© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Xenotransplantation 2015: 22: 144–150 doi: 10.1111/xen.12160
XENOTRANSPLANTATION
Original Article
Induction of PERV antigen in porcine peripheral blood mononuclear cells by human herpesvirus 1 Kim J, Kim JH, Hwang ES. Induction of PERV antigen in porcine peripheral blood mononuclear cells by human herpesvirus 1. Xenotransplantation 2015: 22: 144–150. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. Abstract: Background: Xenotransplantation represents one of alternative candidates for allotransplantation due to the chronic shortage of suitable human tissues; however, many obstacles remain. Expression and release of endogenous retroviral antigens by porcine cells after transplantation may evoke adverse immune responses in human subjects. Here, we examined whether human herpesvirus 1 (HHV-1) could induce the production of porcine endogenous retrovirus (PERV) antigens in porcine peripheral blood mononuclear cells (PBMCs). Methods: Porcine PBMCs were infected with HHV-1 and examined for the production of PERV Gag protein and HHV-1 using antigen-capture ELISA and quantitative real-time polymerase chain reaction (PCR), respectively. Results: HHV-1 infection resulted in a 1.7- to 33.2-fold induction of PERV Gag relative to mock infection controls, compared to a 2.9- to 12.9-fold induction following treatment with PMA. Expression of PERV Gag was detected in porcine PBMCs and PK-15 cells after HHV-1 infection by double immunofluorescence staining of PERV Gag and HHV-1 antigen. The viability of HHV-1-infected porcine PBMCs was significantly lower than that of mock-infected cells. The HHV-1 level in the culture supernatant increased 5.2-fold relative to controls 24-h post-infection, indicative of active replication within these cells; decreased levels of HHV-1 were detected 72-h post-infection. Conclusions: These results suggest that HHV-1 may be capable of infecting transplanted porcine cells, resulting in strong direct induction of PERV antigen.
Introduction
Xenotransplantation represents one of alternative candidates for allotransplantation, in part because of chronic shortages in suitable human transplant materials, including both cells and organs. However, long-term maintenance of transplants remains a significant obstacle, with allo- or xenotransplanted cells and organs often damaged by host immune reactions or as a result of exogenous or endogenous infectious agents. Successful allotransplantation has been achieved through the development of effective immunosuppression regimens and tolerance induction [1], with stronger immunosuppression regi144
Jiyeon Kim,1,2,3 Jung Heon Kim1,a and Eung Soo Hwang1,3 1
Department of Microbiology and Immunology, Seoul National University College of Medicine, 2 BK21 Plus Biomedical Science Project, Seoul National University College of Medicine, 3Institute of Endemic Diseases, Seoul National University Medical Research Center, Seoul, Korea, aPresent address: Department of Microbiology and Immunology, Louisiana State University Health Sciences Center-Shreveport, Shreveport, LA, 71130, USA
Key words: Gag – human herpesvirus – porcine cells – porcine endogenous retrovirus – xenotransplantation Address reprint requests to Eung Soo Hwang, Department of Microbiology and Immunology, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul 110-799, Korea (E-mail: [email protected]) Received 10 June 2014; Accepted 27 January 2015
mens likely required for xenotransplantations. While such strong immunosuppression may minimize transplantation-related immune reactions in recipients, it is also associated with an increased risk of infection, including the reactivation of latent pathogens such as human herpesviruses (HHVs). Porcine endogenous retrovirus (PERV) present in xenografted tissues has the potential to induce immune responses as foreign antigens in the human host. Although most proviruses are unable to replicate as a result of structural defects, they are still capable of expressing viral RNAs and proteins [2]. If endogenous retroviral antigens present in porcine cells were to be produced and
PERV Ag Induction in pig PBMCs by HHV1 presented to the host immune system after xenotransplantation, they may be sufficient to induce potent immune responses, similar to those seen in response to human immunodeficiency virus (HIV) components present during HIV infection [3]. The infectivity of some HHVs to porcine cells has been reported [4–6], suggesting that human recipients of xenografts may be affected by latent pathogens. The potential risk to human recipients posed by transplant-derived pathogens can be minimized by the use of cells/ organs from designated or specific pathogen-free animals; however, the effect of host-derived infectious agents on allo- or xenotransplanted tissues remains poorly understood. Here, we examined whether HHV-1 could induce the expression of PERV antigens in porcine peripheral blood mononuclear cells (PBMCs).
Materials and methods Cells and viruses
PK-15 (ATCC CCL-33) and HEK293 cells (ATCC CRL-1573) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone; Thermo Scientific, Marietta, OH, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone) in a 5% CO2 atmosphere at 37 °C. All animal experiments were approved by the Animal Care and Use Committee (IACUC: 12-0374-C1A2) of the Clinical Research Institute of Seoul National University Hospital, an AAALAC accredited facility consistent with the guidelines set forth by the National Institutes of Health. Porcine peripheral blood was obtained from designated pathogen-free SNU miniature pigs bred at the Center for Animal Resource Development of Seoul National University College of Medicine. Mononuclear cells were collected at the interface after centrifugation for 30 min on a Ficoll-Paque gradient (GE Healthcare, Little Chalfront, UK). PBMCs were cultured in RPMI1640 (Hyclone) containing 10% FBS in a 5% CO2 atmosphere at 37 °C. Human herpesvirus 1 (HHV-1) (herpes simplex virus type 1 [HSV-1]) MacIntyre (ATCC VR-539) and HHV-5 (human cytomegalovirus [HCMV]) Towne (ATCC VR-977) were used. HHV-1 and HHV-5 were purified by ultracentrifugation (Beckman) on a 20% sucrose cushion for 2 h at 100 000 g with SW28. Viruses derived from the same batch were used for all experiments. Viral titers were measured using a conventional plaque assay; HHV-1 copy numbers were determined by real-time polymerase chain reaction (PCR). A multiplicity of infection of 2–5 was used for all experiments. Mock
infection, the treatment of the same media without the purified virus, was included in the experiment of viral infection as a control. Antibodies and reagents
