American Journal of Transplantation 2005; 5: 1837–1847Blackwell Munksgaard

Copyright C© Blackwell Munksgaard 2005

doi: 10.1111/j.1600-6143.2005.00978.x

Pseudotyping of Porcine Endogenous Retrovirus byXenotropic Murine Leukemia Virus in a Pig IsletXenotransplantation Model

Yuri Martinaa, Sunil Kuriana, Stephanie

Cherquia, Gabriel Evanoffa, Carolyn Wilsonb

and Daniel R. Salomona,∗

aDepartment of Molecular and Experimental Medicine,The Scripps Research Institute, La Jolla, California, USAbDivision of Cellular and Gene Therapies, Center forBiologics Evaluation and Research, U.S. Food and DrugAdministration, Bethesda, Maryland, USA∗Corresponding author: Daniel R. Salomon,dsalomon@scripps.edu

The potential of porcine endogenous retrovirus (PERV)as a human pathogen, particularly as a public healthrisk, is a major concern for xenotransplantation. Invitro PERV transmission to human cells is well es-tablished. Evidence from human/pig hematopoieticchimeras in immunodeficient mice suggests PERVtransmission from pig to human cells in vivo. How-ever, recently Yang et al. demonstrated in such amodel that PERV-C, a nonhuman-tropic class, couldbe transmitted via pseudotyping by xenotropic murineleukemia virus (X-MLV). We developed a mouse pigislet xenotransplant model, where pig and human cellsare located in physically separate compartments, todirectly assess PERV transmission from a functionalpig xenograft. X-MLV efficiently pseudotypes all threeclasses of PERV, including PERV-A and -B that areknown to productively infect human cell lines andPERV-C that is normally not infectious for human cells.Pseudotyping also extends PERV’s natural tropism tononpermissive, nonhuman primate cells. X-MLV is ac-tivated locally by the surgical procedure involved inthe tissue transplants. Thus, the presence and activa-tion of endogenous X-MLV in immunodeficient micelimits the clinical significance of previous reports ofin vivo PERV transmission from pig tissues to humancells.

Key words: Pig islets, porcine endogenous retro-virus (PERV), pseudotyping, xenotransplantation,xenotropic MLV

Received 21 March 2005, revised 13 April 2005 and ac-cepted for publication 19 April 2005

Introduction

Xenotransplantation has been proposed as a solution tothe shortage of suitable human donors for transplantation(1). However, it raises the potential risk of transmittingpathogens from the animal donors to the human patients.Whether this risk is restricted to the individual patient orwhether there is also a risk of further spread to the generalpublic remains uncertain. Nonhuman primates have essen-tially been ruled out as organ donors because they can di-rectly transmit several potentially pathogenic viruses to hu-mans (2). Pigs are currently favored for xenotransplantationbased on size, physiological compatibility, ability to do ge-netic engineering as well as cloning and well-establishedanimal husbandry. However, porcine xenografts can alsobe associated with infectious disease risks for humans(3–5). Herd selection and pathogen-free breeding can beused to eliminate most of the known pig microorganisms.Thus the major safety concern remains the presence ofporcine endogenous retroviruses (PERV).

PERV are type-C gammaretroviruses, related to murine,feline and gibbon ape leukemia viruses and are presentin multiple copies in all pig genomes (6–8,19); PERV RNAhas been detected in almost all tissues of otherwisedesignated pathogen-free pigs (9). Both pig primary cells(7,10–12) and pig cell lines (8,13–15) spontaneously releaseinfectious PERV particles. Freshly isolated pig islets, isletsafter transplantation and mitogen-activated porcine periph-eral blood mononuclear cells also release infectious PERVand cellular activation clearly enhances infectious virion ex-pression (11,16,17). Three replication-competent classesof PERV (PERV-A, -B and -C) have been identified in thegenomic DNA of pigs (18). PERV-A and, to a lesser extent,PERV-B, are amphotropic/polytropic in that they have beenreported to infect human cell lines and primary human cellsin vitro (8,11,12,19). PERV-C acts like an ecotropic virus,only infecting certain pig cell lines (18,20).

No evidence of PERV infection has been found in nonhu-man primates (21) or human patients exposed to porcinetissues including pig islet xenotransplantation, extracorpo-real splenic or kidney perfusion (18,22–25). We have pub-lished that a range of nonhuman primate cell lines have twodifferent blocks to productive infection, one at the level ofviral entry and the other a defect in viral particle assembly

1837

Martina et al.

(26). These results raise the question whether nonhumanprimates are a suitable model for assessing PERV infec-tion risks. In the case of the human clinical studies, mostof the data were obtained retrospectively, and there is lim-ited evidence for prolonged pig cell survival or function inthe majority but not all of those studied. On the other hand,we demonstrated with porcine islet xenografts in immun-odeficient mice that infectious PERV was produced at thetransplant site up to 3 months post-transplant, that manytissue compartments contained pig cells that had migratedfrom the transplant to produce PERV viral RNA locally andthat mouse cells in these chimerized tissues were alsopositive for PERV viral DNA (16). Other studies have alsoconfirmed the limited transfer of PERV genomes in trans-planted mice (16,27,28), but no study demonstrated evi-dence of productive viral infection.

The potential of using immunodeficient mice as a modelfor the risk of PERV infection to human cells in vivo wastested by three different groups. Nude mice were trans-planted with either a PERV-producing pig cell line (PK-15)or pig islets in combination with a human tumor xenograftof 293T cells and demonstrated infection of the human tu-mor cells by PERV (27). Another strain of immunodeficientmice was reconstituted with resting and CD3-activatedhuman peripheral blood lymphocytes, transplanted withPERV-producing pig aortic endothelial cells and PERV in-fection of the human cells was documented (29). Finally,a third strain of immunodeficient mice was injected withboth human and pig peripheral blood mononuclear cells andevidence of PERV infection of the human cells documented(30). The conclusion of all three studies was that PERV in-fection could be transmitted to human cells underlining apotential risk of PERV transmission to immunosuppressedhuman xenotransplant patients. Unfortunately, limited im-portance was given to the presence in mice of xenotropicmurine leukemia viruses (X-MLV) and the potential that vi-ral pseudotyping could confound the interpretations of theresults was not tested.

