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Journal of Virology, September 2008, p. 8951-8953, Vol. 82, No. 17
0022-538X/08/$08.00+0     doi:10.1128/JVI.00929-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

LETTER TO THE EDITOR

Human B19 Erythrovirus In Vitro Replication: What's New?

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Human B19 erythrovirus (B19) tropism is almost restricted to erythroid progenitor cells and mediated by receptor and coreceptor interactions: i.e., P antigen (2, 3), ₅β₁ integrin complex (20), and Ku80 antigen (11). Few in vitro models allow production of B19 infectious particles and study of the virus life cycle. The first one involved the use of bone marrow erythroid cells (12, 13, 18). Infection of the UT7 cell line, derived from an erythroleukemia clone (8, 16), has been generally employed (5, 7, 9, 21, 23, 24); however, B19 replication is thus limited to a particular cell clone and at a level that cannot allow mass production of B19. Other erythroleukemia cell lines have been used (1, 10, 19), without demonstrating their clear usefulness in B19 production (4). Thus, the major source of virus is currently obtained from sera of viremic patients.

The recent report by Wong et al. describes B19 replication from CD36⁺ erythroid progenitors (22) obtained from mobilized blood and claims to be "the first description of an ex vivo method to produce large numbers of erythroid progenitor cells that are highly permissive to B19 infection and replication." However, we previously showed that such primary cells constituted a major tool to explore B19 expression and replication, as well as to produce B19 infectious particles.

CD36⁺ erythroid progenitor cells were generated by Freyssinier et al. in 1999 from CD34⁺ peripheral adult or cord blood stem cells (6). The cells consisted of 96% late erythroid burst-forming units and erythroid colony-forming cells: i.e., the main in vivo targets of B19. After infection of these CD36⁺ erythroid progenitor cells with B19 infectious particles, we showed the interaction of NS1 protein with the tumor necrosis factor alpha pathway leading to apoptosis of the infected erythroid progenitor cells (17). Infection of CD36⁺ cells with B19 also allowed expression of VP1 and VP2 capsid proteins in about 50% of the cells 24 h postinfection (14, 17). B19 protein expression was enhanced when CD36⁺ erythroid cells were exposed to low oxygen tension (15). We also showed that hypoxia increases B19 replication, leading to the production of higher titers of B19 infectious particles. This phenomenon resulted, at least in part, from an interaction of the major cellular factor implicated in oxygen tension response, hypoxia-inducible factor 1, with the P6 promoter. All of these studies that showed the usefulness of CD36⁺ erythroid progenitor cells to produce B19 infectious particles as well as to be a relevant model to explore B19 infectious cycle should have been taken into account.

Therefore, we believe that the method described in the study by Wong et al. (22) as published in the March 2008 issue of the Journal of Virology cannot be considered a "new tool" for B19 replication and that the study does not provide original information concerning the in vitro generation of B19 virus by erythroid progenitors.

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6. Freyssinier, J. M., C. Lecoq-Lafon, S. Amsellem, F. Picard, R. Ducrocq, P. Mayeux, C. Lacombe, and S. Fichelson. 1999. Purification, amplification and characterization of a population of human erythroid progenitors. Br. J. Haematol. 106:912-922.[CrossRef][Medline]
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7. Gallinella, G., E. Manaresi, E. Zuffi, S. Venturoli, L. Bonsi, G. P. Bagnara, M. Musiani, and M. Zerbini. 2000. Different patterns of restriction to B19 parvovirus replication in human blast cell lines. Virology 278:361-367.[CrossRef][Medline]
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9. Leruez, M., C. Pallier, I. Vassias, J. F. Elouet, P. Romeo, and F. Morinet. 1994. Differential transcription, without replication, of non-structural and structural genes of human parvovirus B19 in the UT7/EPO cell as demonstrated by in situ hybridization. J. Gen. Virol. 75:1475-1478.[Abstract/Free Full Text]
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10. Miyagawa, E., T. Yoshida, H. Takahashi, K. Yamaguchi, T. Nagano, Y. Kiriyama, K. Okochi, and H. Sato. 1999. Infection of the erythroid cell line, KU812Ep6, with human parvovirus B19 and its application to titration of B19 infectivity. J. Virol. Methods 83:45-54.[CrossRef][Medline]
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11. Munakata, Y., T. Saito-Ito, K. Kumura-Ishii, J. Huang, T. Kodera, T. Ishii, Y. Hirabayashi, Y. Koyanagi, and T. Sasaki. 2005. Ku80 autoantigen as a cellular coreceptor for human parvovirus B19 infection. Blood 106:3449-3456.
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12. Ozawa, K., G. Kurtzman, and N. Young. 1987. Productive infection by B19 parvovirus of human erythroid bone marrow cells in vitro. Blood 70:384-391.[Abstract/Free Full Text]
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13. Ozawa, K., G. Kurtzman, and N. Young. 1986. Replication of the B19 parvovirus in human bone marrow cell cultures. Science 233:883-886.[Abstract/Free Full Text]
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14. Pillet, S., Z. Annan, S. Fichelson, and F. Morinet. 2003. Identification of a nonconventional motif necessary for the nuclear import of the human parvovirus B19 major capsid protein (VP2). Virology 306:25-32.[CrossRef][Medline]
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15. Pillet, S., N. Le Guyader, T. Hofer, F. NguyenKhac, M. Koken, J. T. Aubin, S. Fichelson, M. Gassmann, and F. Morinet. 2004. Hypoxia enhances human B19 erythrovirus gene expression in primary erythroid cells. Virology 327:1-7.[CrossRef][Medline]
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22. Wong, S., N. Zhi, C. Filippone, K. Keyvanfar, S. Kajigaya, K. E. Brown, and N. S. Young. 2008. Ex vivo-generated CD36⁺ erythroid progenitors are highly permissive to human parvovirus B19 replication. J. Virol. 82:2470-2476.[Abstract/Free Full Text]
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23. Zhi, N., I. P. Mills, J. Lu, S. Wong, C. Filippone, and K. E. Brown. 2006. Molecular and functional analyses of a human parvovirus B19 infectious clone demonstrates essential roles for NS1, VP1, and the 11-kilodalton protein in virus replication and infectivity. J. Virol. 80:5941-5950.[Abstract/Free Full Text]
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24. Zhi, N., Z. Zadori, K. E. Brown, and P. Tijssen. 2004. Construction and sequencing of an infectious clone of the human parvovirus B19. Virology 318:142-152.[CrossRef][Medline]