Monoclonal anti-HHV-1 antibody (clone MHSVI116) [7] and fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse IgG (Invitrogen, Carlsbad, CA, USA) were used to detect HHV-1 antigen. Monoclonal anti-HHV-5 antibody, clone 6 IE1, was a gift from E.S. Huang. Goat anti-GAPDH antibody (Calbiocam, Darmstadt, Germany), reactive against both the human and porcine protein, was used as a control. Monoclonal anti-PERV Gag antibodies, clones C14044 and C13088, were produced by conventional hybridoma methods [7] and were specific to the PERV Gag but not to HIV or human endogenous retrovirus Gag. Phorbol-12-myristate-13-acetone (PMA; 50 ng/ml), phytohemagglutinin (PHA; 25 ng/ml), lipopolysaccharide (LPS; 50 ng/ml), and prostaglandin E2 (PGE2; 50 ng/ml) were used as cell activators. All reagents were purchased from Sigma Chemical (St. Louis, MO, USA) unless otherwise stated. Cell extraction
Porcine PBMCs were washed three times with PBS and lysed by incubation in RIPA buffer for 30 min on ice. Cleared supernatants were prepared at 16 000 g by microcentrifugation for 10 min. Protein levels were quantified using a microbicinchoninic acid assay, according to the manufacturer’s instructions (Thermo Scientific). Antigen-capture ELISA
The anti-PERV Gag antibody clone C14044 was used as the capture antibody. Anti-PERV Gag antibody clone C13088 was conjugated to biotin using an EZ-Link NHS-PEO Solid-Phase Biotinylation kit (Thermo Scientific) according to the manufacturer’s instructions and used as a detection antibody in antigen-capture ELISA. ELISA plates (Corning, New York, NY, USA) were coated with 10 lg/ml anti-PERV Gag antibody (C14044) in carbonate buffer, washed with phosphate-buffered saline (PBS), and blocked with 2% skim milk in PBS. Samples were then added to test plates for 1 h, washed with PBS, and incubated with biotin-conjugated anti-PERV Gag antibody (C13088) and peroxidase-conjugated avidin at 37 °C for 1 h. Samples were then washed with PBST (PBS plus 0.05% 145
Kim et al. Tween 20) and reacted with an o-phenylene diamine solution and H2O2 in phosphate citrate buffer for 15 min at room temperature. The enzyme reaction was stopped by adding 2N H2SO4, and the optical density was measured with an ELISA plate reader (Perkin Elmer, Waltham, MA, USA) at A490 nm. Recombinant glutathione S-transferase (GST)-PERV Gag fusion protein produced in Escherichia coli was used as a control. Immunofluorescent staining
Virus-infected cells were washed twice with PBS, coated on spot slides, air dried, and fixed with icecold acetone for 10 min. Cells were then washed with PBST and reacted with the primary antibody, followed by a FITC-conjugated anti-mouse IgG antibody, at room temperature. Fluorescent signals were observed under a fluorescence microscope (Olympus IX-51, Tokyo, Japan). For the double immunofluorescence staining of PERV Gag and HHV-1 antigen, acetone-fixed cells were reacted primarily with anti-HHV-1 antibody (clone MHSVI116) and followed by Alexa Fluor568-conjugated goat anti-mouse IgG (Invitrogen) at room temperature. Cells were then air dried, fixed with ice-cold acetone, and reacted with biotin-conjugated anti-PERV Gag antibody (C13088) and Alexa Fluor488-conjugated avidin (Invitrogen) at room temperature. Cell nuclei were stained with TO-PRO-3 (Invitrogen). Fluorescent signals were observed under a confocal microscope (Fluoview FV1000; Olympus).
Real-time PCR
Real-time PCRs were performed using a Taqman MasterMix (Applied Biosystems, Foster City, CA, USA) containing 10 pmol HHV-1 forward (50 -CC GTCAGCACCTTCATCGA-30 ) and reverse (50 CAGGCCGCTGTCCTTGAT-30 ) primers, 10 pmol HHV-1 Taqman probe, FAM-50 -ATCAC GAGTTTGTCCCCCTGGAGGTGTA-30 -TAMRA, and varying concentrations of HHV-1 DNA in a 20-ll reaction, and run on an ABI PRISM 7900 sequence detection system (Applied Biosystems). HHV-1-specific primers and Taqman probes were synthesized commercially at Bioneer (Seoul, Korea). Real-time PCR was performed under standard reaction conditions, as recommended by Applied Biosystems; 40 cycles of denaturation for 10 s at 95 °C and annealing and extension for 60 s at 60 °C.
Results Establishment of an antigen-capture ELISA for PERV gag
Serially diluted GST-PERV Gag protein was used to establish an antigen-capture ELISA for measuring PERV Gag protein; GST protein was used as the negative control. The lower limit of detection was 1.56 ng/ml recombinant GST-PERV Gag using a cut-off value equal to twice that of the negative control. This method was then used to detect PERV Gag in PK-15 and HEK 293 cell extracts. Serial dilutions of PERV Gag protein produced by PK-15 samples were readily detected by ELISA, while expression in HEK293 cells was below the limit of detection.
Western blot analysis
HCMV IE-1 protein levels in porcine PBMCs after HCMV infection were detected at the indicated time points by Western blot. Protein concentrations were determined using a BCA protein assay kit (Thermo) according to the manufacturer’s instructions. Samples were denatured, run on 10% polyacrylamide gels under reducing conditions, and transferred electrophoretically to PVDF membranes. Membranes were then probed with an antiHCMV IE-1 antibody (1 : 1000 dilution) for 24 h at 4 °C, washed in PBST, and then probed with a horseradish peroxidase-conjugated goat antimouse antibody for 1 h at room temperature as a secondary antibody; an anti-GAPDH antibody was used as a loading control. Signal was developed using an enhanced chemiluminescence (ECL) reagent (Thermo) according to the manufacturer’s instructions.
146
Induction of PERV Gag in PK-15 by cell activators
PK-15 cells were stimulated with PMA, PHA, LPS, or PGE2, and the cell pellet and culture supernatant harvested at 0, 3, and 7 days. Soluble PERV Gag protein (ng/ml) was measured in the culture supernatant, while total PERV Gag production was determined by comparing total PERV Gag concentrations to that of total cell protein extracts. Basal levels of expression in the culture supernatant and cell extract were 4.75 2.46 ng/ml and 0.026 0.006 ng/mg, respectively (Fig. 1). No difference in PERV Gag expression levels was seen three and 7 days after treatment with PHA, LPS, or PGE2; however, when treated with PMA, the amount of PERV Gag in the culture supernatant and the relative amount of PERV Gag in the cell extract increased to 21.76 7.80 ng/ml and
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Fig. 1. Changes in PERV Gag production following treatment with cell activators. PERV Gag levels in the culture supernatant (A) and extract (B) of PK-15 cells stimulated with the indicated activators. PERV Gag was assayed by antigen-capture ELISA and expressed as total PERV Gag in the culture supernatant (ng/ml) and in the cell extract relative to total protein (ng/mg). Results are expressed as means standard error of three independent experiments.
