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Journal of Clinical Microbiology, September 2004, p. 4300-4302, Vol. 42, No. 9
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.9.4300-4302.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Potential Screening Assay for Undetectable Viruses on the Basis of Their Capacity To Induce Alpha Interferon Production

Barbara Schmidt,1 Brittany Ashlock,1 Frank Neipel,2 and Jay A. Levy1*

Department of Medicine, Division of Hematology/Oncology, University of California, San Francisco, San Francisco, California,1 Institute of Clinical and Molecular Virology, German National Reference Centre for Retroviruses, Erlangen, Germany2

Received 18 March 2004/ Returned for modification 28 April 2004/ Accepted 13 May 2004


   ABSTRACT
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High interferon production by plasmacytoid dendritic cells (PDC) was unexpectedly noted after their coculture with CD4+ cells from a healthy donor whose cells subsequently showed human herpesvirus type 6 and 7 infections. This release of interferon was not observed with uninfected normal CD4+ cells. Induction of PDC interferon production could help screen for covert virus infections.


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During hematopoiesis, stem cells give rise to two dendritic cell precursors: myeloid and plasmacytoid dendritic cells (PDC). The latter cells are the major source of alpha interferons (IFN-{alpha}). In studying the mechanism of interferon induction by viruses and virus-infected cells, peripheral blood mononuclear cells (PBMC) were isolated from buffy coat bags obtained from healthy blood donors (Blood Centers of the Pacific, San Francisco, Calif.) by Ficoll-Hypaque gradient separation (Sigma Diagnostics Inc., St. Louis, Mo.) (1). PDC were purified from PBMC by using a BDCA-4 cell isolation kit (Miltenyi Biotec, Auburn, Calif.). Usually, 0.5 x 106 to 1 x 106 PDC were obtained from 500 x 106 PBMC. After a two-column separation, these cells were >95% pure as determined by flow cytometry. Isolated PDC were cultivated in RPMI 1640 medium containing 10% heat-inactivated (56°C, 30 min) fetal bovine serum, 2 mM glutamine, and 1% antibiotics (penicillin, 100 U/ml; streptomycin, 100 µg/ml), supplemented with 10 ng of interleukin-3 (R&D Systems, Minneapolis, Minn.) per ml. They were plated at a density of 104 cells/well in 96-well flat-bottom plates and then cocultivated with either UV-irradiated herpes simplex virus type 1 (HSV-1) (106 PFU/ml) or phytohemagglutinin (PHA)-stimulated CD4+ cells (6 x 104), infected with human immunodeficiency virus type 1 SF33 (HIV-1SF33) (6) for 2 days. Uninfected CD4+ cells served as negative controls. Supernatant was removed after 24 h and analyzed for IFN-{alpha} by enzyme-linked immunosorbent assay (Biosource International, Camarillo, Calif.). All assays were performed in duplicate.

Uninfected CD4+ cells only induced a mean IFN-{alpha} level of 8.2 pg/ml (0.9 to 15.5 pg/ml) in 30 separate assays. Unexpectedly, CD4+ cells obtained from one buffy coat donor (internal lab code, NL16176) induced significantly higher levels (mean, 2,818.5 pg/ml; 95% confidence level; 1,692.4 to 3,944.8 pg/ml; P = 0.0004 by the Mann-Whitney test) (Fig. 1). These IFN-{alpha} levels were comparable to HSV-1- and HIV-infected CD4+ cells, inducing 2,087.5 pg/ml (95% confidence level; 1,497.5 to 2,677.6 pg/ml) and 1,561.4 pg/ml (1,174.6 to 1,948.2 pg/ml), obtained in 17 and 31 separate assays, respectively.



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  		FIG. 1. IFN-{alpha} release by PDC after exposure to uninfected CD4+ cells from normal blood donors, CD4+ cells derived from donor NL16176 (see the text), CD4+ cells infected (inf.) with HIV-1SF33 (1) for 2 days, and irradiated HSV-1. Bars represent mean values.

 
When purified CD4+ cells from donor NL16176 were cultivated separately for 14 days, a cytopathic effect was observed with cell ballooning and multinucleated syncytia formation (Fig. 2A). This effect was reproduced three times by transferring filtered supernatant (0.2-µm-pore-size disposable syringe filters; Schleicher & Schuell, Keene, N.H.) to PHA-stimulated CD4+ cells from different blood donors. These newly infected CD4+ cells induced similar high levels of IFN-{alpha}. Electron microscopy revealed numerous enveloped viral particles inside and outside the cells, consistent with the size (diameter, approximately 180 nm) and appearance of herpesviruses (Fig. 2B). No virus budding was observed.



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  		FIG. 2. Identification of the viral pathogen in CD4+ cells of donor NL16176. (A) Light microscopy of cytopathic changes observed 2 weeks after PHA stimulation. (B) Electron microscopy of herpesvirus particles after the third passage of cell-free supernatant to CD4+ cells of a different donor. (C) Multiplex PCR containing primers for HHV-6 and HHV-7 (fragment sizes of 781 and 432 bp, respectively) for the original CD4+ cell culture (lane 1), passages 1 to 3 (lanes 2 to 4), an Epstein-Barr virus-containing B-lymphoblastoid cell line (lane 5), and the water negative control (lane 6). The M lane shows control size markers.