MLV is divided into receptor classes on the basis of theirtropism and receptor interference: (i) ecotropic virusesinfect only murine cells, (ii) amphotropic and polytropicviruses infect both murine and nonmurine cells and (iii)xenotropic viruses infect nonmurine cells (31,32). It iswell established that MLV can pseudotype different retro-viruses and retroviral-based vectors in murine cellular sys-tems (33–35). In addition, xenotropic MLV (X-MLV) areactivated by immunosuppression and graft versus host dis-ease, presumably as a consequence of cell activation (36–38). Pseudotyping of PERV by X-MLV in vivo was recentlydemonstrated in a complex model of ‘triple chimeric’ im-munodeficient mice transgenic for the production of sev-eral pig cytokines (39). These mice were transplanted withboth human fetal liver and thymic xenografts and also re-constituted with pig bone marrow cells derived from a spe-cial breed of pigs selected because they do not produce ahuman-tropic PERV but produce ecotropic PERV-C. After

25 weeks of human/pig/mouse chimerism human bonemarrow cells were infected in vivo with both PERV-C andX-MLV. Moreover, PERV-C and X-MLV could be transmittedto a human tumor cell line in cocultures with explanted hu-man cells but only X-MLV was carried forward in secondarycultures. The conclusions were that X-MLV could pseudo-type a class of PERV that would normally not infect humancell lines in culture but that this was not efficient based onthe extended time frame required (i.e. 25 weeks) and thefact that only 5 of 20 animals demonstrated pseudotyping.

In the present study, we extend our original studies ofPERV infectious risk in our model of immunodeficient miceusing xenotransplantation of freshly isolated pig islets asa source of both human-tropic PERV-A and -B, as well asecotropic PERV-C. We demonstrate that X-MLV is presentin NOD/SCID mice, the virus is activated by transplanta-tion and within 3 weeks of transplantation can efficientlypseudotype PERV genomes of all three classes in vivointo both transplanted pig islets and human 293T tumorcell xenografts. We conclude that xenotropic MLV can effi-ciently expand the tropism of all the known PERV classes.

Materials and Methods

Animals and cell lines

Mouse Strains: NODlt/szSCID, FVB/Nj and C57/Bl6. All studies were ap-proved by the Scripps Animal Care and Use Committee. Cell lines: HUVEC(ATCC: CRL-1730, human umbilical endothelial vein cells), 293T/17 (ATCC:CRL-11268, fetal human kidney cell line), VERO (ATCC: CCL-81, Africangreen monkey kidney cells), COS (ATCC: CRL-1651, African green monkeykidney cells), HeLa (ATCC: CCL-13, human liver cell line) and NIH3T3 (ATCC:CRL-1658, mouse fibroblast cells). All cells were grown in DMEM exceptfor HUVEC cells grown in Endothelial Growth Media-2 (EGM-2, Cambrex,Baltimore, MD). Pig islets were obtained from a mixed strain comprisingYorkshire, Duroc and Pietran (S&S Farms, Ramona, CA) provided as a giftby MicroIslet, Inc. (San Diego, CA).

Transplantation

NOD/SCID mice were transplanted with Matrigel (BD Biosciences Palo Alto,CA) templates, containing 105 293T cells or 105 HUVEC cells or 103 pig isletsunder the skin of mice. Three weeks post transplant, Matrigel templateswere explanted and digested with collagenase (1.6 mg/mL at 37◦C × 2 h)to release contained cells.

In vitro coculture of porcine and human cells

Coculture experiments were carried out as previously described (16). Af-ter 4 days coculture, 293T-neomycin-resistant cells were selected with0.8 mg/mL of neomycin for up to 3 weeks. Cell and supernatant sampleswere collected at 1, 2 and 3 weeks. Genomic DNA and total RNA wereextracted from cells, while supernatants were spun down (1700 rpm ×5 min) and filtered (0.45 lm filter). Polybrene (BD Biosciences, Palo Alto,CA) was used at 4 lg/mL to infect fresh 293T cells in culture.

DNA/RNA extraction and reverse transcription

DNA was extracted using the DNeasy tissue kit (Qiagen, Valencia, CA) fromcell cultures while Trizol (Invitrogen, Carlsbad, CA) was used to extract DNAand RNA from the tissue samples. Tissues were manually ground withTeflon grinders, extracted with Trizol and total RNA was finished on Qiagen

1838 American Journal of Transplantation 2005; 5: 1837–1847

X-MLV Pseudotyping of PERV

RNeasy columns. DNA was eliminated using in situ DNase I treatment.Equal amounts of total RNA, determined spectrophotometrically by 260/280nm ratios, were reverse transcribed with SuperScript Choice System (Invit-rogen). After reverse transcription, the enzyme was inactivated, sampleswere diluted in water to 100 lL final volume and 10 lL was used for eachwell in the RT-qPCR. Control reactions without reverse transcriptase wereperformed for each sample to assess the complete removal of genomicDNA by the DNAse treatment.

Quantitative PCR and RT-PCR

Quantitative PCR and RT-PCR of PERV envelope sequences used amaster mix containing TaqGold, MgCl2, dNTP and PCR gold buffer.Forward/reverse primers and probes were added at a final concentra-tion of 0.1 lM each. 10 lL of each sample was added to 40 lLof master mix and each sample was run in triplicate. Standardswere serial dilutions of PERV-A (EMBL y12238), -B (EMBL y12239)and -C env (GeneBank AF038600) plasmid DNA. Primer sequenceswere forward PERV-A env 5′-CCTACCAGTTATAATCAATTTAATTATGGC-3′,reverse PERV-A env 5′-TGACAGCTTATTTGCTTATTTCGTAC-3′; forwardPERV-B env 5′-TTCTGTAGGAGATGGAGCTGC-3′, reverse PERV-B env5′-CCGGAATTGACAAAGGAGAATTTT-3′; forward PERV-C env 5′-CTGACCT-GGATTAGAACTGG-3′ and reverse PERV-C env 5′-TTTTCCTTTCTCAGTGA-AACT-3′. Probe sequences were PERV-A 5′-CATGGGAGATGGAAAGATT-GGCAACAGCG-3′; PERV-B 5′-CGATGGAGACTGGAAATGGCCGATCTCTC-3′ and PERV-C 5′-AGCCCCAAGTGCTCTCCTTCAGA-3′. Specific primersand a TaqMan probe for 18S ribosomal RNA were used as con-trols and for normalization (ABI Primer Express, Foster City, CA).QPCR for pig and mouse centromeric DNA was performed usingthe iQ SYBR Green Supermix system (BioRad, Hercules, CA). DNA(5 ng for the pig islets samples or 500 ng for the other cell sam-ples) was amplified with the following: pig centromeric forward 5′-AGCCATGCTGCATGTAATGC-3′ and reverse 5′-GGAGCGTGGCCCAAT-3′;mouse centromeric forward 5′-ATTTAGAAATGTCCACTGTAGGACGT-3′ andreverse 5′-TGTGCATTTCTCATTTTTCACGTTT-3′. PCR was performed in a50 lL reaction volume containing 300 nM of each primer. Cycling param-eters were 2 min/50◦C, 10 min/95◦C and 40 cycles of 15 sec/95◦C and1.5 min/60◦C. To create standards, pig or mouse DNA was serially dilutedassuming that each cell contain ≈7 pg of genomic DNA. A parallel reac-tion for 18S rRNA was done for each to check quality and to normalize.The sensitivity of the assay was defined experimentally as 10 copies per500 000 cells (3.3 lg DNA), which gives >99.99% confidence of detecting≥10 copies.