0.20 0.16 ng/mg at day 3, respectively; and 132.79 60.84 ng/ml and 0.51 0.09 ng/mg at day 7, respectively (Fig. 1). These results show that only PMA induced the production of PERV Gag in PK-15 cells. Effect of herpesvirus infection on the induction of PERV Gag in porcine PBMCs
Porcine PBMCs were infected with HHV-1 at a multiplicity of infection of 2–5, and the culture supernatants harvested on days 0, 2, and 6; total PERV Gag protein in the culture supernatant was then measured by antigen-capture ELISA. As PMA was found to be the strongest inducer of PERV Gag in PK-15, PMA-treated porcine PBMCs were used as a positive control. Basal PERV Gag expression was 0.071 0.025 ng/ml and 0.22 0.032 ng/ml on days 2 and 6, respectively. Following treatment with PMA, the quantity of PERV Gag in the culture supernatant increased to 0.21 0.16 ng/ml and 2.85 0.68 ng/ml on days 2 and 6, respectively. PERV Gag was detected following HHV-5 infection, with cells expressing 0.23 0.10 ng/ml and 0.16 0.041 ng/ml, respectively. Expression was considerably increased following HHV-1 infection, with cells expressing 0.12 0.06 ng/ml and 7.33 0.69 ng/ml PERV Gag on days 2 and 6, respectively (Fig. 2A). These results showed that HHV-1, but not HHV-5, could induce the production of PERV Gag in porcine PBMCs. To determine whether PERV Gag expression was directly induced by HHV-1 infection, HHV-1 -infected cells were stained serially with an antiHHV-1 antibody and Alexa Fluor568-conjugated anti-mouse IgG, and biotin-conjugated anti-PERV
Gag and Alexa Fluor488-conjugated avidin, and observed under a confocal microscope. Expression of PERV Gag was not observed in mock-infected PBMCs, but was weakly detected in mock-infected PK-15 cells. After HHV-1 infection PERV Gag stained intensely in both PBMCs and PK-15 cells, with staining intensity closely mirroring that of HHV-1 antigen production (Fig. 2B). To determine the reason for the negligible PERV Gag induction by HHV-5, the infectivity of HHV-5 to porcine PBMCs was examined by Western blot analysis. HHV-5 IE-1 expression was not observed in any of the HHV-5-infected PBMCs (Fig. 2C), indicating that HHV-5 was unable to infect porcine PBMCs. Infectivity of HHV-1 to porcine PBMCs
Porcine PBMCs were infected with HHV-1, and the changes in PBMC cell counts were monitored. The number of HHV-1-infected porcine PBMCs dropped markedly from day 1 post-infection, compared to that of mock-infected cells (Fig. 3A). Immunofluorescence staining of PBMCs infected with HHV-1 demonstrated that the HHV-1infected PBMCs expressed the respective antigen throughout the course of the experiment (Fig. 3B). To quantify HHV-1 production in infected cells, HHV-1 was harvested from porcine PBMC culture supernatants after HHV-1 infection on days 0, 1, 2, 3, and 7 post-infection, and analyzed by real-time PCR. Marked increases in HHV-1 copy number were evident between days 1 and 2 postinfection (3.5 2.3 and 18.1 0.6, respectively), with copy number peaking 3 days post-infection (19.4 3.3) and decreasing thereafter (Fig. 3C). These data suggest that HHV-1 can infect porcine 147
Kim et al. A
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PBMCs, replicate effectively in them, and release virions into the culture supernatant. Discussion
HHV-1 and HHV-5 infections are prevalent worldwide [8,9]. These viruses establish life-long latency after primary infection and can be reactivated by 148
Fig. 2. Induction of PERV Gag in porcine PBMCs following human herpesvirus challenge. (A) Detection of PERV Gag protein in the culture supernatant of SNU miniature pig PBMCs after infection with HHV-1. PERV Gag concentrations were analyzed by antigen-capture ELISA, and expressed as total PERV Gag (ng/ml); the culture supernatants of PBMCs after treatment with PMA and medium alone were used as the positive and negative controls, respectively. Results are expressed as means standard error of three independent experiments. (B) Double immunofluorescence staining of PERV Gag and HHV-1 antigen in SNU miniature pig PBMCs and PK-15 cells after infection with HHV-1. HHV-1 antigen was probed with an anti-HHV-1 antibody and Alexa Fluor568-conjugated anti-mouse IgG (red), PERV Gag with a biotin-conjugated anti-PERV Gag antibody and Alexa Fluor 488-conjugated avidin (green), and nuclei with TO-PRO-3 (blue), and observed under a confocal microscope. PERV Gag was not detected in mock-infected PBMCs, but was weakly detected in mock-infected PK-15 cells. PERV Gag stained intensely in both PBMCs and PK-15 cells after HHV-1 infection; the staining intensity closely mirrored that of HHV-1 antigen production. Representative photographs were taken from at least three fields per sample in two separate experiments. (C) Western blot analysis of HHV-5 IE-1 expression in porcine PBMCs after HHV-5 infection. P indicates the positive control, which is the extract of human cells infected with HHV-5 in the same condition as porcine PBMCs.
various conditions such as stresses, high fever, some hormonal changes, immune suppression, and other factors. Here, we showed that HHV-1 could efficiently infect porcine PBMCs, resulting in increased production of PERV Gag (Fig. 2A). Induction of PERV Gag by HHV-1 was demonstrated using double immunofluorescence staining, with expression of PERV Gag found only in PBMCs harboring HHV-1 antigen; this co-localization was particularly strong among HHV-1-infected PK-15 cells (Fig. 2B). While HHV-5 has been shown to infect some porcine cell types [4–6], HHV-5 was unable to infect porcine PBMCs (Fig. 2C). This lack of infectivity likely accounts for the failure of HHV-5 to induce PERV Gag expression. HHV-1 infection significantly reduced the cell count compared to mock infection; however, HHV-1 was able to replicate within infected cells, leading to a marked increase in the production of viral progeny (Fig. 3A). Taken together, these data show that increased detection of PERV Gag was not simply the result of cell lysis due to HHV-1 infection, as baseline expression of PERV Gag in mock-infected cells was consistently below detection limits (Fig. 2B). As with HHV-1, PERV can also be present in the human host after xenotransplantation, as long as a suitable porcine cell population remains. While no diseases associated with PERV have yet been reported, circumstantial evidence such as serologically PERV-negative patients with extra-
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Fig. 3. Infectivity of HHV-1 to porcine PBMCs. (A) Changes in porcine PBMC cell numbers following HHV-1 infection. Results are expressed as means standard error of two independent experiments. (B) Detection of HHV-1 antigen in porcine PBMCs by immunofluorescence assay after infection with HHV-1. Green fluorescence was observed only in PBMCs infected with HHV-1. Images are representative of two independent experiments. (C) Quantitation of HHV-1 in the culture supernatant of porcine PBMCs after infection with HHV-1. Results are expressed as means standard error of two independent experiments.