 
To identify the viral pathogen, 1 µg of DNA was isolated from the CD4+ cells showing the cytopathic effects using a QIAGEN DNA Mini kit (QIAGEN Inc., Valencia, Calif.) and then subjected to PCRs for human herpesvirus type 6 (HHV-6) and HHV-7. HHV-6 was amplified with primers VGP1 (5'-GTGGCGTTAAGACGGATTGT-3') and VGP1r (5'-ATCTATCCCTCGACTGCTTC-3'). The PCR was performed at 95°C for 4 min, followed by 35 cycles of 94°C for 20 s, 55°C for 30 s, and 72°C for 1 min with a final extension of 72°C for 4 min. HHV-7 was amplified with HHV-7p1 (5'-CGCTCTTCTAAACTAATTGTATCC-3') and HHV-7p1R (5'-GCTACGAGAACGAATCAAAAC-3') by using the same PCR conditions. The amplification products were at 781 and 432 bp, respectively. DNA isolated from an Epstein-Barr virus-containing B-lymphoblastoid cell line was used as a negative control. The CD4+ cells from donor NL16176 were positive for both viruses. Sequencing of the PCR products confirmed more than 99% homology with two HHV-6B strains (GenBank accession numbers AB021506 and AF157706) and two HHV-7 strains (U43400 and AF037218), respectively.

Dual infection was confirmed by immunofluorescence, by using sera with selective reactivity against either HHV-6 or HHV-7 to stain CD4+ cells from the third passage. Viral kinetics were analyzed by subjecting supernatants from different passages to a multiplex PCR containing primers for both viruses. HHV-7 was gradually overgrown by HHV-6 in subsequent passages (Fig. 2C). None of the supernatants or cells were positive for HSV-1 and -2, cytomegalovirus, and Epstein-Barr virus (data not shown). To check the frequency of HHV-6 and -7 reactivation, supernatants of cultured CD4+ cells from 11 different blood donors were analyzed 2 weeks after PHA stimulation. None tested positive in the multiplex PCR.

HHV-6 and -7 are beta-herpesviruses known for their latency in the human host after primary infection. The seroprevalence is greater than 95% (5). Both viruses are frequently reactivated together in immunocompromised patients (2) but rarely in the immunocompetent host. In vitro, HHV-7 (but not HHV-6B) can be reactivated from PBMC by T-cell stimulation; however, latent HHV-6B can be recovered after cells are infected with HHV-7 (3). This phenomenon most likely occurred to the PHA-stimulated CD4+ cells from donor NL16176. Viral outgrowth was not observed with CD4+ cells from other donors, suggesting that donor NL16176 did not control herpesvirus replication very well at that time. NL16176 was identified as a healthy male donor, without any indication of immunosuppression. HHV-6 and -7 could no longer be detected in this donor's CD4+ cells 4 months later. Unfortunately, it remains unclear which herpesvirus came first, because no early serum was available. However, serological testing showed signs of HHV-6 reactivation (HHV-6 to immunoglobulin G [IgG] at 1:256, IgM negative; HHV-7 to IgG at 1:32, IgM unspecific). Therefore, we believe this case reflects an acute HHV-7 infection.

Importantly, this viral infection was discovered by chance through PDC interferon production. The effect was observed before the CD4+ cells showed any cytopathic changes. The interferon induction was only noted when viral outgrowth was observed, suggesting that latent virus infection would not induce interferon production by PDC. When cell-free HHV-6 and -7 was used for interferon induction, only one out of three virus preparations, derived from CD4+ cells with prominent cytopathic effects, were active. This finding is consistent with reports from others, noting interferon induction only with high titers of virus (4). Thus, virus-infected cells appear to be the main inducers of interferon production, as we have observed with CD4+ cells and other cell types infected by HIV-1 (Schmidt et al., unpublished observation). We expect a similar phenomenon will be observed with other replicating viruses, particularly herpesviruses. This experimental setting may be an attractive approach in the search for undetected known and unknown viruses as a possible cause of human diseases (e.g., multiple sclerosis, rheumatoid arthritis, and acute lymphoproliferative disorders).


   ACKNOWLEDGMENTS
 
These studies were supported by the UCSF California AIDS Research Center (C-ARC), the Universitywide AIDS Research Program (UARP), and a fellowship to B. Schmidt from the Max Kade Foundation.

The UV-irradiated HSV-1 stock was provided by Yong-Jun Liu, DNA Research Institute, Palo Alto, California. The follow-up sample of donor NL16176 was provided by Nora Hirschler, Blood Centers of the Pacific. We thank Jo Dee Fish at the Gladstone Institute for technical assistance with the electron microscopy, Kyle Bonneau for support in sequencing, and Ann Murai and Kaylynn Peter for assistance in preparation of the manuscript.

None of the authors have a commercial or other association that might pose a conflict of interest.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Medicine, Division of Hematology/Oncology, University of California School of Medicine, San Francisco, CA 94143-1270. Phone: (415) 476-4071. Fax: (415) 476-8365. E-mail: jalevy{at}itsa.ucsf.edu. Back


   REFERENCES
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  3. Katsafanas, G. C., E. C. Schirmer, L. S. Wyatt, and N. Frenkel. 1996. In vitro activation of human herpesvirus 6 and 7 from latency. Proc. Natl. Acad. Sci. USA 93:9788-9792.[Abstract/Free Full Text]
  4. Kikuta, H., A. Nakane, H. Lu, Y. Taguchi, T. Minagawa, and S. Matsumoto. 1990. Interferon induction by human herpesvirus 7 in mononuclear cells. J. Infect. Dis. 162:35-38.[Medline]
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Journal of Clinical Microbiology, September 2004, p. 4300-4302, Vol. 42, No. 9
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.9.4300-4302.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.




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