PCR and RT-PCR for xenotropic MLV envelope

Degenerate primers for xenotropic MLV envelope (X-MLV) were de-signed based on the sequence in PubMed (GI: AAA63287 (40)).Primer sequences: forward 5′- KCTACTGTGSCWMWTGGGGMTG-3′,reverse 5′-CAGGCACAGCCAGCAYTCYTGNGT-3′. Expected size of the am-plicon was 550 bp. Each reaction contained 5 nM of each primer. The cyclingparameters were 35 cycles 94◦C × 10 s, 55◦C × 30 s, 72◦C × 30 s andone cycle at 72◦C × 10 minutes. PCR products were detected by ethidiumbromide staining. Negative and positive extraction controls were includedin each experiment.

Retrovirus infection assays

Fresh target cells were plated 24 h before infection, grown overnight toapproximately 50% confluency and infected with filtered, cell-free super-natants in the presence of polybrene at 4 lg/mL. The media was changedafter 4 h incubation. Cells were passed subconfluently at least twice aweek and at the designated time points cells were harvested, washed inphosphate-buffered saline (PBS) and frozen in liquid nitrogen prior to extrac-tion of DNA and RNA.

Results

PERV transmission in vitro from pig islets to

293T cells

To characterize the infective properties of the three classesof PERV produced by pig islets, we analyzed the resultsof coculturing freshly harvested pig islets with the hu-man embryonic kidney cell line, 293T, stably transfectedwith a neomycin resistance gene (293TNeo). After 4 daysof coculture the pig islets were removed and remaining293TNeo were placed in neomycin-containing culture me-dia. Weekly for 3 weeks, cells were harvested and an-alyzed by quantitative TaqMan PCR and RT-PCR for thepresence of all three classes of PERV envelopes: PERV-A, -B and -C. As shown in Figure 1A, PERV-A and -Benv sequences are detected at the 3 week time point inboth DNA and RNA. We were not able to detect PERV-C(data not shown). These in vitro PERV-infected cells are re-ferred to in subsequent experimentals as 293TNeoPERV.We confirmed that neomycin selection killed all the origi-nal pig cells in coculture by a negative result with quanti-tative PCR for centromeric pig DNA (data not shown). Totest if 293TNeoPERV cells were productively infected, cell-free supernatants were harvested at weekly intervals for3 weeks and used to infect fresh human 293T. Only PERV-Awas detected (Figure 1B), no PERV-B or PERV-C. In con-clusion, freshly isolated pig islets release both PERV-A and-B that infect 293T in vitro, though a sustained produc-tive infection can only be documented for PERV-A in thissystem.

PERV transmission in vivo from transplanted pig

islets to human cells

To test if pig islets transmit PERV in our in vivo xenotrans-plantation model, we transplanted severely immunodefi-cient NOD/SCID mice with freshly isolated pig islets, 293Tcells and the primary human endothelial cell line, HUVEC,in three separate locations in the same animal (Figure 2, de-sign 1). Negative controls were NOD/SCIDs transplantedwith 293T and HUVECs but not pig islets. Cells were trans-planted in Matrigel templates in physically separate subcu-taneous spaces in the perispinous region on opposite sidesof the spine. After 4 weeks templates were explanted,digested enzymatically and DNA/RNA was extracted. Ap-proximately equivalent copy numbers of PERV-A, -B and -Cwere detected by qPCR on genomic DNA and total RNAin both HUVEC and 293T from the animals cotransplantedwith pig islets (Table 1). It is important to note that ex-planted human cell preparations were consistently nega-tive by qPCR for pig centromeric DNA sequences indicat-ing no contamination of these results by microchimerismof pig cells. Thus, transplantation of pig islets in vivo leadsto transmission of PERV-A, -B and -C to human cells.

To test the potential that xenotropic murine leukemia virus(X-MLV) might pseudotype PERV in this model, we de-signed degenerate PCR primers for X-MLV env sequences.

American Journal of Transplantation 2005; 5: 1837–1847 1839

Martina et al.

Figure 1: (A) PERV transmission from pig islets to 293T cells.

Pig islets and 293-Neo cells were cocultured for 4 days; afterthis time 293-Neo cells were selected in neomycin and analyzedafter 1, 2 and 3 weeks in culture by qPCR and RT-qPCR withPERV-specific envelope primers (PERV-A, -B or -C). The results areshown as PERV env copy number per 0.5 lg of DNA or total RNA(black bars PERV-A DNA; white bars PERV-B DNA; striped barsPERV-A RNA; dotted bars PERV-B RNA). The results are represen-tative of three experiments and standard deviations are shown.(B) Productive PERV-A infection in fresh 293T cells. Cell-free su-pernatants from cocultured pig islets and 293T-Neo cells were col-lected after 1, 2 or 3 weeks in culture. These supernatants werethen used to infect fresh 293T human cells that were analyzed forPERV-A infection 3 days later. Data are shown as PERV-A env copynumber per 0.5 lg of genomic DNA with standard deviations.

As shown in Table 1, all cell preparations transplanted in theNOD/SCID mice (human and pig) were positive for X-MLVin both DNA and RNA. As controls, the untransplanted hu-man cells (data not shown) and freshly isolated pig isletswere consistently negative by PCR for X-MLV. The difficultyin interpreting these positive results for X-MLV infection invivo is the likelihood that the explanted templates also con-tain mouse cells. Indeed, by qPCR using specific primersfor mouse centromeric DNA, we estimated that 40–60%of the cells in our samples were host-derived mouse cells

(data not shown). To determine whether pig islets are pro-ductively infected by X-MLV after transplantation and thatX-MLV is able to pseudotype the PERV produced by pigcells, we next did an experiment with pig islets that werestably transfected with a Zeocin antibiotic resistance gene(Figure 2, design 2).

X-MLV infection of transplanted pig islets and

pseudotyping of naturally produced PERV

Freshly harvested pig islets transfected with the Zeocin re-sistance gene (PigZeo) were transplanted into a NOD/SCIDand explanted after 3 weeks. Cells explanted with the tem-plate, including the PigZeo islet cells, were selected withhigh-dose Zeocin for 4 days. High dose was defined as 3-fold greater than the dose required to kill 100% of NIH 3T3cells in culture (data not shown). Genomic DNA and totalRNA were extracted for analysis. The cell-free supernatantwas used to infect fresh 293T cells and to extract viral RNA.

