Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract 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 HighWire Citing Articles via Google Scholar Google Scholar Articles by Bente, D. A. Articles by Rico-Hesse, R. Search for Related Content PubMed PubMed Citation Articles by Bente, D. A. Articles by Rico-Hesse, R.

 Previous Article  |  Next Article 

Journal of Virology, November 2005, p. 13797-13799, Vol. 79, No. 21
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.21.13797-13799.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Dengue Fever in Humanized NOD/SCID Mice

Dennis A. Bente,¹ Michael W. Melkus,² J. Victor Garcia,² and Rebeca Rico-Hesse^1*

Southwest Foundation for Biomedical Research, 7620 NW Loop 410, San Antonio, Texas 78227,¹ University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, Texas 75390²

Received 5 July 2005/ Accepted 8 August 2005

Top Abstract Text References

 
The increased transmission and geographic spread of dengue fever (DF) and its more severe presentation, dengue hemorrhagic fever (DHF), make it the most important mosquito-borne viral disease of humans (50 to 100 million infections/year) (World Health Organization, Fact sheet 117, 2002). There are no vaccines or treatment for DF or DHF because there are no animal or other models of human disease; even higher primates do not show symptoms after infection (W. F. Scherer, P. K. Russell, L. Rosen, J. Casals, and R. W. Dickerman, Am. J. Trop. Med. Hyg. 27:590-599, 1978). We demonstrate that nonobese diabetic/severely compromised immunodeficient (NOD/SCID) mice xenografted with human CD34⁺ cells develop clinical signs of DF as in humans (fever, rash, and thrombocytopenia), when infected in a manner mimicking mosquito transmission (dose and mode). These results suggest this is a valuable model with which to study pathogenesis and test antidengue products.

Top Abstract Text References

 
Dengue virus infections in humans can be subclinical or can cause illnesses ranging from a mild, flu-like syndrome with rash and some hemorrhagic manifestations (dengue fever [DF]) to a severe and sometimes fatal disease, with coagulopathy, capillary leakage, and hypovolemic shock (dengue hemorrhagic fever [DHF]). The development of dengue vaccines and antivirals is complicated by the viruses ' diversity: they would have to protect against four different serotypes and numerous genotypes within each serotype. We have shown that dengue viruses differ in their rates of infection and replication in primary human cells, and these rates vary from one donor to another (3). In addition, secondary infection by a heterologous virus enhances disease severity, and multivalent vaccine preparations could prove dangerous to vaccinees if they induce subneutralizing levels of antibodies (5). Thus, it is hypothesized that viral and host (genetic and prior immune status) factors contribute to dengue pathogenesis, but there has been no in vivo system in which to measure their contributions (9).

Little is understood about the events that lead to DF or DHF after an infected mosquito (mainly Aedes aegypti) bites and injects virus into the human epidermis. The main targets for viral infection and replication seem to be dendritic cells (DCs) and macrophages, mainly Langerhans and monocyte-derived DCs (7, 13). A full repertoire of DCs develops in nonobese diabetic/severely compromised immunodeficient (NOD/SCID) mice transplanted with human cord blood hematopoietic progenitor (CD34⁺) cells (4, 8). The NOD/SCID strain of mice lacks T and B cells and has defects in natural killer cell function and antigen-presenting cell development and function and genetically lacks C5, resulting in a deficiency in hemolytic complement; it therefore provides an excellent environment for reconstitution with human hematopoetic cells and tissues.

All animal procedures were reviewed and approved by our Institutional Animal Care and Use Committees.