corporeal pig organs perfusion and butchers exposed to pig tissues [10–12], along with several clinical trials [13,14] suggest that PERV poses a low risk during xenotransplantation. However, foreign antigens present in porcine tissues would be capable of inducing strong immune responses,
making adverse responses to these antigens a legitimate concern [3]. Porcine endogenous retrovirus Gag was induced in PK-15 cells treated with PMA in a time-dependent manner, but not when treated with PHA, LPS, or PGE2. This result is similar to that of Scobie et al. [15] in which the expression of PERV constructs increased following treatment with phorbol derivatives, but not in the presence of cyclosporin A, prednisolone, TNF-alpha, IFNgamma, oestradiol, or dexamethasone. Other studies failed to detect increased PERV production in pig hepatocytes after PHA or PMA treatments [16]; this discrepancy was likely the result of differences in the tissues and cell types used [17]. In our study, PBMCs infected with HHV-1 displayed a high level of activated PERV when compared with PBMCs activated by PMA, although it was not proven that this increased PERV Gag expression would be sufficient to produce a host antibody response. We performed co-culture experiment with HHV-1-infected PBMCs and 293 cells to see whether PERV produced from PBMCs infected with HHV-1 could infect human susceptible cells. As expected 293 cells showed cytopathic effect (CPE) after the co-cultivation and we could not maintain the cultures for the experiment of the detection of PERV in 293 cells (Data not shown. Figure was attached in Supplement file). This result revealed that HHV-1 could infect porcine PBMCs and produce its progeny, which resulted in the induction of CPE in co-cultured 293 cells. Further research is necessary to determine the full range of cell types and stimuli capable of inducing PERV expression in exogenous hosts. An analysis of PERV-specific antibodies in recipients of pig islets did not provide evidence of PERV transmission, as none of the patients’ sera reacted to both Gag and Env; however, some patients exhibited reactivity against one of the two proteins [14]. While the mechanism of this antibody formation is unknown, potential causes include cross-reactive antigenic epitopes in retroviral proteins [18], or carbohydrates in glycoproteins [19]. The porcine PBMCs used in this study have a relatively short lifespan in vitro, which limits the potential applications of our findings to monitoring of PERV Gag in patients after xenotransplantation. However, our demonstration of PERV Gag induction by HHV-1 infection suggests the possibility of long-lived xenografted cells being affected by reactivated HHV-1. Such a result further highlights the need for persistent control of latent pathogens among transplant recipients. 149
Kim et al. Acknowledgments
This study was supported by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI13C0954). We thank Eun Young Choi and Jisung Kim for their excellent technical assistance. Author contributions
JK performed antigen-capture ELISA and virus experiments and drafted manuscript. JHK made PERV-specific antibodies and established and performed a part of antigen-capture ELISA. ESH designed research, interpreted data, and revised the manuscript. References 1. VINCENTI F, KIRK AD. What’s next in the pipeline. Am J Transplant 2008; 8: 1972–1981. 2. DENNER J. Immunosuppression by retroviruses: implications for xenotransplantation. Ann NY Acad Sci 1998; 862: 75–86. € A et al. Antibody 3. GAINES H, von SYDOW M, SONNERBORG response in primary human immunodeficiency virus infection. Lancet 1987; 329: 1249–1253. 4. KIM JH, JUNG ES, KWON Y et al. Infection of porcine cells with human herpesviruses. Transplant Proc 2010; 42: 2134–2137. € L, SINZGER C et al. Efficiency of 5. MILLARD AL, HABERLI porcine endothelial cell infection with human cytomegalovirus depends on both virus tropism and endothelial cell vascular origin. Xenotransplantation 2010; 17: 274– 287. 6. GHIELMETTI M, MILLARD AL, HAEBERLI L et al. Human CMV infection of porcine endothelial cells increases adhesion receptor expression and human leukocyte recruitment. Transplantation 2009; 87: 1792–1800. 7. CHA CY, HWANG ES, KOOK YH. Production and characterization of monoclonal antibodies specific to herpes simplex viruses. J Korean Soc Microbiol 1988; 23: 505– 515. 8. NAHMIAS AJ, LEE FK, BECKMAN-NAHMIAS S. Sero-epidemiological and -sociological patterns of herpes simplex virus infection in the world. Scand J Infect Dis Suppl 1990; 69: 19–36.
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9. BATE SL, DOLLARD SC, CANNON MJ. Cytomegalovirus seroprevalence in the United States: the national health and nutrition examination surveys, 1988-2004. Clin Infect Dis 2010; 50: 1439–1447. 10. NICUOLO GD, D’ALESSANDRO A, ANDRIA B et al. Longterm absence of porcine endogenous retrovirus infection in chronically immunosuppressed patients after treatment with the porcine cell–based Academic Medical Center bioartificial liver. Xenotransplantation 2010; 17: 431–439. 11. PARADIS K, LANGFORD G, LONG Z et al. Search for crossspecies transmission of porcine endogenous retrovirus in patients treated with living pig tissue. Science 1999; 285: 1236–1241. 12. TACKE S, BODUSCH K, BERG A et al. Sensitive and specific detection methods for porcine endogenous retroviruses applicable to experimental and clinical xenotransplantation. Xenotransplantation 2001; 8: 125–135. 13. HENEINE W, TIBELL A, SWITZER WM et al. No evidence of infection with porcine endogenous retrovirus in recipients of porcine islet-cell xenografts. Lancet 1998; 352: 695–699. 14. GARKAVENKO O, CROXSON MC, IRGANG M et al. Monitoring for presence of potentially xenotic viruses in recipients of pig islet xenotransplantation. J Clin Microbiol 2004; 42: 5353–5356. 15. SCOBIE L, TAYLOR S, LOGAN NA et al. Characterization of germline porcine endogenous retroviruses from Large White pig. J Gen Virol 2004; 85: 2421–2428. 16. NYBERG SL, HIBBS JR, HARDIN JA et al. Influence of human fulminant hepatic failure sera on endogenous retroviral expression in pig hepatocytes. Liver Transpl 2000; 6: 76–84. 17. BITTMANN I, MIHICA D, PLESKER R et al. Expression of porcine endogenous retroviruses (PERV) in different organs of a pig. Virology 2012; 433: 329–336. 18. GRANT RF, WINDSOR SK, MALINAK CJ et al. Characterization of infectious type D retrovirus from baboons. Virology 1995; 207: 292–296. € J, DAVIDSON EA, TEICH NM et al. Heterophil 19. LOWER human antibodies recognize oncovirus envelope antigens: epidemiological parameters and immunological specificity of the reaction. Virology 1981; 109: 409–417.