Both genomic DNA and total RNA were positive for PERV-A, -B, -C and X-MLV envelope sequences. RT-qPCR and RT-PCR of the culture supernatants confirmed the presence ofPERV and X-MLV released into the media by PigZeo isletcells. To confirm the success of the Zeocin selection forpig islet cells, the genomic DNA was tested for mousecentromeric sequences by qPCR. PigZeo DNA was nega-tive for the presence of mouse centromeric DNA in threeexperiments with triplicate wells for each (Table 2).

Fresh human 293T were exposed to the cell-free super-natants from the PigZeo-X-MLV-infected islet cells and cul-tured for 7 days. Genomic DNA analysis by qPCR revealedall three classes of PERV as well as X-MLV (Table 2). Theseresults indicate that X-MLV is able to efficiently pseudotypeand mediate infection by all three PERV classes.

In vivo infection of a transplanted PERV-infected

human cell line by X-MLV demonstrates the impact of

xenotropic viral pseudotyping on changing the

natural tropism of PERV

In the previous experiments, the source of pseudotypedPERV are X-MLV-infected pig islet cells. To expand our stud-ies, we tested the effects of X-MLV infection on pseudotyp-ing PERV produced by a human cell line infected with PERV-A. Infection was tested against both permissive human celllines and nonpermissive, nonhuman primate cells.

As already described above, 293TNeo cells were co-cultured in vitro with pig islets, then selected for 3 weeks inneomycin and found to be positive for PERV-A and PERV-B(Figure 1). These cells, 293TNeoPERV, were capable ofproducing infectious particles of PERV-A but did not pro-duce infectious PERV-B virions. 293TNeoPERV cells werethen transplanted into NOD/SCID mice (Figure 2, Design 3).After 3 weeks the cells were explanted and cultivated for3 weeks with neomycin selection to eliminate all mousecells. qPCR and RT-qPCR analysis revealed that both DNA

1840 American Journal of Transplantation 2005; 5: 1837–1847

X-MLV Pseudotyping of PERV

Figure 2: Flow chart illustrating the experimental designs used to demonstrate X-MLV pseudotyping of PERV viruses. Differenttypes of cells were transplanted (step 1) in NOD/SCID mice in different locations subcutaneously. After 3 weeks, cells were explantedand selected in appropriate media (step 2). Finally in step 3 genomic DNA and total RNA from explanted cells were analyzed by qPCRand RT-qPCR. Where applicable cell-free supernatants were used to infect fresh 293T cells and (for experimental design 3) VERO, COS,HeLa and NIH3T3 cells.

and RNA were positive for PERV-A, -B and X-MLV envelopesequences.

The potential that these cells were productively infectedwas assayed by exposing fresh 293T to cell-free super-natants. PERV-A and X-MLV but not PERV-B were detected(data not shown). The RT-qPCR results of the supernatantsalso revealed only PERV-A and X-MLV (Table 3). We namedthis new 293TNeo cell line, PERV (X-MLV).

To determine whether X-MLV can pseudotype and expandthe tropism of PERV-A, cell-free supernatants from thePERV (X-MLV) line were used to infect a set of cell lineschosen for different permissivity for PERV and X-MLV infec-tion. The PERV nonpermissive African green monkey lines,VERO and COS (26), were infected with supernatants fromthe PERV (X-MLV) as well as a high-titer PERV-A producing293T cell line (14-220) and the 293TNeoPERV cells. DNAwas analyzed by qPCR.

As expected, 14-220 and 293TNeoPERV cell supernatantsdid not transmit PERV-A (Figure 3A) to VERO or COS. Incontrast, PERV (X-MLV) supernatants transmitted PERV-A (Figure 3A) as demonstrated at the 3 day, 1 week and2 week time points. It is also clear that PERV-A copy num-bers increase as a function of time in culture. These resultsare consistent with the conclusion that X-MLV is produc-tively pseudotyping PERV-A.

To confirm that X-MLV was passed by cell-free super-natants from the PERV (X-MLV) to VERO and COS cells,we tested genomic DNA 3 weeks post-infection by PCR

for X-MLV envelope sequences. The cells exposed to thePERV (X-MLV) supernatants were positive for X-MLV, whilethose exposed to the 14-220 and 293TNeoPERV super-natants were negative (Figure 3B).

As positive controls, we infected the highly PERV-permissive human 293T and HeLa cell lines with thesame three supernatants. All the combinations were pos-itive for PERV-A and X-MLV was transmitted by the PERV(X-MLV) cell supernatant (data not shown). NIH3T3 thatare not permissive for PERV or X-MLV, were used as nega-tive controls. All combinations with NIH3T3 were negativefor PERV-A and X-MLV envelope sequences after infection(data not shown).

Sequence analysis of the X-MLV envelopes and virus

activation

Are the X-MLV classes present in the NOD/SCID miceresponsible for the infections of the PigZeo islets andPERV (X-MLV) cells and for the subsequent pseudotyp-ing of PERV? We sequenced the PCR products of theX-MLV envelope covered by our primer set that ampli-fies the N-terminal region from aa. 126 to aa. 314. Thiswas done with genomic DNA from NOD/SCIDs, PigZeoislet cells, PERV (X-MLV), NIH3T3 and two other mousestrains, C57/Bl6 and FVB/Nj. Alignments of a referencexenotropic MLV envelope sequence from a transgenicstrain of NOD/SCID mice (Gene Bank Accession number:AAR12224) with X-MLV sequences from our NOD/SCIDmice, the PigZeo islets and the PERV (X-MLV) cells show100% homology (Figure 4). These results confirm that theX-MLV virus present in the NOD/SCID mice was the virus

American Journal of Transplantation 2005; 5: 1837–1847 1841

Martina et al.

Ta

ble

1:

Ana

lysi

sof

PE

RV

tran

smis

sion

invi

vofr

ompi

gis

lets

tohu

man

cells

DN

AR

NA

Sam

plea

X-M

LVP

ER

VA

bP

ER

VB

bP

ER

VC

bP

ig-c

ent

X-M

LVP

ER

VA

bP

ER

VB

bP

ER

VC

b

Pig

isle

tsN

EG

c1.

5±

0.3

×10

52.

7±

0.3

×10

52.

3±

0.7

×10

58.

7±

0.1

×10

2dN

EG

2.1

±0.

6×

103

1.7

±0.

4×

103

1.3

±0.

1×

103

293T

(−)e

+N

Df

ND

ND

ND

g+

ND

ND

ND

HU

VE

C(−

)+

ND

ND

ND

ND

g+

ND

ND

ND

293T

(+)h

+93

.6±

6.4

27.8

5±

1151

.7±

4N

Dg

+12

.55

±1.

89

±1

10.7

±0.

8H

UV

EC

(+)

+27

.7±

6.1

12.8

±0.

411

.1±

0.6

ND

g+

4.07

±0.

