1 Virology Laboratory, UPRES-JE 2437, Nantes University Hospital and UFR Sciences Pharmaceutiques, 9 quai Moncousu, 44093 Nantes Cedex, France
2 Haematology Department, Nantes University Hospital, France
3 Virology Laboratory, UPRES-EA2387, Pitie-Salpetriere, Paris University Hospital, France
4 Cell and Gene Therapy Unit, Nantes University Hospital, France
Correspondence
Berthe-Marie Imbert-Marcille
bmimbert{at}sante.univ-nantes.fr
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ABSTRACT |
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MAIN TEXT |
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The origin of monocyte infection has not yet been described and previous attempts to induce HHV-6 reactivation in in vitro semi-solid cultures of CD34+ cells have failed (Luppi et al., 1999). The use of liquid media to perform stem-cell culture has been described to favour virus replication in cells infected with high titres of hCMV strains (Maciejewski et al., 1992
; Movassagh et al., 1996
). In order to further our understanding of herpesvirus reactivation, we thus assessed whether HHV-6 infection occurs naturally (e.g. in the absence of exogenous superinfection) during in vitro culture of myeloid progenitor cells in liquid media supplemented with a combination of cytokines that are involved in haematopoiesis.
After informed and written consent, peripheral blood progenitor cells (PBPCs) were collected from ten patients undergoing leukapheresis after stem-cell mobilization with 5 µg granulocyte colony-stimulating factor (G-CSF) kg1 day1 (Neupogen; Amgen) for 5 days. Highly purified peripheral CD34+ cells were isolated by using the CliniMACS cell-selection system (Miltenyi Biotec) (Schumm et al., 1999). Purity was 99 %, except for culture no. 2 (96 %), as determined by flow cytometry. HHV-6 serostatus was determined by ELISA (HHV-6 IgG EIA; Biotrin).
Ex vivo expansion of 106 CD34+ PBPCs was carried out for 1421 days in 10 ml STEM ALPHA.AG medium (Stem Alpha) supplemented with 10 ng interleukin 1 (IL1), IL3, IL6, granulocytemacrophage colony-stimulating factor (GM-CSF), G-CSF and stem-cell factor (SCF) ml1. Every 7 days, non-adherent cells were removed and counted: 106 cells were replated in fresh medium and aliquots of 5x1051x106 cells were kept at 80 °C for subsequent molecular analysis. When available, additional aliquots were removed at the end of the culture period for cytospins (with MayGrünwaldGiemsa staining) and co-cultivation with peripheral blood mononuclear cells from an HHV-6-seronegative donor. Among the 11 ex vivo expansions (Table 1), six (cultures 0, 2, 4, 5, 9 and 10) were maintained for 21 days and exhibited an increase in total nucleated cells (TNCs) at the end of the culture period that ranged from three- to 216-fold (median was 68-fold). The others could not be expanded beyond 2 weeks (cultures 1, 3, 6, 7 and 8) and showed a 0·5- to sixfold increase in TNCs at the end of the culture period (median was onefold). As expected, a higher number of mature cells was obtained after 21 days; as cytokines were added to the media, these mature cells were mostly monocytes and immature granulocytes. These results are in accordance with those of a previous study that was conducted in liquid medium without serum (Mahe et al., 1996
). The fact that there was little or no expansion in some of the cultures was not related to age, sex or underlying disease of patients.
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Among the 10 cultures from HHV-6-seropositive patients, half expressed the spliced HHV-6 U100 mRNA (Fig. 1; Table 1
). The early, 371 bp, unspliced form was amplified at day 14 in three cultures: two of these cultures were stopped on that day (cultures 6 and 7) and a PCR inhibitor was detected at day 21 in the other culture (culture 10). The late, 238 bp, spliced form, which is indicative of a complete replication cycle, was detected at the end of the culture period in cultures 8 and 9. These data were confirmed in a second set of experiments and, because cultures were not performed at the same time, the possibility of viral cross-contamination can be excluded. HHV-6 DNA was not amplified from unexpanded CD34+ cells, as reported in another study for one-third of CD34+ samples (Luppi et al., 1999
). HHV-6 DNA was, however, detected in all mRNA-positive samples that were also evaluated for DNA amplification (cultures 810). This suggests indirectly that viral DNA was present in small amounts and became detectable with our PCR method during the differentiation process only when the DNA load increased. As our PCR methodology has previously been used in a clinical study to assess active infection (Imbert-Marcille et al., 2000
), this shows that samples were obtained from patients who were not actively infected at the time of PBPC collection. The length of amplified DNA fragments allowed us to confirm that HHV-6 strains were B variants in all cases. Our data thus demonstrate for the first time, and in the absence of in vitro cell infection, that HHV-6 can enter a replication cycle during haematopoietic differentiation. This last point was confirmed in culture 10, the only one for which a sufficient amount of cells was obtained to perform two complementary tests. The first one was a co-culture of a sample from day 21 with fresh peripheral blood mononuclear cells from an HHV-6-seronegative donor, which led to the recovery of infectious virus. This was confirmed by the appearance of the characteristic cytopathic effect and expression of a late HHV-6 antigen after 2 weeks co-culture (Fig. 2
). The second test was a real-time PCR that was performed as described previously (sensitivity, 10 copies per reaction) (Gautheret-Dejean et al., 2002
) with each sequential sample of this CD34+ expansion. An 18-fold increase in HHV-6 viral load was observed during week 3 of culture (401 copies in 106 cells at day 7, 416 copies in 106 cells at day 14 and 7296 copies in 106 cells at day 21), which is indicative of genome replication.
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ACKNOWLEDGEMENTS |
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Received 26 May 2004;
accepted 23 July 2004.
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