B

0.01 0.1 1 10 100 1000 NS1 Ag concentration (mg/ml)

No. of days after onset of fever

average over period of time

FIG. 1. NS1 antigen circulation in plasma during acute DENV infections. (A) Standard curves of the DENV NS1 antigen-capture ELISA. The sensitivity of the assay was assessed for each of the four serotypes using the corresponding purified secreted form of the NS1 protein. The positive threshold, indicated by a dotted line, represents twice the mean value of the negative controls. The detection sensitivity of the ELISA is about 0.5 ng/ml for type 1 and 2, 1 ng/ml for type 3, and 4 ng/ml for type 4. (B) Percentage of NS1-positive plasma samples in panels of DENV-infected patients. Infections occurred during the epidemics of DENV-1 and -2 in French Guiana in 1996-99, and of DENV-3 in Guadeloupe in 2000-01. Specimens were recovered within the acute phase of the disease (between the onset of fever, day 0, till day 6) or during the convalescent phase (day 10 onwards).

comprising 100—200 individuals were constituted within the Pasteur Institute Network during epidemics involving DENV-1 and -2 in French Guiana in 1996— 99, and DENV-3 in Guadeloupe in 2000-2001 (collaboration with J.-L. Cartel, Y. Sanchis and F. Saintpere). Sera were recovered during the acute phase (usually less than a week after the onset of fever, set at day 0) and the convalescent phase (most frequently day 10 onwards). Acute sera were tested for viraemia by RT-PCR or virus isolation, and by MAC-ELISA for DENV-specific IgM detection. Some of the convalescent sera were tested by ELISA for the quantification of both IgM and IgG, or by HI in order to identify primary from secondary infections. The vast majority of the sera recovered between day 0 and day 9 were found to be positive in NS1 antigen, irrespective of the viral serotype involved in the infection (Fig. 1B) (Alcon et al 2002). Over the period of day 0 to day 6, the percentage of positive sera varied from 50% to 100%, with a mean value as of 87% for type 1, 70% for type 2, and 77% for type 3 (Fig. 1B). For type 4, only a limited number of sera were available due to a lower incidence of this particular serotype of DENV. Out of 14 blood samples collected in Vietnam within a week after the onset of fever (kindly provided by VTQ Huong), 11 were positive in NS1 antigen, representing 79% of the samples tested. As expected, none of the convalescent sera recovered after day 15 had detectable levels of NS1, presumably once infected cells have been cleared by the immune system, and the protein degraded or opsonized by specific antibodies. Not surprisingly, the NS1 antigen-capture assay showed a very high correlation with detection of virus by RT-PCR, up to 90% for type 1, 87% for type 2 and 95% for type 3 (data not shown). In addition, the NS1 protein could still be detected several days after viraemia had become undetectable, suggesting that NS1 secretion may occur at a higher magnitude than virus particle release or that the half-life of the NS1 protein may be substantially longer than virions as the immune system acts to eliminate the infection.

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