 2002 11. 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 12. Harrison I, Takeuchi Y, Bartosch B, et al: Determinants of high titer in recombinant porcine endogenous retroviruses. J Virol 78:13871, 2004 13. Zhang ZG: Pig breeds in China. Shanghai: Shanghai Scientific and Technical Publishers; 1986 14. Patience C, Takeuchi Y, Weiss RA: Infection of human cells by an endogenous retrovirus of pigs. Nat Med 3:282, 1997 15. Yu P, Zhang L, Li SF, et al: Transmission of porcine endogenous retrovirus to human cells in nude mouse. Acta Virol 52:257, 2008