Supporting Information
Additional Supporting Information may be found in the online version of this article: Figure S1. Cytopathic effect (CPE) in 293 cells after co-culture with PBMCS treated with PMA (or) HHV-1.
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Xenotransplantation 2015: 22: 144–150 doi: 10.1111/xen.12160
XENOTRANSPLANTATION
Original Article
Induction of PERV antigen in porcine peripheral blood mononuclear cells by human herpesvirus 1 Kim J, Kim JH, Hwang ES. Induction of PERV antigen in porcine peripheral blood mononuclear cells by human herpesvirus 1. Xenotransplantation 2015: 22: 144–150. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. Abstract: Background: Xenotransplantation represents one of alternative candidates for allotransplantation due to the chronic shortage of suitable human tissues; however, many obstacles remain. Expression and release of endogenous retroviral antigens by porcine cells after transplantation may evoke adverse immune responses in human subjects. Here, we examined whether human herpesvirus 1 (HHV-1) could induce the production of porcine endogenous retrovirus (PERV) antigens in porcine peripheral blood mononuclear cells (PBMCs). Methods: Porcine PBMCs were infected with HHV-1 and examined for the production of PERV Gag protein and HHV-1 using antigen-capture ELISA and quantitative real-time polymerase chain reaction (PCR), respectively. Results: HHV-1 infection resulted in a 1.7- to 33.2-fold induction of PERV Gag relative to mock infection controls, compared to a 2.9- to 12.9-fold induction following treatment with PMA. Expression of PERV Gag was detected in porcine PBMCs and PK-15 cells after HHV-1 infection by double immunofluorescence staining of PERV Gag and HHV-1 antigen. The viability of HHV-1-infected porcine PBMCs was significantly lower than that of mock-infected cells. The HHV-1 level in the culture supernatant increased 5.2-fold relative to controls 24-h post-infection, indicative of active replication within these cells; decreased levels of HHV-1 were detected 72-h post-infection. Conclusions: These results suggest that HHV-1 may be capable of infecting transplanted porcine cells, resulting in strong direct induction of PERV antigen.
Introduction
Xenotransplantation represents one of alternative candidates for allotransplantation, in part because of chronic shortages in suitable human transplant materials, including both cells and organs. However, long-term maintenance of transplants remains a significant obstacle, with allo- or xenotransplanted cells and organs often damaged by host immune reactions or as a result of exogenous or endogenous infectious agents. Successful allotransplantation has been achieved through the development of effective immunosuppression regimens and tolerance induction [1], with stronger immunosuppression regi144
Jiyeon Kim,1,2,3 Jung Heon Kim1,a and Eung Soo Hwang1,3 1
Department of Microbiology and Immunology, Seoul National University College of Medicine, 2 BK21 Plus Biomedical Science Project, Seoul National University College of Medicine, 3Institute of Endemic Diseases, Seoul National University Medical Research Center, Seoul, Korea, aPresent address: Department of Microbiology and Immunology, Louisiana State University Health Sciences Center-Shreveport, Shreveport, LA, 71130, USA
Key words: Gag – human herpesvirus – porcine cells – porcine endogenous retrovirus – xenotransplantation Address reprint requests to Eung Soo Hwang, Department of Microbiology and Immunology, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul 110-799, Korea (E-mail: [email protected]) Received 10 June 2014; Accepted 27 January 2015
mens likely required for xenotransplantations. While such strong immunosuppression may minimize transplantation-related immune reactions in recipients, it is also associated with an increased risk of infection, including the reactivation of latent pathogens such as human herpesviruses (HHVs). Porcine endogenous retrovirus (PERV) present in xenografted tissues has the potential to induce immune responses as foreign antigens in the human host. Although most proviruses are unable to replicate as a result of structural defects, they are still capable of expressing viral RNAs and proteins [2]. If endogenous retroviral antigens present in porcine cells were to be produced and
PERV Ag Induction in pig PBMCs by HHV1 presented to the host immune system after xenotransplantation, they may be sufficient to induce potent immune responses, similar to those seen in response to human immunodeficiency virus (HIV) components present during HIV infection [3]. The infectivity of some HHVs to porcine cells has been reported [4–6], suggesting that human recipients of xenografts may be affected by latent pathogens. The potential risk to human recipients posed by transplant-derived pathogens can be minimized by the use of cells/ organs from designated or specific pathogen-free animals; however, the effect of host-derived infectious agents on allo- or xenotransplanted tissues remains poorly understood. Here, we examined whether HHV-1 could induce the expression of PERV antigens in porcine peripheral blood mononuclear cells (PBMCs).
