Limited infection without evidence of replication by porcine endogenous retrovirus in guinea pigs

Takele Argaw, Winston Colon-Moran and Carolyn A. Wilson

Laboratory of Immunology and Virology, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, 8800 Rockville Pike, HFM-725, Bethesda, MD 20892, USA

Correspondence
Carolyn Wilson
wilsonc{at}cber.fda.gov


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Porcine endogenous retrovirus (PERV) may potentially be transmitted through porcine xenotransplantation products administered to humans. This study examined the feasibility of using guinea pigs as a model to characterize the in vivo infectivity of PERV. To enhance the susceptibility of guinea pigs to retroviral infection or genomic integration, moderate physiological or immunological changes were induced prior to exposing the animals to PERV. Quantitative PERV-specific PCR performed on all tested samples resulted in either undetectable or very low copy numbers of proviruses, even in animals possessing PERV-specific antibody responses. The low copy number of viral DNA detected suggests that PERV infected a limited number of cells. However, PERV DNA levels did not increase over time, suggesting no virus replication occurred. These results in the guinea pig are similar to previous observations of non-human primate cells that allow PERV infection but do not support PERV replication in vitro.


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The supply of human organs is insufficient to meet the demand for transplantation and this has led some members of the scientific and medical communities to consider use of organs from animals, particularly from the pig (Lai et al., 2002; Tucker et al., 2002). Although data from clinical trials in pig-to-human xenotransplantation have not provided evidence for transmission of porcine endogenous retrovirus (PERV) (Loss et al., 2001; Paradis et al., 1999; Patience et al., 1998; Switzer et al., 2001), the possibility of cross-species transmission of PERV as well as other pig-derived infectious agents is still of concern in xenotransplantation procedures.

PERV is a gammaretrovirus present in the pig genome in multiple copies (Todaro et al., 1974). Several studies have demonstrated that porcine primary cells and continuous cell lines can release PERV virions that replicate in cells from pig, cat, mink and human (Martin et al., 2000; Patience et al., 1997; Takeuchi et al., 1998; Wilson et al., 1998). However, no studies, retrospective or prospective, have found evidence for transmission of PERV from porcine xenotransplantation products to human recipients (Heneine et al., 1998; Paradis et al., 1999; Patience et al., 1998; Schumacher et al., 2000; Tacke et al., 2001). Additionally, studies of PERV performed in small animals and non-human primates have been unsuccessful in finding a model that supports PERV replication (Specke et al., 2001; Switzer et al., 2001). The notable exceptions have been two studies in immunodeficient mice implanted with porcine islets, where evidence of limited PERV transmission to murine tissues was observed (Deng et al., 2000; van der Laan et al., 2000). In contrast, unpublished data presented by Dr David Onions at a meeting of the FDA's Subcommittee on Xenotransplantation: 13 January 2000 (Xenotransplantation Committee, 2000) provided evidence from an animal model where up to 70 000 copies of PERV DNA were detected in the spleen of guinea pigs several weeks after exposure to PERV in an immunization protocol. This preliminary finding suggested that guinea pigs might support active virus replication, in contrast to the negative results reported by Specke et al. (2001). However, there were some key experimental differences in how the animals were treated in these two studies. In particular, the unpublished data were based on an experiment meant to immunize the animals, while the study by Specke and coworkers did not use an immunizing strategy. Therefore, we sought to examine whether and under what circumstances guinea pigs may provide a feasible model to assess the in vivo replication properties of PERV.

In the studies described here, outbred strain Hartley guinea pigs (HARLAN), 2–3 weeks old, were used. The Institutional Animal Care and Use Committee (CBER/FDA) reviewed and approved all experiments.

The virus isolate used in all experiments was PERV-NIH, derived originally from NIH mini pigs as previously described (Wilson et al., 1998). Serial passage through HEK 293 cells resulted in a virus producer line capable of generating viral titres of >105 ml-1. The MoMLV-based retroviral vector genome G1BgSvN (McLachlin et al., 1993), encoding the bacterial lacZ gene, was introduced to generate the virus used in our experiments: PERV-NIH-16' {beta}-gal. These cells produce a mixture of PERV-NIH virions and pseudovirions composed of PERV-NIH core and envelope surrounding the MoMLV-based G1BgSvN genome. The presence of the G1BgSvN genome allows use of histochemical staining for lacZ activity to determine the infectious titre of viral stocks used in this study as previously described (Wilson & Eiden, 1991).