92.

23±

0.1

3.18

±0.

1P

igis

lets

(TX

)i+

4.5

±0.

5×

104

9.3

±0.

8×

104

7.5

±0.

5×

104

Not

avai

labl

e+

6.9

±0.

3×

102

6.8

±0.

5×

102

4.7

±0.

2×

102

a Dat

are

pres

ent

four

anim

als

per

cond

ition

intw

odi

ffer

ent

expe

rimen

ts.

bD

ata

expr

esse

das

PE

RV

env

copy

num

ber

per

0.5

lg

ofnu

clei

cac

id(D

NA

orR

NA

);st

anda

rdde

viat

ions

are

show

n.c N

EG

=no

ampl

icon

sde

tect

edby

PC

Ron

ethi

dium

-sta

ined

gels

.dD

ata

expr

esse

das

estim

ated

num

ber

ofce

llson

5ng

ofge

nom

icD

NA

(see

Mat

eria

lsan

dM

etho

dsse

ctio

nfo

rde

tails

)e(−

)rep

rese

nts

anim

als

tran

spla

nted

with

293T

and

HU

VE

Cbu

tno

pig

isle

ts.

f ND

=no

tde

tect

edas

defin

edby

the

Bio

Rad

iQC

ycle

rso

ftw

are.

gD

ata

expr

esse

das

estim

ated

num

ber

ofce

llson

500

ngof

geno

mic

DN

A;s

ensi

tivity

ofth

eas

say

is2

copi

esin

500

ngD

NA

(18S

rRN

Ase

quen

ces

wer

eus

edas

PC

Rco

ntro

l;se

eM

ater

ials

and

Met

hods

sect

ion

for

deta

ils).

h(+

)rep

rese

nts

anim

als

tran

spla

nted

with

pig

isle

ts,2

93T

and

HU

VE

C.

i TX

=tr

ansp

lant

edpi

gis

lets

.

Ta

ble

2:

Ana

lysi

sof

293T

cells

expo

sed

toP

igZe

oce

ll-fr

eesu

pern

atan

ts

DN

AR

NA

Sam

ple

X-M

LVP

ER

VA

aP

ER

VB

aP

ER

VC

aM

ur-c

ent

X-M

LVP

ER

VA

aP

ER

VB

aP

ER

VC

a

293T

NE

Gb

ND

cN

DN

DN

otdo

neN

EG

ND

ND

ND

Pig

Zeo

+6.

3±

0.5

×10

43.

1±

1.5

×10

44.

8±

0.3

×10

4N

Dd

+1.

6±

0.2

×10

32.

4±

0.3

×10

32.

9±

0.1

×10

3

293T

+P

igZe

o+

4.5

±0.

2×

102

1.3

±0.

2×

102

1.3

±0.

1×

102

Not

done

a Dat

aex

pres

sed

asP

ER

Ven

vco

pynu

mbe

rpe

r0.

5lg

ofnu

clei

cac

id(D

NA

orR

NA

);st

anda

rdde

viat

ions

are

show

n.bN

EG

=no

ampl

icon

sde

tect

edby

PC

Ron

ethi

dium

-sta

ined

gels

.c N

D=

not

dete

cted

asde

fined

byth

eB

ioR

adiQ

Cyc

ler

soft

war

e.dD

ata

expr

esse

das

estim

ated

num

ber

ofm

urin

ece

llson

500

ngge

nom

icD

NA

(pig

cent

rom

eric

and

18S

rRN

Ase

quen

ces

wer

eus

edas

PC

Rco

ntro

l;se

eM

ater

ials

and

Met

hods

sect

ion)

.

1842 American Journal of Transplantation 2005; 5: 1837–1847

X-MLV Pseudotyping of PERV

Table 3: Analysis of 293TNeoPERV and 293TNeoPERVX-MLV cells and supernatants

DNA RNA

Sample X-MLV PERV Aa PERV Ba PERV Ca X-MLV PERV Aa PERV Ba PERV Ca

14-220 NEGb 2.5 ± 0.2 × 106 NDc ND NEG 8.2 ± 0.3 × 104 ND ND293TNeoPERV NEG 3.9 ± 0.1 × 104 8.3 ± 0.1 × 102 ND NEG 1.5 ± 0.1 × 104 1.1 ± 0.3 × 102 NDPERV (X-MLV) + 3.8 ± 0.1 × 104 1 ± 0.1 × 102 ND + 1.1 ± 0.9 × 104 1.9 ± 0.4 × 102 NDSupernatantd Viral RNAe

293TNeoPERV NEG 2 ± 0.2 × 103 ND NDPERV (X-MLV) + 2.2 ± 0.6 × 103 ND NDaData expressed as PERV env copies per 0.5 lg of nucleic acid (DNA and total RNA); standard deviations are shown.bNEG = no evidence of amplicon by PCR.cND = not detected as defined by the BioRad iQCycler software.dCell-free supernatant.eData expressed as PERV env copies per mL of cell-free supernatant (viral RNA extracted using Qiagen Viral Kit).

Figure 3: (A) Infection of VERO (black bars) and COS (white bars) monkey cells with cell-free supernatants from a high-titer

PERV-A producing cell line (14-220), 293NeoPERV cells and PERV (X-MLV) cells. Values are expressed as number of PERV-A envcopies per 0.5 lg of genomic DNA. Cells were analyzed at three different time points to monitor for productive infection (3 days,1 week and 2 weeks post-infection). (B) PCR analysis of VERO and COS cells for the presence of X-MLV env after infection with cell-freesupernatants from 14-220, 293TNeoPERV or PERV (X-MLV) cells. The results showing infection by PERV (X-MLV) of the nonpermissivenonhuman primate cells demonstrate the expansion of PERV tropism by X-MLV pseudotyping.

responsible for the infection of human and pig cells in vivoafter transplantation.

Finally, X-MLV RNA transcripts were not detected by RT-PCR in normal skin, spleen and liver samples taken frommultiple mice of the NOD/SCID, FVB/Nj and C57/Bl6 strains(Table 4). Therefore, we investigated the activation ofX-MLV transcription in our xenotransplantation model withthe hypothesis that surgical tissue injury induced X-MLV.Three days after a full thickness skin incision in one flank,the mice were sacrificed and total RNA was extracted from

the surgical site as well as the unmanipulated skin of theopposite flank. All the unmanipulated skin tissues werenegative by RT-PCR for X-MLV env , while the surgical sitetissues were positive (Table 4). These results suggest thatthe surgical incision was sufficient to transcriptionally ac-tivate the X-MLV genomes in both immunodeficient andimmunocompetent mice. We also analyzed the activationof X-MLV in spleen cells in culture by the cellular mitogen,PHA. The PHA-activated splenocytes and unactivated con-trols were harvested after 72 h, total RNA was extractedand RT-PCR for X-MLV performed. As shown in Table 4,

American Journal of Transplantation 2005; 5: 1837–1847 1843

Martina et al.

