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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ABSTRACT |
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MAIN TEXT |
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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 In contrast, unpublished data presented by Dr David Onions at a meeting of the FDA's Subcommittee on Xenotransplantation: 13 January 2000 (Xenotransplantation Committee, 2000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 In particular, the unpublished data were based on an experiment meant to immunize the animals, while the study by Specke and coworkers did not use an immunizing strategy.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), 23 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'
-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 (610 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 100115 % and 5560 % 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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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.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 8 July 2003;
accepted 30 September 2003.
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