In a preliminary study two groups of animals (n=16) were exposed to 2x106 PERV-NIH producer cells or virus-containing supernatant. These animals were killed at different time points post-inoculation (up to a total of 4 weeks post-initial inoculation). Gross examination upon necropsy did not reveal any pathological changes. Genomic DNA was isolated from tissues and subjected to PCR analysis combined with Southern blotting (Wilson et al., 1998). Similar to the results of Specke et al. (2001), only rare positive results were obtained, without consistent positive results at different time points (data not shown). These results indicated that intact guinea pigs were not readily infected with PERV; we therefore sought to determine other experimental conditions that may enhance the susceptibility of guinea pigs to PERV infection.

Chemical stimulation or mild liver damage induces hepatocyte proliferation, increasing susceptibility to retroviral vector infection (Forbes et al., 1998; Kitten et al., 1997). To assess whether a similar regimen would increase susceptibility to PERV, we exposed guinea pigs to allyl alcohol (AA) (0·05 ml kg-1 intraperitoneally, i.p.), an agent known to induce mild liver necrosis (Werlich et al., 1999; Yin et al., 1999) prior to i.p. inoculation of PERV. Animals were inoculated i.p. with two doses of virus (4x105 blue-forming units, b.f.u.) at 24 and 48 h after AA administration. At each end time point (see Fig. 1) four animals were killed. As controls, two animals inoculated with PERV, without AA treatment, and one animal inoculated with complete DMEM only, were killed at each end time point. To measure the effectiveness of AA in inducing hepatocyte proliferation, we administered 50 mg kg-1 of BrdU (Sigma) to guinea pigs (n=16) 24 and 48 h post AA injection and 2 h prior to killing. Samples of liver tissues were randomly excised during postmortem examination and preserved for histochemistry and histopathological analysis. Liver sections (6–10 µm) were obtained from American HistoLabs (Gaithersburg, MD, USA) and subjected to immunohistochemical staining using a BrdU in situ detection kit (BD, BioSciences PharMinegen). The staining for BrdU-positive cells, as a measure of proliferative response, revealed a mean of 7·9±2·2 % positive hepatocytes in liver samples obtained from AA-treated animals killed 3 days post viral exposure (n=4) compared to 1·6±0·4 % positive hepatocytes in liver samples obtained from untreated animals (n=2). Additionally, a preliminary study performed on three animals to assess the effect of AA treatment on liver function resulted in a 100–115 % and 55–60 % increase in blood level of liver enzymes (aspartate transaminase and alanine transaminase) at 24 h and 48 h post AA administration, respectively, relative to untreated controls, indicating liver damage.



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Fig. 1. Experimental design for guinea pig infectivity studies. (A) Induction of hepatocyte proliferation with AA followed by two exposures to virus; (B) enhancing the immunity to PERV by injecting disrupted virus mixed with CFA. Treatments are shown as indicated: D, day; Wk, week; AA, allyl alcohol; V, virus; No, no treatment; CFA, complete Freund's adjuvant. All inoculations of virus only were i.p.

 
PERV pol-specific real-time quantitative PCR (qPCR) was performed, as previously described (Argaw et al., 2002), on genomic DNA (gDNA) isolated from tissue samples obtained from animals killed at the indicated time points post-treatment. Within each tissue type, the analysis yielded variable results, ranging from 0 to 433 copies of viral DNA per 100 ng gDNA tested. Generally, the highest detectable copy numbers of the virus and the greatest number of PERV-positive tissues per animal were observed at early time points, 3–7 days post viral exposure, decreasing with later time points (Table 1). Treatment of guinea pigs with AA did not induce an increase in infectivity in animals with evidence of liver damage, although in other studies mild and transient liver damage has resulted in proliferation of hepatocytes that eased retrovirus cellular transduction and augmented transgene expression (Kitten et al., 1997; Rettinger et al., 1994).


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Table 1. Quantitative PCR analysis and biodistribution of PERV DNA in AA-treated and untreated animals