CD34⁺ cells were isolated from human cord blood with immunomagnetic beads and transplanted into preconditioned mice (325 rads) as we have previously described (4, 8). Mice were analyzed for reconstitution with human cells (CD45⁺) and for the presence of human DCs (defined as CD11c⁺ or CD123⁺ lineage negative, HLA-DR⁺) in peripheral blood by flow cytometry using human-specific antibodies, as we have previously described (4, 8). We obtained high levels of reconstitution and DC development in multiple organs in these mice: human CD45⁺ cells in whole blood before inoculation (8+ weeks posttransplant) ranged from 0.9 to 43% (n = 18; median, 14%). A total of 18 reconstituted NOD/SCID mice were inoculated subcutaneously with approximately 4.7 log₁₀ PFU (7.7 log₁₀ genome equivalents) of dengue serotype 2 strain K0049 (Southeast Asia genotype; C6/36 cell passage 3); this virus was selected because it grows to high levels in human DCs in vitro (3). Six reconstituted and 15 nonreconstituted NOD/SCID mice (same genetic background but not receiving human CD34⁺ cells) served as negative controls (inoculated with saline, C6/36 cell culture supernatant, or virus). The most dramatic signs of dengue infection in humanized mice were erythema and thrombocytopenia (Fig. 1a), as occurs in humans. We measured erythema using a DermaSpectrometer (Cortex Technology, Denmark) while mice were anesthesized; when compared to nonreconstituted, infected mice, erythema mean values were significantly different on days 1 through 8 (all pairwise t tests, P < 0.05; reconstituted mouse range, –1.36 to 8.44; nonreconstituted mouse range, –5.35 to –1.51). A marked decrease in platelets was measurable on day 8 postinoculation and was statistically highly significant (Mann-Whitney test, P < 0.001) when comparing six infected, reconstituted mice (range, 320,000 to 594,000/mm³) to three nonreconstituted, infected and six reconstituted, noninfected mice (normal mouse range, 600,000 to 1,200,000/mm³; range for our negative controls, 610,000 to 1,251,000/mm³). Rash was visible on days 2 to 4 in the majority (eight of nine) of infected, reconstituted mice and continued through day 14 in some mice (Fig. 1c). Viremias were measured in sera (25- to 50-µl retro-orbital bleed on alternate days) by quantitative, real-time reverse transcription-PCR (RT-PCR) (Fig. 1b) (11); virus levels peaked on days 2 to 6 postinoculation (range, 4.2 to 5.4 log₁₀ genome equivalents/ml). A rise in body temperature followed the viremia, and temperature returned to normal levels on day 10. For reconstituted mice, changes in temperature were statistically significant by analysis of variance (P < 0.02); for nonreconstituted mice, changes in temperature were not significant by analysis of variance (P >> 0.05). Nonreconstituted, infected NOD/SCID mice showed consistently lower viremias (undetectable by day 10; range, 3.6 to 4.1 log₁₀ genome equivalents/ml) and, as noted above, showed no rash, rise in temperature, or decrease in platelets and therefore served as negative controls for statistical analyses throughout. Body weight decreased dramatically in some reconstituted, infected animals (20% loss in four mice); there were no gastrointestinal abnormalities on necropsy, and weight loss seemed to be due to lethargy or lack of eating at the time of viremia or fever. Several organs were tested for viral positive- and negative-strand RNA template (surrogate for replicating virus) by quantitative, real-time RT-PCR (3) at different times postinfection in reconstituted and nonreconstituted mice. Only the reconstituted, infected mice had dengue virus RNA in various tissues on day 8: spleen (six of six mice), liver (three of six mice), and skin (one of six mice) had positive-strand RNA, while negative-strand RNA was detectable in spleen (two of six mice) and skin (one of six mice) (Fig. 2). These data are consistent with viral replication in this model.

View larger version (41K): [in this window] [in a new window]

FIG. 1. Signs of dengue virus infection in humanized NOD/SCID mice. (a) Thrombocytopenia and erythema in infected mice by day 8 (means and standard errors of the means); platelet levels in nonreconstituted, infected and noninfected ("normal"; n = 9) versus reconstituted, infected (n = 6); and erythema development in the latter group. O.D., optical density. (b) Viremia and fever on days 1 to 18 (mean and standard error) in mice reconstituted with human CD34+ cells. (c) Comparison of rashes on the backs of shaved mice. The nonreconstituted, infected mouse is on the left, and the reconstituted, infected mouse is on the right (both day 7 postinfection).