Materials and methods Cells and viruses
PK-15 (ATCC CCL-33) and HEK293 cells (ATCC CRL-1573) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone; Thermo Scientific, Marietta, OH, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone) in a 5% CO2 atmosphere at 37 °C. All animal experiments were approved by the Animal Care and Use Committee (IACUC: 12-0374-C1A2) of the Clinical Research Institute of Seoul National University Hospital, an AAALAC accredited facility consistent with the guidelines set forth by the National Institutes of Health. Porcine peripheral blood was obtained from designated pathogen-free SNU miniature pigs bred at the Center for Animal Resource Development of Seoul National University College of Medicine. Mononuclear cells were collected at the interface after centrifugation for 30 min on a Ficoll-Paque gradient (GE Healthcare, Little Chalfront, UK). PBMCs were cultured in RPMI1640 (Hyclone) containing 10% FBS in a 5% CO2 atmosphere at 37 °C. Human herpesvirus 1 (HHV-1) (herpes simplex virus type 1 [HSV-1]) MacIntyre (ATCC VR-539) and HHV-5 (human cytomegalovirus [HCMV]) Towne (ATCC VR-977) were used. HHV-1 and HHV-5 were purified by ultracentrifugation (Beckman) on a 20% sucrose cushion for 2 h at 100 000 g with SW28. Viruses derived from the same batch were used for all experiments. Viral titers were measured using a conventional plaque assay; HHV-1 copy numbers were determined by real-time polymerase chain reaction (PCR). A multiplicity of infection of 2–5 was used for all experiments. Mock
infection, the treatment of the same media without the purified virus, was included in the experiment of viral infection as a control. Antibodies and reagents
Monoclonal anti-HHV-1 antibody (clone MHSVI116) [7] and fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse IgG (Invitrogen, Carlsbad, CA, USA) were used to detect HHV-1 antigen. Monoclonal anti-HHV-5 antibody, clone 6 IE1, was a gift from E.S. Huang. Goat anti-GAPDH antibody (Calbiocam, Darmstadt, Germany), reactive against both the human and porcine protein, was used as a control. Monoclonal anti-PERV Gag antibodies, clones C14044 and C13088, were produced by conventional hybridoma methods [7] and were specific to the PERV Gag but not to HIV or human endogenous retrovirus Gag. Phorbol-12-myristate-13-acetone (PMA; 50 ng/ml), phytohemagglutinin (PHA; 25 ng/ml), lipopolysaccharide (LPS; 50 ng/ml), and prostaglandin E2 (PGE2; 50 ng/ml) were used as cell activators. All reagents were purchased from Sigma Chemical (St. Louis, MO, USA) unless otherwise stated. Cell extraction
Porcine PBMCs were washed three times with PBS and lysed by incubation in RIPA buffer for 30 min on ice. Cleared supernatants were prepared at 16 000 g by microcentrifugation for 10 min. Protein levels were quantified using a microbicinchoninic acid assay, according to the manufacturer’s instructions (Thermo Scientific). Antigen-capture ELISA
The anti-PERV Gag antibody clone C14044 was used as the capture antibody. Anti-PERV Gag antibody clone C13088 was conjugated to biotin using an EZ-Link NHS-PEO Solid-Phase Biotinylation kit (Thermo Scientific) according to the manufacturer’s instructions and used as a detection antibody in antigen-capture ELISA. ELISA plates (Corning, New York, NY, USA) were coated with 10 lg/ml anti-PERV Gag antibody (C14044) in carbonate buffer, washed with phosphate-buffered saline (PBS), and blocked with 2% skim milk in PBS. Samples were then added to test plates for 1 h, washed with PBS, and incubated with biotin-conjugated anti-PERV Gag antibody (C13088) and peroxidase-conjugated avidin at 37 °C for 1 h. Samples were then washed with PBST (PBS plus 0.05% 145
Kim et al. Tween 20) and reacted with an o-phenylene diamine solution and H2O2 in phosphate citrate buffer for 15 min at room temperature. The enzyme reaction was stopped by adding 2N H2SO4, and the optical density was measured with an ELISA plate reader (Perkin Elmer, Waltham, MA, USA) at A490 nm. Recombinant glutathione S-transferase (GST)-PERV Gag fusion protein produced in Escherichia coli was used as a control. Immunofluorescent staining
Virus-infected cells were washed twice with PBS, coated on spot slides, air dried, and fixed with icecold acetone for 10 min. Cells were then washed with PBST and reacted with the primary antibody, followed by a FITC-conjugated anti-mouse IgG antibody, at room temperature. Fluorescent signals were observed under a fluorescence microscope (Olympus IX-51, Tokyo, Japan). For the double immunofluorescence staining of PERV Gag and HHV-1 antigen, acetone-fixed cells were reacted primarily with anti-HHV-1 antibody (clone MHSVI116) and followed by Alexa Fluor568-conjugated goat anti-mouse IgG (Invitrogen) at room temperature. Cells were then air dried, fixed with ice-cold acetone, and reacted with biotin-conjugated anti-PERV Gag antibody (C13088) and Alexa Fluor488-conjugated avidin (Invitrogen) at room temperature. Cell nuclei were stained with TO-PRO-3 (Invitrogen). Fluorescent signals were observed under a confocal microscope (Fluoview FV1000; Olympus).
Real-time PCR
Real-time PCRs were performed using a Taqman MasterMix (Applied Biosystems, Foster City, CA, USA) containing 10 pmol HHV-1 forward (50 -CC GTCAGCACCTTCATCGA-30 ) and reverse (50 CAGGCCGCTGTCCTTGAT-30 ) primers, 10 pmol HHV-1 Taqman probe, FAM-50 -ATCAC GAGTTTGTCCCCCTGGAGGTGTA-30 -TAMRA, and varying concentrations of HHV-1 DNA in a 20-ll reaction, and run on an ABI PRISM 7900 sequence detection system (Applied Biosystems). HHV-1-specific primers and Taqman probes were synthesized commercially at Bioneer (Seoul, Korea). Real-time PCR was performed under standard reaction conditions, as recommended by Applied Biosystems; 40 cycles of denaturation for 10 s at 95 °C and annealing and extension for 60 s at 60 °C.
Results Establishment of an antigen-capture ELISA for PERV gag
Serially diluted GST-PERV Gag protein was used to establish an antigen-capture ELISA for measuring PERV Gag protein; GST protein was used as the negative control. The lower limit of detection was 1.56 ng/ml recombinant GST-PERV Gag using a cut-off value equal to twice that of the negative control. This method was then used to detect PERV Gag in PK-15 and HEK 293 cell extracts. Serial dilutions of PERV Gag protein produced by PK-15 samples were readily detected by ELISA, while expression in HEK293 cells was below the limit of detection.
Western blot analysis
HCMV IE-1 protein levels in porcine PBMCs after HCMV infection were detected at the indicated time points by Western blot. Protein concentrations were determined using a BCA protein assay kit (Thermo) according to the manufacturer’s instructions. Samples were denatured, run on 10% polyacrylamide gels under reducing conditions, and transferred electrophoretically to PVDF membranes. Membranes were then probed with an antiHCMV IE-1 antibody (1 : 1000 dilution) for 24 h at 4 °C, washed in PBST, and then probed with a horseradish peroxidase-conjugated goat antimouse antibody for 1 h at room temperature as a secondary antibody; an anti-GAPDH antibody was used as a loading control. Signal was developed using an enhanced chemiluminescence (ECL) reagent (Thermo) according to the manufacturer’s instructions.