 
We next performed a study based on the unpublished results of D. Onions (Xenotransplantation Committee, 2000) to see whether an initial immunizing dose of PERV might influence the outcome of infection when animals were later inoculated with live PERV. While typically immunization would be expected to prevent or reduce infectivity, it has also been postulated that virus-specific antibodies may enhance infectivity (Morens, 1994; Olsen, 1993). The previous PERV study in guinea pigs (Xenotransplantation Committee, 2000) detected 3000–70 000 copies of viral DNA per 106 cells in spleen tissue isolated from guinea pigs immunized with complete Freund's adjuvant (CFA)-treated PERV followed by a booster dose with live PERV. To determine whether an anti-PERV antibody response may enhance virus replication, we first immunized guinea pigs to PERV by inoculating subcutaneously with an equal volume mixture of CFA and PERV virions (5x105 b.f.u.). Four weeks later, animals were inoculated with the same dose level of live PERV in the absence of adjuvant. Animals were bled and killed at the time points indicated in Table 2 and Fig. 1, to collect serum and different tissue samples for immunoassay and DNA extraction. While low levels of viral DNA were detected in some samples at 8 and 12 weeks after the initial immunization (4 and 8 weeks after live PERV inoculation), no DNA was detected in any samples examined from animals killed 16 weeks post-inoculation (Table 2).


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Table 2. Tissue distribution of PERV pol DNA in tissues of animals exposed to virus (5x105 infectious virions) after immunization with PERV and complete Freund's adjuvant (CFA) subcutaneously

 
Guinea pig plasma samples were analysed for the presence of PERV-specific antibodies by Western blot assay against whole virion lysate, as previously described (Tacke et al., 2001). Anti-SSAV p30 goat serum (lot 81S000315, NCI repository, Quality Biotech) served as a positive control to run the immunoassay. Analysis of anti-p27 PERV antibody demonstrated that the immunization strategy was effective: specific anti-PERV antibodies were detected in animals inoculated with PERV, but not in control uninoculated animals (data not shown). Immunological modulation has also been reported to enhance the infectivity of some viruses via antibody-dependent enhancement (ADE) of infection (reviewed by Morens, 1994; Olsen, 1993) whereby the entry of virus–antibody complexes into cells results in significantly increased virus infectivity. This phenomenon has been experimentally correlated with enhanced infectivity of several viruses, including dengue virus (Morens & Halstead, 1990), Ross river virus (Lidbury & Mahalingam, 2000), HIV (Kozlowski et al., 1995), and feline infectious peritonitis virus (Olsen, 1993). In our experiment, animals were immunized with PERV mixed with CFA, to determine whether ADE of PERV infection occurs. However, we observed that presence of anti-PERV antibodies did not enhance PERV infection of guinea pigs, as demonstrated by the lack of differences in viral DNA copy number per tissue detected in animals treated with CFA or untreated prior to inoculation with live PERV (Table 2).

Viral inoculation of naïve animals may lead to persistent, transient, or no evidence of infection (Jilbert et al., 1998; Zinkernagel, 1996). We found that exposure of guinea pigs to PERV-NIH produces only a transient low-level viral infection, as measured by detection of viral DNA most consistently at early time points, and with decreased frequency and copy number at later time points after viral inoculation (Tables 1, and 2). In addition, gross examination of organs during necropsy and limited histopathological examinations did not reveal any virus-induced gross or microscopic lesions in any animals examined.

The detection of PERV viral DNA in some tissues indicates that PERV may be able to infect guinea pig cells. Either tightly controlled suppression of virus replication or a potent host clearance mechanism against PERV may explain the reduced levels of viral DNA detected at later time points. The latter interpretation is supported by the durable humoral immunity observed in animals (data not shown) during the time-course of the experiment (16 weeks).

In a recent study of non-human primate cells exposed to PERV, we found that PERV infection was restricted, resulting in low copy numbers of viral DNA and lack of virus replication (Ritzhaupt et al., 2002). The results reported here from the guinea pig studies, where only a low copy number of viral DNA was detected, implies that a similar mechanism restricting virus replication may operate in guinea pigs. Although it is out of the scope of this study, the adaptation of viral stocks by serial passage in guinea pig tissues might increase the replication capacity of PERV.

Apart from two reports (Deng et al., 2000; van der Laan et al., 2000) showing limited transmission of PERV to immunodeficient mice, no other conventional laboratory small animal had been previously well investigated for susceptibility to PERV infection. After a preliminary study on intact animals, we have investigated whether immunological or physiological manipulations can increase susceptibility to PERV replication in guinea pigs. Our study clearly demonstrates that guinea pigs are refractory to PERV replication.


   ACKNOWLEDGEMENTS
 
We thank Oliver Zill and Elizabeth Skelton for providing expert technical assistance. We are also indebted to the animal care unit of the Division of Veterinary Service of CBER/FDA for their valuable laboratory animal services and excellent animal care. We thank Drs Eda Bloom and Steven Bauer for manuscript review and valuable comments.


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Received 8 July 2003; accepted 30 September 2003.



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