View larger version (16K): [in this window] [in a new window]

FIG. 2. Comparison of dengue virus RNA in reconstituted, infected NOD/SCID mice (hu-SCID) and nonreconstituted, infected NOD/SCID mice (SCID) on day 8. Virus positive-strand RNA was estimated in each organ (samples in triplicate), using quantitative, real-time RT-PCR, as described in reference 3. The dotted line reflects the limit of detection: approximately 60 genome equivalents per organ sample.

Others have used transplanted SCID or inbred AG129 or A/J mice to show variable thrombocytopenia, paralysis, or death after injection of massive amounts (8 log₁₀ PFU, intravenous) of cell-culture-passaged dengue virus or smaller amounts (4 to 6 log₁₀ PFU, intraperitoneal) of mouse-adapted dengue virus; however, these mice did not show signs of dengue disease as in humans (1, 2, 6, 10). The model described here is therefore highly relevant to human infection and can now be used to test antiviral preparations and vaccine attenuation. It may eventually also prove useful for understanding the immunopathogenesis (antibody-dependent enhancement of infection and cross-reactive T-cell activation) of DHF, after transplantation of other factors or tissues, to obtain a functional adaptive immune system.

 
We thank R. Wolf and staff at the University of Texas Health Science Center at San Antonio for animal facilities and care, A. Padgett-Thomas and M. del P. Martin for transplantation of mice, and M. Sharp and A. Hopstetter for statistics and graphics. Cord blood samples were obtained through the Department of Obstetrics and Gynecology's Tissue Procurement Facility of the University of Texas Southwestern Medical Center at Dallas (NIH grant HD-011149).

Funding was provided by The Ellison Medical Foundation and NIH grants AI50123 (to R.R.-H.) and CA82055 (to J.V.G.).

 
* Corresponding author. Mailing address: Department of Virology and Immunology, Southwest Foundation for Biomedical Research, 7620 NW Loop 410, San Antonio, TX 78227. Phone: (210) 258-9681. Fax: (210) 258-9776. E-mail: rricoh{at}sfbr.org.

Top Abstract Text References

 

1
1. An, J., J. Kimura-Kuroda, Y. Hirabayashi, and K. Yasui. 1999. Development of a novel mouse model for dengue virus infection. Virology 263:70-77.[CrossRef][Medline]
2
2. Blaney, J. E., Jr., C. T. Hanson, K. A. Hanley, B. R. Murphy, and S. S. Whitehead. 4 October 2004, posting date. Vaccine candidates derived from a novel infectious cDNA clone of an American genotype dengue virus type 2. BMC Infect. Dis. 4:39. [Online.] doi:10.1186/1471-2334-4-39.[CrossRef][Medline]
3
3. Cologna, R., P. M. Armstrong, and R. Rico-Hesse. 2005. Selection for virulent dengue viruses occurs in humans and mosquitoes. J. Virol. 79:853-859.[Abstract/Free Full Text]
4
4. Cravens, P. D., M. W. Melkus, A. Padgett-Thomas, M. Islas-Ohlmayer, M. del P. Martin, and J. V. Garcia. 2005. Development and activation of human dendritic cells in vivo in a xenograft model of human hematopoiesis. Stem Cells 23:264-278.[Abstract/Free Full Text]
5
5. Halstead, S. B., and J. Deen. 2002. The future of dengue vaccines. Lancet 360:1243-1245.[CrossRef][Medline]
6
6. Johnson, A. J., and J. T. Roehrig. 1999. New mouse model for dengue virus vaccine testing. J. Virol. 73:783-786.[Abstract/Free Full Text]
7
7. Kwan, W. H., A. M. Helt, C. Maranon, J. B. Barbaroux, A. Hosmalin, E. Harris, W. H. Fridman, and C. G. Mueller. 2005. Dendritic cell precursors are permissive to dengue virus and human immunodeficiency virus infection. J. Virol. 79:7291-7299.[Abstract/Free Full Text]
8
8. Palucka, A. K., J. Gatlin, J. P. Blanck, M. W. Melkus, S. Clayton, H. Ueno, E. T. Kraus, P. Cravens, L. Bennett, A. Padgett-Thomas, F. Marches, M. Islas-Ohlmayer, J. V. Garcia, and J. Banchereau. 2003. Human dendritic cell subsets in NOD/SCID mice engrafted with CD34+ hematopoietic progenitors. Blood 102:3302-3310.[Abstract/Free Full Text]
9
9. Scherer, W. F., P. K. Russell, L. Rosen, J. Casals, and R. W. Dickerman. 1978. Experimental infection of chimpanzees with dengue viruses. Am. J. Trop. Med. Hyg. 27:590-599.
10
10. Shresta, S., J. L. Kyle, P. Robert Beatty, and E. Harris. 2004. Early activation of natural killer and B cells in response to primary dengue virus infection in A/J. mice. Virology 319:262-273.[CrossRef][Medline]
11
11. Wang, W.-K., T.-L. Sung, Y.-C. Tsai, C.-L. Kao, S.-M. Chang, and C.-C. King. 2002. Detection of dengue virus replication in peripheral blood mononuclear cells from dengue virus type 2-infected patients by a reverse transcription-real-time PCR assay. J. Clin. Microbiol. 40:4472-4478.[Abstract/Free Full Text]
12
12. Reference deleted.
13
13. Wu, S. J., G. Grouard-Vogel, W. Sun, J. R. Mascola, E. Brachtel, R. Putvatana, M. K. Louder, L. Filgueira, M. A. Marovich, H. K. Wong, A. Blauvelt, G. S. Murphy, M. L. Robb, B. L. Innes, D. L. Birx, C. G. Hayes, and S. S. Frankel. 2000. Human skin Langerhans cells are targets of dengue virus infection. Nat. Med. 6:816-820.[CrossRef][Medline]

