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Flaviviruses are major arthropod-borne human pathogens responsible for life-threatening encephalitis, hepatitis and haemorrhagic fevers. These enveloped, single-stranded, positive-sense RNA viruses encode a polyprotein precursor of about 3400 amino acids, processed into three structural and seven non-structural proteins. The non-structural glycoprotein NS1 is essential for flavivirus viability. During host-cell infection in vitro, NS1 is found associated with intracellular organelles as a requisite for its role in viral replication, or is transported to the cell surface where it may trigger specific signalling pathways. In addition, a secreted form of the protein is released from flavivirus-infected mammalian cells. We have previously shown that the NS1 protein circulates during the acute phase of the disease in the plasma of patients infected with dengue virus type 1 and have extended our retrospective studies to dengue type 2 and type 3 cohorts, confirming the value of the NS1 antigen as an alternative diagnostic marker. Interestingly, detection of the NS1 protein in yellow fever virus and West Nile virus infections suggests that NS1 secretion is a hallmark of human flavivirus infections. The objectives of our current studies are to define the biological properties of the secreted form of the NS1 protein, to evaluate its possible contribution to viral pathogenesis, and to validate this protein as a candidate target for passive immunoprophylaxis against flaviviruses.

The pathogenesis of dengue hemorrhagic fever (DHF) is not well known, but the role of host factors has been suggested. The level of immunoreactive circulating and cell-generated tumor necrosis factor alpha (TNF alpha) was studied in 35 patients with DHF; its relationship with virus isolation and/or genome detection by reverse transcription polymerase chain reaction (RT-PCR) and specific antibodies were detected by hemagglutination inhibition (HI). Large variation of TNF alpha plasma levels was obtained in dengue-infected patients at the same stage of the disease and at the same day after infection. Most of the patients (14 out of 17 patients) who displayed augmented spontaneous in vitro production of TNF alpha by heparinized whole-blood culture compared with controls also had elevated levels of TNF alpha in the plasma. The TNF alpha values in lipopolysaccharide and phytohemagglutinin heparinized whole-blood cultures were not higher in patients than in controls, but low TNF alpha levels were obtained in three out of 30 patients. An inverse correlation was observed between spontaneous in vitro TNF alpha production and viral replication, which raises the issue of the antiviral effect of TNF alpha in dengue infection. The results do not support the hypothesis of the role of antibody-dependent enhancement giving rise to increased viremic titers and production of TNF alpha in patients. The present study demonstrates the activation of the TNF alpha-producing cells in dengue-infected patients and suggests further investigation to define the mechanism and the role of TNF alpha in the pathogenesis of dengue virus infection.

A reverse transcription/polymerase chain reaction (RT/PCR) protocol for the rapid detection and identification of flaviviruses was developed using a set of universal oligonucleotide primers. These primers correspond to sequences in the 3' non-coding region and in the NS5 gene which are highly conserved among the mosquito-borne flaviviruses. The sequences of the resulting amplified products were analysed for dengue 1, dengue 2, dengue 3, dengue 4, Japanese encephalitis, West Nile, yellow fever and Zika viruses, and compared with the published sequences of other flaviviruses. The 291-297 nucleotides corresponding to the C-terminus of NS5 gene showed 56 to 76% similarity, whereas the 3' non-coding region (190 to 421 nucleotides) showed only 20 to 36% similarity. Genetic classification of the Zika virus supported its traditional serological grouping. Recombinant plasmids containing the flavivirus sequences were used in a nucleic acid hybridization test to identify the RT/PCR products derived from viral RNA extracted from experimentally infected mosquitoes. The plasmids were dotted on a strip of nitrocellulose membrane and incubated with the RT/PCR product labelled with digoxigenin during the PCR step. This is a valuable method for the rapid and specific identification of mosquito-borne flaviviruses in biological specimens and for subsequent sequence analysis.