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Induction of PERV Gag in PK-15 by cell activators
PK-15 cells were stimulated with PMA, PHA, LPS, or PGE2, and the cell pellet and culture supernatant harvested at 0, 3, and 7 days. Soluble PERV Gag protein (ng/ml) was measured in the culture supernatant, while total PERV Gag production was determined by comparing total PERV Gag concentrations to that of total cell protein extracts. Basal levels of expression in the culture supernatant and cell extract were 4.75 2.46 ng/ml and 0.026 0.006 ng/mg, respectively (Fig. 1). No difference in PERV Gag expression levels was seen three and 7 days after treatment with PHA, LPS, or PGE2; however, when treated with PMA, the amount of PERV Gag in the culture supernatant and the relative amount of PERV Gag in the cell extract increased to 21.76 7.80 ng/ml and
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Fig. 1. Changes in PERV Gag production following treatment with cell activators. PERV Gag levels in the culture supernatant (A) and extract (B) of PK-15 cells stimulated with the indicated activators. PERV Gag was assayed by antigen-capture ELISA and expressed as total PERV Gag in the culture supernatant (ng/ml) and in the cell extract relative to total protein (ng/mg). Results are expressed as means standard error of three independent experiments.
0.20 0.16 ng/mg at day 3, respectively; and 132.79 60.84 ng/ml and 0.51 0.09 ng/mg at day 7, respectively (Fig. 1). These results show that only PMA induced the production of PERV Gag in PK-15 cells. Effect of herpesvirus infection on the induction of PERV Gag in porcine PBMCs
Porcine PBMCs were infected with HHV-1 at a multiplicity of infection of 2–5, and the culture supernatants harvested on days 0, 2, and 6; total PERV Gag protein in the culture supernatant was then measured by antigen-capture ELISA. As PMA was found to be the strongest inducer of PERV Gag in PK-15, PMA-treated porcine PBMCs were used as a positive control. Basal PERV Gag expression was 0.071 0.025 ng/ml and 0.22 0.032 ng/ml on days 2 and 6, respectively. Following treatment with PMA, the quantity of PERV Gag in the culture supernatant increased to 0.21 0.16 ng/ml and 2.85 0.68 ng/ml on days 2 and 6, respectively. PERV Gag was detected following HHV-5 infection, with cells expressing 0.23 0.10 ng/ml and 0.16 0.041 ng/ml, respectively. Expression was considerably increased following HHV-1 infection, with cells expressing 0.12 0.06 ng/ml and 7.33 0.69 ng/ml PERV Gag on days 2 and 6, respectively (Fig. 2A). These results showed that HHV-1, but not HHV-5, could induce the production of PERV Gag in porcine PBMCs. To determine whether PERV Gag expression was directly induced by HHV-1 infection, HHV-1 -infected cells were stained serially with an antiHHV-1 antibody and Alexa Fluor568-conjugated anti-mouse IgG, and biotin-conjugated anti-PERV
Gag and Alexa Fluor488-conjugated avidin, and observed under a confocal microscope. Expression of PERV Gag was not observed in mock-infected PBMCs, but was weakly detected in mock-infected PK-15 cells. After HHV-1 infection PERV Gag stained intensely in both PBMCs and PK-15 cells, with staining intensity closely mirroring that of HHV-1 antigen production (Fig. 2B). To determine the reason for the negligible PERV Gag induction by HHV-5, the infectivity of HHV-5 to porcine PBMCs was examined by Western blot analysis. HHV-5 IE-1 expression was not observed in any of the HHV-5-infected PBMCs (Fig. 2C), indicating that HHV-5 was unable to infect porcine PBMCs. Infectivity of HHV-1 to porcine PBMCs
Porcine PBMCs were infected with HHV-1, and the changes in PBMC cell counts were monitored. The number of HHV-1-infected porcine PBMCs dropped markedly from day 1 post-infection, compared to that of mock-infected cells (Fig. 3A). Immunofluorescence staining of PBMCs infected with HHV-1 demonstrated that the HHV-1infected PBMCs expressed the respective antigen throughout the course of the experiment (Fig. 3B). To quantify HHV-1 production in infected cells, HHV-1 was harvested from porcine PBMC culture supernatants after HHV-1 infection on days 0, 1, 2, 3, and 7 post-infection, and analyzed by real-time PCR. Marked increases in HHV-1 copy number were evident between days 1 and 2 postinfection (3.5 2.3 and 18.1 0.6, respectively), with copy number peaking 3 days post-infection (19.4 3.3) and decreasing thereafter (Fig. 3C). These data suggest that HHV-1 can infect porcine 147
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PBMCs, replicate effectively in them, and release virions into the culture supernatant. Discussion
HHV-1 and HHV-5 infections are prevalent worldwide [8,9]. These viruses establish life-long latency after primary infection and can be reactivated by 148
Fig. 2. Induction of PERV Gag in porcine PBMCs following human herpesvirus challenge. (A) Detection of PERV Gag protein in the culture supernatant of SNU miniature pig PBMCs after infection with HHV-1. PERV Gag concentrations were analyzed by antigen-capture ELISA, and expressed as total PERV Gag (ng/ml); the culture supernatants of PBMCs after treatment with PMA and medium alone were used as the positive and negative controls, respectively. Results are expressed as means standard error of three independent experiments. (B) Double immunofluorescence staining of PERV Gag and HHV-1 antigen in SNU miniature pig PBMCs and PK-15 cells after infection with HHV-1. HHV-1 antigen was probed with an anti-HHV-1 antibody and Alexa Fluor568-conjugated anti-mouse IgG (red), PERV Gag with a biotin-conjugated anti-PERV Gag antibody and Alexa Fluor 488-conjugated avidin (green), and nuclei with TO-PRO-3 (blue), and observed under a confocal microscope. PERV Gag was not detected in mock-infected PBMCs, but was weakly detected in mock-infected PK-15 cells. PERV Gag stained intensely in both PBMCs and PK-15 cells after HHV-1 infection; the staining intensity closely mirrored that of HHV-1 antigen production. Representative photographs were taken from at least three fields per sample in two separate experiments. (C) Western blot analysis of HHV-5 IE-1 expression in porcine PBMCs after HHV-5 infection. P indicates the positive control, which is the extract of human cells infected with HHV-5 in the same condition as porcine PBMCs.