---

Journal of Virology, November 2005, p. 13797-13799, Vol. 79, No. 21
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.21.13797-13799.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Prestwood, T. R., Prigozhin, D. M., Sharar, K. L., Zellweger, R. M., Shresta, S. (2008). A Mouse-Passaged Dengue Virus Strain with Reduced Affinity for Heparan Sulfate Causes Severe Disease in Mice by Establishing Increased Systemic Viral Loads. J. Virol. 82: 8411-8421 [Abstract] [Full Text]  
• Sariol, C. A., Munoz-Jordan, J. L., Abel, K., Rosado, L. C., Pantoja, P., Giavedoni, L., Rodriguez, I. V., White, L. J., Martinez, M., Arana, T., Kraiselburd, E. N. (2007). Transcriptional Activation of Interferon-Stimulated Genes but Not of Cytokine Genes after Primary Infection of Rhesus Macaques with Dengue Virus Type 1. CVI 14: 756-766 [Abstract] [Full Text]  
• Srikiatkhachorn, A., Ajariyakhajorn, C., Endy, T. P., Kalayanarooj, S., Libraty, D. H., Green, S., Ennis, F. A., Rothman, A. L. (2007). Virus-Induced Decline in Soluble Vascular Endothelial Growth Receptor 2 Is Associated with Plasma Leakage in Dengue Hemorrhagic Fever. J. Virol. 81: 1592-1600 [Abstract] [Full Text]  
• Clyde, K., Kyle, J. L., Harris, E. (2006). Recent Advances in Deciphering Viral and Host Determinants of Dengue Virus Replication and Pathogenesis. J. Virol. 80: 11418-11431 [Full Text]  
• Shresta, S., Sharar, K. L., Prigozhin, D. M., Beatty, P. R., Harris, E. (2006). Murine Model for Dengue Virus-Induced Lethal Disease with Increased Vascular Permeability.. J. Virol. 80: 10208-10217 [Abstract] [Full Text]  

This Article Abstract 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 HighWire Citing Articles via Google Scholar Google Scholar Articles by Bente, D. A. Articles by Rico-Hesse, R. Search for Related Content PubMed PubMed Citation Articles by Bente, D. A. Articles by Rico-Hesse, R.