Figure 3: Continued.

Figure 4: Alignment of

X-MLV env sequences

obtained from different

mouse strains or cell

samples. PCR amplifica-tion was done using degen-erate primers for X-MLVenv sequences. Primer de-sign was based on thexenotropic MLV envelopesequence contained in thePubMed database from aa.129 to 314 N-terminal re-gion. Highlighted boxes in-dicate the regions highlyconserved among the dif-ferent aligned sequences.

X-MLV was activated in the PHA-treated splenocytes fromall of the three mouse strains tested.

Discussion

We have studied an in vivo mouse model of pig islet xeno-transplantation and demonstrated PERV transmission tomouse tissues (16). It is important to emphasize that trans-mission of PERV to mouse cells in the same mouse can-not involve pseudotyping by the X-MLV of the host mouse.

Therefore, the issue of pseudotyping by X-MLV in immun-odeficient mouse models such as ours is only extant whenhuman cells are also cotransplanted with PERV-producingpig cells. In this context, the published in vivo models(27,29,30,39) for pig to human transmission of PERV arebased on creating hematopoietic chimerism in the immun-odeficient animals or by intraperitoneal injection of eitherpig peripheral blood cells or endothelial cells. In contrast,we have focused on vascularized pig islet xenotransplantsas the source of PERV and in the present study takenadvantage of a template transplantation technology to

1844 American Journal of Transplantation 2005; 5: 1837–1847

X-MLV Pseudotyping of PERV

Table 4: Analysis of X-MLV activation in three different mousestrains

X-MLV

Tissue Mouse strain DNA RNA

Skin UnmanipulatedC57/Bl6 + −FVB + −NOD/SCID + −

IncisedC57/Bl6 + +FVB + +NOD/SCID + +

Spleen No PHA activationC57/Bl6 + −FVB + −NOD/SCID + −

PHA activationC57/Bl6 + +FVB + +NOD/SCID + +

implant human and pig cells in physically separate loca-tions. However, in common with all these experimentalmodels for clinical xenotransplants is the potential thatmurine endogenous retroviruses, specifically xenotropicMLV, could pseudotype and transmit PERV to human cellsin a fashion that does not correctly reflect the risk of PERVtransmission in a human patient. Indeed, a recent publica-tion from Yang et al. (39) demonstrated that the nonhuman-tropic class of PERV, PERV-C, can be pseudotyped by X-MLV and infect human cells in bone marrow hematopoieticchimeras created in immunodeficient mice transgenic fora number of pig growth factors. The obvious limitation ofthe Yang study is that long-term hematopoietic chimerismmay not represent the same set of risk factors as a vascu-larized pig islet xenograft in a single site. Moreover, the useof PERV C that is not human tropic, is an elegant means oftesting for pseudotyping but does not model the situationwhere infectious virions of human-tropic PERV classes, Aand B, will be present.

X-MLV viruses have been characterized previously to infecta variety of species ranging from human to nonhuman pri-mates and porcine cells (41). X-MLV are also well knownto pseudotype other murine endogenous retroviruses (42)as well as viruses such as HIV-1 (35) or Avian Leukosis andsarcoma viruses (43). In the present study, we demon-strate in vivo infection of human cell lines and pig isletcells by X-MLV after transplantation into immunodeficientNOD/SCID mice. Yang and coworkers made a similar ob-servation. However, they suggested that human cells werenot able to efficiently recover X-MLV in subsequent cul-tures and that the presence of pig cells was mandatory forefficient recovery in this setting. In contrast, we demon-strate that X-MLV can be efficiently recovered from thehuman cell lines, HUVEC and 293T in the absence of pigcells. In the case of the explanted 293TNeo cultures, wealso recovered X-MLV after antibiotic selection in the ab-

sence of any mouse or pig cells and transferred X-MLV withinfectious supernatants to a series of both human and non-human primate cell lines.

PERV pseudotyping by X-MLV was demonstrated in twodifferent ways. First, we used 293T cells to analyze thetropism of the three PERV classes released from pigislets explanted from immunodeficient mice. Second, wedemonstrate transmission of PERV-A produced by a 293Thuman cell line infected with both PERV-A and X-MLV toAfrican green monkey VERO and COS cells that are notpermissive for PERV (26). The pig islet cells infected withX-MLV are transmitting not only PERV-A but also PERV-B and the nonxenotropic PERV-C in vivo (Figure 2,design 2; Table 2). Similarly, 293T cells infected withboth PERV-A and X-MLV transmit PERV-A to the X-MLV-permissive/PERV-A nonpermissive cell lines, VERO andCOS. Thus, the results of both sets of experiments areconsistent with the conclusion that X-MLV acquired in vivoafter xenotransplantation can efficiently pseudotype PERV.

Evidence suggests that some infectious classes of PERVare actually A/C envelope recombinants (44). We have datawith RT-PCR that pig islets in culture and after transplan-tation in the NOD/SCID mice also produce PERV A/C virusand can infect 293T cells. As previously documented forother tissues, we found that freshly harvested pig isletDNA was negative for A/C sequences. Interestingly, theDNA of pig islets after transplantation in the NOD/SCIDmice is positive for A/C recombinants as well as X-MLVconsistent with the conclusion that PERV A/C is pseudo-typed by X-MLV in vivo and that in turn infects the pig isletcells. These results indicate that transplanted pig cells arealso subject to the impact of pseudotyping on changingPERV tropism.

It is interesting that mRNA transcripts for X-MLV werenot detectable in unmanipulated mouse tissues despitethe evidence that X-MLV was consistently transmitted tothe xenografts. It is well established that transcription ofmurine gammaretroviruses is activated by immunologi-cal events such as graft versus host disease and mixedlymphocyte cultures (36–38,45). In the present study, wedemonstrate that surgical tissue trauma created during im-plantation of the Matrigel templates is sufficient to acti-vate X-MLV transcription. This is consistent with our previ-ous observation that PERV transcription is enhanced in pigislets following xenotransplantation (16). Clearly, whateverproinflammatory milieu is created in a surgical site will alsoimpact on the activation state of the endogenous retroviruswithin the pig cells in addition to the endogenous retroviruswithin the murine cells.