various conditions such as stresses, high fever, some hormonal changes, immune suppression, and other factors. Here, we showed that HHV-1 could efficiently infect porcine PBMCs, resulting in increased production of PERV Gag (Fig. 2A). Induction of PERV Gag by HHV-1 was demonstrated using double immunofluorescence staining, with expression of PERV Gag found only in PBMCs harboring HHV-1 antigen; this co-localization was particularly strong among HHV-1-infected PK-15 cells (Fig. 2B). While HHV-5 has been shown to infect some porcine cell types [4–6], HHV-5 was unable to infect porcine PBMCs (Fig. 2C). This lack of infectivity likely accounts for the failure of HHV-5 to induce PERV Gag expression. HHV-1 infection significantly reduced the cell count compared to mock infection; however, HHV-1 was able to replicate within infected cells, leading to a marked increase in the production of viral progeny (Fig. 3A). Taken together, these data show that increased detection of PERV Gag was not simply the result of cell lysis due to HHV-1 infection, as baseline expression of PERV Gag in mock-infected cells was consistently below detection limits (Fig. 2B). As with HHV-1, PERV can also be present in the human host after xenotransplantation, as long as a suitable porcine cell population remains. While no diseases associated with PERV have yet been reported, circumstantial evidence such as serologically PERV-negative patients with extra-
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Fig. 3. Infectivity of HHV-1 to porcine PBMCs. (A) Changes in porcine PBMC cell numbers following HHV-1 infection. Results are expressed as means standard error of two independent experiments. (B) Detection of HHV-1 antigen in porcine PBMCs by immunofluorescence assay after infection with HHV-1. Green fluorescence was observed only in PBMCs infected with HHV-1. Images are representative of two independent experiments. (C) Quantitation of HHV-1 in the culture supernatant of porcine PBMCs after infection with HHV-1. Results are expressed as means standard error of two independent experiments.
corporeal pig organs perfusion and butchers exposed to pig tissues [10–12], along with several clinical trials [13,14] suggest that PERV poses a low risk during xenotransplantation. However, foreign antigens present in porcine tissues would be capable of inducing strong immune responses,
making adverse responses to these antigens a legitimate concern [3]. Porcine endogenous retrovirus Gag was induced in PK-15 cells treated with PMA in a time-dependent manner, but not when treated with PHA, LPS, or PGE2. This result is similar to that of Scobie et al. [15] in which the expression of PERV constructs increased following treatment with phorbol derivatives, but not in the presence of cyclosporin A, prednisolone, TNF-alpha, IFNgamma, oestradiol, or dexamethasone. Other studies failed to detect increased PERV production in pig hepatocytes after PHA or PMA treatments [16]; this discrepancy was likely the result of differences in the tissues and cell types used [17]. In our study, PBMCs infected with HHV-1 displayed a high level of activated PERV when compared with PBMCs activated by PMA, although it was not proven that this increased PERV Gag expression would be sufficient to produce a host antibody response. We performed co-culture experiment with HHV-1-infected PBMCs and 293 cells to see whether PERV produced from PBMCs infected with HHV-1 could infect human susceptible cells. As expected 293 cells showed cytopathic effect (CPE) after the co-cultivation and we could not maintain the cultures for the experiment of the detection of PERV in 293 cells (Data not shown. Figure was attached in Supplement file). This result revealed that HHV-1 could infect porcine PBMCs and produce its progeny, which resulted in the induction of CPE in co-cultured 293 cells. Further research is necessary to determine the full range of cell types and stimuli capable of inducing PERV expression in exogenous hosts. An analysis of PERV-specific antibodies in recipients of pig islets did not provide evidence of PERV transmission, as none of the patients’ sera reacted to both Gag and Env; however, some patients exhibited reactivity against one of the two proteins [14]. While the mechanism of this antibody formation is unknown, potential causes include cross-reactive antigenic epitopes in retroviral proteins [18], or carbohydrates in glycoproteins [19]. The porcine PBMCs used in this study have a relatively short lifespan in vitro, which limits the potential applications of our findings to monitoring of PERV Gag in patients after xenotransplantation. However, our demonstration of PERV Gag induction by HHV-1 infection suggests the possibility of long-lived xenografted cells being affected by reactivated HHV-1. Such a result further highlights the need for persistent control of latent pathogens among transplant recipients. 149
Kim et al. Acknowledgments
This study was supported by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI13C0954). We thank Eun Young Choi and Jisung Kim for their excellent technical assistance. Author contributions
JK performed antigen-capture ELISA and virus experiments and drafted manuscript. JHK made PERV-specific antibodies and established and performed a part of antigen-capture ELISA. ESH designed research, interpreted data, and revised the manuscript. References 1. VINCENTI F, KIRK AD. What’s next in the pipeline. Am J Transplant 2008; 8: 1972–1981. 2. DENNER J. Immunosuppression by retroviruses: implications for xenotransplantation. Ann NY Acad Sci 1998; 862: 75–86. € A et al. Antibody 3. GAINES H, von SYDOW M, SONNERBORG response in primary human immunodeficiency virus infection. Lancet 1987; 329: 1249–1253. 4. KIM JH, JUNG ES, KWON Y et al. Infection of porcine cells with human herpesviruses. Transplant Proc 2010; 42: 2134–2137. € L, SINZGER C et al. Efficiency of 5. MILLARD AL, HABERLI porcine endothelial cell infection with human cytomegalovirus depends on both virus tropism and endothelial cell vascular origin. Xenotransplantation 2010; 17: 274– 287. 6. GHIELMETTI M, MILLARD AL, HAEBERLI L et al. Human CMV infection of porcine endothelial cells increases adhesion receptor expression and human leukocyte recruitment. Transplantation 2009; 87: 1792–1800. 7. CHA CY, HWANG ES, KOOK YH. Production and characterization of monoclonal antibodies specific to herpes simplex viruses. J Korean Soc Microbiol 1988; 23: 505– 515. 8. NAHMIAS AJ, LEE FK, BECKMAN-NAHMIAS S. Sero-epidemiological and -sociological patterns of herpes simplex virus infection in the world. Scand J Infect Dis Suppl 1990; 69: 19–36.
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Supporting Information
Additional Supporting Information may be found in the online version of this article: Figure S1. Cytopathic effect (CPE) in 293 cells after co-culture with PBMCS treated with PMA (or) HHV-1.
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