						Sylvie Pillet* Serge Fichelson Frédéric Morinet Laboratory of Virology CHU of Saint-Etienne Saint Priest en Jarez 42270, France
						* Phone: 33 4 77 82 81 22, Fax: 33 4 77 82 84 60, E-mail: sylvie.pillet{at}univ-st-etienne.fr

Authors’ Reply

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We appreciate Dr. Pillet's interest in our recent report published in the journal.

Since our discoveries that B19 parvovirus (B19V) inhibited human erythroid colony formation and that the virus could be propagated in human bone marrow (4-6), numerous approaches have been undertaken to culture the virus in vitro. In our current paper, we cite (3, 5, 6, 10-12) the findings of other groups concerning the use of human peripheral blood or hematopoietic cells derived from bone marrow or fetal liver for this purpose. However, in general, neither primary cells nor cell lines have been efficient for B19V propagation. In unselected bone marrow, blood, and fetal liver, only a subset of cells is permissive for B19V infection. These primary cells have not been useful to generate the virus in practical quantities. Indeed, as Dr. Pillet states, erythroid colony-forming cells and CD36⁺ erythroid progenitors generated from circulating CD34⁺ cells (1, 2), while employed in B19V infectivity assays (7-10), have not been assessed for their abilities to generate large quantities of virus. The lack of an efficient and sensitive culture system has been a serious obstacle in B19V research.

Based on modification of recent advances in obtaining relatively pure populations of erythroid progenitor cells (EPCs), we established a simplified cell culture system which is highly permissive to B19V infection. In comparison with previously published papers, our study focused on (i) large-scale ex vivo generation of pure CD36⁺ EPCs, (ii) determination of the differentiation stage of erythroid progenitors that would be optimal for B19V infection, and (iii) assessment of the capacity of such a system to generate large quantities of B19V.

As described in the Materials and Methods section of our paper, we first established a procedure for ex vivo generation of a large number of EPCs by modifying the Freyssinier method, which was used by Pillet et. al in their studies (7, 8). Specifically, our culture system does not contain interleukin-6 but includes growth factors that were not present in the Freyssinier method (hydrocortisone, ferrous sulfate, ferrous nitrate, and erythropoietin used throughout the entire culture period). As a result, we were able to amplify our cells >200-fold by day 8 and 800,000-fold by day 19 (Fig. 1). (Amplification of CD34⁺ cells by the Freyssinier method was significantly less efficient.) The EPCs generated ex vivo without additional manipulation after initial placement in culture were >90% CD36⁺ on day 8 (and designated CD36⁺ EPCs in our paper), whereas the Freyssinier method required column purification to obtain a pure population of CD36⁺ cells. By immunophenotype, our cells were predominantly CD36⁺/globoside⁺ by day 5 (see Fig. 2 in our article), while this proportion was only 28.8% ± 8.2% recovered on day 7 by the Freyssinier method (see Table 3 in their article [1]).

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FIG. 1. Amplification of EPCs from human CD34+ cells isolated from granulocyte colony-stimulating factor-mobilized peripheral blood stem cells. Cells were cultured in a serum-free expansion medium supplemented with growth factors. After 4 days of culture in expansion medium, 1 volume of cell culture was expanded into 4 volumes with fresh medium. Otherwise, the cultured cells were cryopreserved and revived when needed. Total numbers of viable cells were determined at different times using a standard hemocytometer averaging four to five fields. The results shown are representative of one set of CD34+ cells (day 0 cells) and one set of cells frozen on day 4 (day 4 cells) and revived.