Our results support the primary conclusion that immun-odeficient NOD/SCID mice are not a suitable model toevaluate the potential risk of PERV transmission from pigcells and tissues to human cells. In fact, the presenceand activation of X-MLV mediates an effective expansion

American Journal of Transplantation 2005; 5: 1837–1847 1845

Martina et al.

of PERV tropism, due to X-MLV pseudotyping, producingfalse-positive results for PERV infection. Thus, it is rea-sonable to consider the possibility that pseudotyping byhuman endogenous or exogenous retrovirus, with a con-sequent expansion of PERV tropism, could be observedin xenotransplanted human patients. For example, MuLVretroviral particles can be pseudotyped by the incorporationof full length, naturally occurring simian immunodeficiencyvirus env proteins (46). On the other hand, only a truncatedversion of HIV env is capable of pseudotyping MuLV viri-ons, though the potential of this occurring in a natural stateis unknown (47). There is only one study suggesting thatPERV virions do not efficiently package human endoge-nous retrovirus sequences (48). Thus, there is a possibil-ity that PERV could be pseudotyped in human patients bycomplementation with human endogenous retrovirus en-velope sequences encoded by human endogenous retro-virus (HERV-W) (49,50), HERV-E (51) or HERV-K (52) andexpressed as functional proteins in different human tis-sues. Whether the same could occur by exogenous retro-viruses such as HIV and HTLV is still an open question. Thefact that these latter viruses are not gammaretroviruseswould suggest that this may not be an efficient processbut in a patient with a stable, PERV-producing xenograft,the question would still be whether even an inefficient pro-cess could have biological and possibly pathological conse-quences for the host.

Acknowledgments

We greatly appreciate the technical advice provided by Dr. Clive Patiencespecifically with respect to primer sequences for X-MLV envelope and pro-tocols kindly shared by Winston Colon-Moran. The work was funded byNIH grant R01 AI52349-03 (DRS and CW) and DK07022-25 (YM) and bythe Molly Baber Research Fund. This study is supported by NIH grant R01AI52349-03 (DRS and CW), DK07022-25 (YM), Molly Baber Research Fund.

References

1. Sachs DH, Sykes M, Robson SC, Cooper DK. Xenotransplanta-tion. Adv Immunol 2001; 79: 129–223.

2. Allan JS. The risk of using baboons as transplant donors. Exoge-nous and endogenous viruses. Ann N Y Acad Sci 1998; 862: 87–99.

3. Chapman LE, Folks TM, Salomon DR, Patterson AP, EggermanTE, Noguchi PD. Xenotransplantation and xenogeneic infections.N Engl J Med 1995; 333: 1498–1501.

4. Bach FH, Fishman JA, Daniels N et al. Uncertainty in xenotrans-plantation: individual benefit versus collective risk. Nat Med 1998;4: 141–144.

5. Fishman JA. Infection and xenotransplantation. Developingstrategies to minimize risk. Ann N Y Acad Sci 1998; 862: 52–66.

6. Tristem M, Kabat P, Lieberman L, Linde S, Karpas A, Hill F. Char-acterization of a novel murine leukemia virus-related subgroupwithin mammals. J Virol 1996; 70: 8241–8246.

7. Akiyoshi DE, Denaro M, Zhu H, Greenstein JL, Banerjee P, Fish-man JA. Identification of a full-length cDNA for an endogenousretrovirus of miniature swine. J Virol 1998; 72: 4503–4507.

8. Patience C, Takeuchi Y, Weiss RA. Infection of human cells by anendogenous retrovirus of pigs. Nat Med 1997; 3: 282–286.

9. Clemenceau B, Lalain S, Martignat L, Sai P. Porcine endogenousretroviral mRNAs in pancreas and a panel of tissues from specificpathogen-free pigs. Diabetes Metab 1999; 25: 518–525.

10. McIntyre MC, Kannan B, Solano-Aguilar GI, Wilson CA, BloomET. Detection of porcine endogenous retrovirus in cultures offreshly isolated porcine bone marrow cells.[erratum appears inXenotransplantation. 2003 Sep;10(5):478 Note: McIntyre MatizaC [corrected to MyIntyre Maritza C]]. Xenotransplantation 2003;10: 337–342.

11. Wilson CA, Wong S, Muller J, Davidson CE, Rose TM, Burd P.Type C retrovirus released from porcine primary peripheral bloodmononuclear cells infects human cells. J Virol 1998; 72: 3082–3087.

12. Martin U, Kiessig V, Blusch JH et al. Expression of pig endoge-nous retrovirus by primary porcine endothelial cells and infectionof human cells. Lancet 1998; 352: 692–694.

13. Todaro GJ, Benveniste RE, Lieber MM, Sherr CJ. Characteriza-tion of a type C virus released from the porcine cell line PK(15).Virology 1974; 58: 65–74.

14. Moennig V, Frank H, Hunsmann G, Ohms P, Schwarz H, SchaferW. C-type particles produced by a permanent cell line from aleukemic pig. II. Physical, chemical, and serological characteriza-tion of the particles. Virology 1974; 57: 179–188.

15. Armstrong JA, Porterfield JS, De Madrid AT. C-type virus particlesin pig kidney cell lines. J Gen Virol 1971; 10: 195–198.

16. van der Laan LJ, Lockey C, Griffeth BC et al. Infection by porcineendogenous retrovirus after islet xenotransplantation in SCIDmice. Nature 2000; 407: 90–94.

17. Tacke SJ, Specke V, Denner J. Differences in release and determi-nation of subtype of porcine endogenous retroviruses producedby stimulated normal pig blood cells. Intervirology 2003; 46: 17–24.

18. Takeuchi Y, Patience C, Magre S et al. Host range and interfer-ence studies of three classes of pig endogenous retrovirus. J Virol1998; 72: 9986–9991.

19. Le Tissier P, Stoye JP, Takeuchi Y, Patience C, Weiss RA. Twosets of human-tropic pig retrovirus. Nature 1997; 389: 681–682.

20. Wilson CA, Wong S, VanBrocklin M, Federspiel MJ. Extendedanalysis of the in vitro tropism of porcine endogenous retrovirus.J Virol 2000; 74: 49–56.

21. Specke V, Schuurman HJ, Plesker R et al. Virus safety in xeno-transplantation: first exploratory in vivo studies in small laboratoryanimals and non-human primates. Transpl Immunol 2002; 9: 281–288.

22. Paradis K, Langford G, Long Z et al. Search for cross-speciestransmission of porcine endogenous retrovirus in patients treatedwith living pig tissue. The XEN 111 Study Group. Science 1999;285: 1236–1241.

23. Levy MF, Crippin J, Sutton S et al. Liver allotransplantation afterextracorporeal hepatic support with transgenic (hCD55/hCD59)porcine livers: clinical results and lack of pig-to-human transmis-sion of the porcine endogenous retrovirus. Transplantation 2000;69: 272–280.

24. Pitkin Z, Mullon C. Evidence of absence of porcine endogenousretrovirus (PERV) infection in patients treated with a bioartificialliver support system. Artif Organs 1999; 23: 829–833.

