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Am. J. Trop. Med. Hyg., 80(1), 2009, pp. 61-65
Copyright © 2009 by The American Society of Tropical Medicine and Hygiene

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SHORT REPORT


An Automated Dengue Virus Microneutralization Plaque Assay Performed in Human Fc{gamma} Receptor-expressing CV-1 Cells

W. W. Shanaka, I. Rodrigo, Danielle C. Alcena, Robert C. Rose, Xia Jin, AND Jacob J. Schlesinger*
Departments of Pathology and Laboratory Medicine, Microbiology and Immunology, and Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York

 

ABSTRACT

We describe microneutralization assays that used automated 96-well enzyme-linked immunospot (ELI-SPOT) readout instrumentation to measure human anti-dengue virus (DENV) antibodies in CV-1 cells that were stably transfected to express human Fc{gamma}RIIA (CD32) using conventional Vero cells as a comparator. Classic plaque reduction neutralization test (PRNT) end-point titers were determined by probit analysis. Neutralization titers against DENV measured in CV-1 transfectants were expressed in terms of both conventional 50% to 90% PRNT end-point titers and differential infectivity of antibody-treated virus in control and CD32-expressing CV-1 cells. Significantly reduced PRNT titers and strikingly heightened infectivity (up to 100-fold) of antibody-treated DENV was observed in CV-1 CD32 transfectants compared with that observed in control CV-1 or Vero cells. Because DENVs may preferentially replicate in CD32-expressing monocytes/macrophages and dendritic cells, in vivo, it is possible that CD32 introduced into a conventional DENV neutralization assay might provide results that better correlate with protection.

Received June 25, 2008. Accepted for publication September 19, 2008.

Acknowledgments: We thank Dr. Eric Henchal (AFRIMS, Bangkok, Thailand) and Ms. Gladys Sather (CDC, Puerto Rico) for human dengue immune sera.

Financial support: This work was funded by the Pediatric Dengue Vaccine Initiative of the International Vaccine Institute, Awards TR 03/04 (J.J.S) and TR16 (X.J.).

* Address correspondence to Jacob J. Schlesinger, University of Rochester Medical Center, Box 689, 601 Crittenden Blvd, Rochester, NY 14642. E-mail: Jacob_schlesinger{at}urmc.rochester.edu

Authors’ addresses: W. W. Shanaka I. Rodrigo, Danielle C. Alcena, Robert C. Rose, Xia Jin, and Jacob J. Schlesinger, University of Rochester Medical Center, Box 689, 601 Crittenden Blvd., Rochester, NY 14642, E-mail: jacob_schlesinger{at}urmc.rochester.edu.






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