Second, it should be emphasized that cell surface expression of CD36 does not confer permissivity to B19V. Under the culture conditions of our study, cells continuously differentiated from CD34⁺ hematopoietic stem cells through EPCs to mature red blood cells. They were highly permissive for B19V infection only in a narrow window from days 5 to 9 and then gradually lost permissivity after day 10—despite remaining CD36 positive. Based on these data, we concluded that day 8 CD36⁺ EPCs were optimal for viral infection, amplified to a reasonable scale (>200-fold) and fully expressing (87%) the B19V receptor, globoside, on the cell surface.

Finally, we assessed viral production of ex vivo-generated CD36⁺ EPCs. We showed that after 8 days in culture, nearly 80% of the population was infected by B19V and viral DNA replication increased 100- to 1,000-fold.

In summary, we believe that our results indeed are novel in their differences from previously published descriptions of B19V propagation in blood cells and in describing a relatively simple, easily reproduced, and highly productive system for B19V culture in vitro that should be useful to generate large quantities of virus for the study of this human pathogen and potentially also for vaccine production.

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1. Freyssinier, J. M., C. Lecoq-Lafon, S. Amsellem, F. Picard, R. Ducrocq, P. Mayeux, C. Lacombe, and S. Fichelson. 1999. Purification, amplification and characterization of a population of human erythroid progenitors. Br. J. Haematol. 106:912-922.[CrossRef][Medline]
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2. Giarratana, M. C., L. Kobari, H. Lapillonne, D. Chalmers, L. Kiger, T. Cynober, M. C. Marden, H. Wajcman, and L. Douay. 2005. Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells. Nat. Biotechnol. 23:69-74.[CrossRef][Medline]
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5. Ozawa, K., G. Kurtzman, and N. Young. 1986. Replication of the B19 parvovirus in human bone marrow cell cultures. Science 233:883-886.[Abstract/Free Full Text]
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6. Ozawa, K., G. Kurtzman, and N. Young. 1987. Productive infection by B19 parvovirus of human erythroid bone marrow cells in vitro. Blood 70:384-391.[Abstract/Free Full Text]
7
7. Pillet, S., Z. Annan, S. Fichelson, and F. Morinet. 2003. Identification of a nonconventional motif necessary for the nuclear import of the human parvovirus B19 major capsid protein (VP2). Virology 306:25-32.[CrossRef][Medline]
8
8. Pillet, S., G. N. Le, T. Hofer, F. NguyenKhac, M. Koken, J. T. Aubin, S. Fichelson, M. Gassmann, and F. Morinet. 2004. Hypoxia enhances human B19 erythrovirus gene expression in primary erythroid cells. Virology 327:1-7.[CrossRef][Medline]
9
9. Sol, N., J. Le Junter, I. Vassias, J. M. Freyssinier, A. Thomas, A. F. Prigent, B. B. Rudkin, S. Fichelson, and F. Morinet. 1999. Possible interactions between the NS-1 protein and tumor necrosis factor alpha pathways in erythroid cell apoptosis induced by human parvovirus B19. J. Virol. 73:8762-8770.[Abstract/Free Full Text]
10
10. Sugawara, H., R. Motokawa, H. Abe, M. Yamaguchi, Y. Yamada-Ohnishi, J. Hirayama, H. Sakata, S. Sato, N. Kamo, K. Ikebuchi, and H. Ikeda. 2001. Inactivation of parvovirus B19 in coagulation factor concentrates by UVC radiation: assessment by an in vitro infectivity assay using CFU-E derived from peripheral blood CD34⁺ cells. Transfusion 41:456-461.[CrossRef][Medline]
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11. Takahashi, T., K. Ozawa, K. Takahashi, S. Asano, and F. Takaku. 1990. Susceptibility of human erythropoietic cells to B19 parvovirus in vitro increases with differentiation. Blood 75:603-610.[Abstract/Free Full Text]
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12. Yaegashi, N., H. Shiraishi, T. Takeshita, M. Nakamura, A. Yajima, and K. Sugamura. 1989. Propagation of human parvovirus B19 in primary culture of erythroid lineage cells derived from fetal liver. J. Virol. 63:2422-2426.[Abstract/Free Full Text]

						Neal S. Young* Ning Zhi Susan Wong National Heart, Lung, and Blood Institute National Institutes of Health 9000 Rockville Pike Bethesda, Maryland 20892
						* Phone: (301) 496-5093, Fax: (301) 496-8396, E-mail: youngn{at}nhlbi.nih.gov

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Journal of Virology, September 2008, p. 8951-8953, Vol. 82, No. 17
0022-538X/08/$08.00+0     doi:10.1128/JVI.00929-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Pillet, S. Articles by Wong, S. Search for Related Content PubMed PubMed Citation Articles by Pillet, S. Articles by Wong, S.