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Enzyme-Linked Immunosorbent Assay Specific to Dengue Virus Type 1 Nonstructural Protein NS1 Reveals Circulation of the Antigen in the Blood during the Acute Phase of Disease in Patients Experiencing Primary or Secondary Infections

Sophie Alcon,¹ Antoine Talarmin,² Monique Debruyne,³ Andrew Falconar,⁴ Vincent Deubel,^1, and Marie Flamand^1*

Unité des Arbovirus et Virus des Fièvres Hémorragiques, Institut Pasteur, 75724 Paris Cedex 15,¹ Laboratoire Pasteur-Cerba, Zone Industrille des Bethunes, 95310 Saint-Ouen l'Aumône, France,³ Centre National de Référence pour la Surveillance de la Dengue et de la Fièvre Jaune, Institut Pasteur de la Guyane, Cayenne, French Guiana,² Department of Infectious Disease Epidemiology, Imperial College of Medicine, University of London, St. Mary's Campus, London W2 1PG, United Kingdom⁴

Received 11 April 2001/ Returned for modification 20 May 2001/ Accepted 24 October 2001

During flavivirus infection in vitro, nonstructural protein NS1 is released in a host-restricted fashion from infected mammalian cells but not vector-derived insect cells. In order to analyze the biological relevance of NS1 secretion in vivo, we developed a sensitive enzyme-linked immunosorbent assay (ELISA) to detect the protein in the sera of dengue virus-infected patients. The assay was based on serotype 1 NS1-specific mouse and rabbit polyclonal antibody preparations for antigen immunocapture and detection, respectively. With purified dengue virus type 1 NS1 as a protein standard, the sensitivity of our capture ELISA was less than 1 ng/ml. When a panel of patient sera was analyzed, the NS1 antigen was found circulating from the first day after the onset of fever up to day 9, once the clinical phase of the disease is over. The NS1 protein could be detected even when viral RNA was negative in reverse transcriptase-PCR or in the presence of immunoglobulin M antibodies. NS1 circulation levels varied among individuals during the course of the disease, ranging from several nanograms per milliliter to several micrograms per milliliter, and peaked in one case at 50 µg/ml of serum. Interestingly, NS1 concentrations did not differ significantly in serum specimens obtained from patients experiencing primary or secondary dengue virus infections. These findings indicate that NS1 protein detection may allow early diagnosis of infection. Furthermore, NS1 circulation in the bloodstream of patients during the clinical phase of the disease suggests a contribution of the nonstructural protein to dengue virus pathogenesis.

Present address: Unité de Biologie des Infections Virales Emergentes, Institut Pasteur, 69365 Lyon Cedex 7, France.

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