25. Dinsmore JH, Manhart C, Raineri R, Jacoby DB, Moore A. Noevidence for infection of human cells with porcine endogenousretrovirus (PERV) after exposure to porcine fetal neuronal cells.Transplantation 2000; 70: 1382–1389.

26. Ritzhaupt A, Van Der Laan LJ, Salomon DR, Wilson CA. Porcineendogenous retrovirus infects but does not replicate in nonhu-man primate primary cells and cell lines. J Virol 2002; 76: 11312–11320.

1846 American Journal of Transplantation 2005; 5: 1837–1847

X-MLV Pseudotyping of PERV

27. Clemenceau B, Jegou D, Martignat L, Sai P. Microchimerism andtransmission of porcine endogenous retrovirus from a pig cell lineor specific pathogen-free pig islets to mouse tissues and humancells during xenografts in nude mice. Diabetologia 2002; 45: 914–923.

28. Deng YM, Tuch BE, Rawlinson WD. Transmission of porcineendogenous retroviruses in severe combined immunodeficientmice xenotransplanted with fetal porcine pancreatic cells. Trans-plantation 2000; 70: 1010–1016.

29. McKane BW, Ramachandran S, Yang J, Xu XC, MohanakumarT. Xenoreactive anti-Galalpha(1,3)Gal antibodies prevent porcineendogenous retrovirus infection of human in vivo. Hum Immunol2003; 64: 708–717.

30. Kuddus RH, Metes DM, Nalesnik MA, Logar AJ, Rao AS,Fung JJ. Porcine cell microchimerism but lack of produc-tive porcine endogenous retrovirus (PERV) infection in naiveand humanized SCID-beige mice treated with porcine periph-eral blood mononuclear cells. Transpl Immunol 2004; 13: 15–24.

31. Weiss RA. Cellular Receptors and Viral Glycoproteins Involvedin Retrovirus Entry. New York: Plenum Press New York,1993.

32. Miller AD. Cell-surface receptors for retroviruses and implicationsfor gene transfer. Proc Natl Acad Sci U S A 1996; 93: 11407–11413.

33. Patience C, Takeuchi Y, Cosset FL, Weiss RA. Packaging ofendogenous retroviral sequences in retroviral vectors producedby murine and human packaging cells. J Virol 1998; 72: 2671–2676.

34. Quinonez R, Sutton RE. Lentiviral vectors for gene delivery intocells. DNA Cell Biol 2002; 21: 937–951.

35. Lusso P, di Marzo Veronese F, Ensoli B et al. Expanded HIV-1cellular tropism by phenotypic mixing with murine endogenousretroviruses. Science 1990; 247: 848–852.

36. Todaro GJ, Arnstein P, Parks WP, Lennette EH, Huebner RJ. Atype-C virus in human rhabdomyosarcoma cells after inoculationinto NIH Swiss mice treated with antithymocyte serum. Proc NatlAcad Sci U S A 1973; 70: 859–862.

37. Suzuki T, Yanagihara K, Yoshida K, Seido T, Kuga N. Infectiousmurine type-C viruses released from human cancer cells trans-planted into nude mice. Gann 1977; 68: 99–106.

38. Hirsch MS, Phillips SM, Solnik C, Black PH, Schwartz RS, Car-penter CB. Activation of leukemia viruses by graft-versus-hostand mixed lymphocyte reactions in vitro. Proc Natl Acad Sci U SA 1972; 69: 1069–1072.

39. Yang YG, Wood JC, Lan P et al. Mouse retrovirus mediatesporcine endogenous retrovirus transmission into human cells inlong-term human-porcine chimeric mice. J Clin Invest 2004; 114:695–700.

40. Massey AC, Coppola MA, Thomas CY. Origin of pathogenic de-terminants of recombinant murine leukemia viruses: analysis ofBxv-1-related xenotropic viruses from CWD mice. J Virol 1990;64: 5491–5499.

41. Gilbert MA, Charreau B, Vicart P, Paulin D, Nandi PK. Mechanismof entry of a xenotropic MMuLV-derived recombinant retrovirusinto porcine cells using the expression of the reporter nlslacZgene. Arch Virol 1992; 124: 57–67.

42. Besmer P, Baltimore D. Mechanism of restriction of ecotropicand xenotropic murine leukemia viruses and formation of pseu-dotypes between the two viruses. J Virol 1977; 21: 965–973.

43. Levy JA. Murine xenotropic type C viruses. III. Phenotypic mixingwith avian leukosis and sarcoma viruses. Virology 1977; 77: 811–825.

44. Oldmixon BA, Wood JC, Ericsson TA et al. Porcine endogenousretrovirus transmission characteristics of an inbred herd of minia-ture swine. J Virol 2002; 76: 3045–3048.

45. Levy JA, Datta SK, Schwartz RS. Recovery of xenotropic virus butnot ecotropic virus during graft-versus-host reaction in mice. ClinImmunol Immunopathol 1977; 7: 262–268.

46. Indraccolo S, Minuzzo S, Feroli F, Mammano F, Calderazzo F,Chieco-Bianchi L et al. Pseudotyping of Moloney leukemia virus-based retroviral vectors with simian immunodeficiency virus en-velope leads to targeted infection of human CD4+ lymphoid cells.Gene Ther 1998; 5: 209–217.

47. Lee MK, Seo JK, Kim HK, Cho JH, Poo H, Kim KL. A vector sys-tem for introducing foreign HIV-1 env genes and pseudotyping ofMuLV particles with the recombinant HIV-1 envelope proteins foranti-HIV-1 assay. Antiviral Res 2002; 53: 99–111.

48. Suling K, Quinn G, Wood J, Patience C. Packaging of human en-dogenous retrovirus sequences is undetectable in porcine en-dogenous retrovirus particles produced from human cells. Virol-ogy 2003; 312: 330–336.

49. Perron H, Lazarini F, Ruprecht K et al. Human endogenous retro-virus (HERV)-W ENV and GAG proteins: physiological expressionin human brain and pathophysiological modulation in multiple scle-rosis lesions. J Neurovirol 2005; 11: 23–33.

50. Christensen T. Association of human endogenous retroviruseswith multiple sclerosis and possible interactions with herpesviruses. Rev Med Virol 2005.

51. Bessis D, Moles JP, Basset-Seguin N, Tesniere A, Arpin C,Guilhou JJ. Differential expression of a human endogenous retro-virus E transmembrane envelope glycoprotein in normal, psoriaticand atopic dermatitis human skin. Br J Dermatol 2004; 151: 737–745.

52. Flockerzi A, Burkhardt S, Schempp W, Meese E, Mayer J. Humanendogenous retrovirus HERV-K14 families: status, variants, evo-lution, and mobilization of other cellular sequences. J Virol 2005;79: 2941–2949.

American Journal of Transplantation 2005; 5: 1837–1847 1847