Abstract
Current methods for dengue virus quantitation are either time consuming, technically demanding or costly. As an alternative, the commercial enzyme immunoassay Platelia™ Dengue NS1 AG (Bio-Rad Laboratories) was used to monitor semiquantitatively dengue virus replication in cultured cells. The presence of NS1 protein was evaluated in supernatants from Vero and C6/36 HT cells infected with dengue virus. The amount of NS1 detected in the supernatants of infected cells was proportional to the initial MOI used and to the time of post infection harvest. This immunoassay was also able to detect the presence of NS1 in the supernatants of infected human macrophages. Inhibition of dengue virus replication in C6/36 HT cells treated with lysosomotropic drugs was readily monitored with the use of this assay. These results suggest that the Platelia™ Dengue NS1 AG kit can be used as a fast and reliable surrogate method for the relative quantitation of dengue virus replication in cultured cells.
Highlights
Dengue is one of the most important arthropod-borne viral diseases in tropical and subtropical areas around the world and represents a serious public health in several countries of America, Asia and Africa
The amount of NS1 protein secreted by dengue virus (DEN) infected cells correlates with the multiplicity of infection To evaluate the convenience of the PlateliaTM Dengue NS1 AG enzyme immunoassay for the quantitation of NS1 protein in the culture medium of infected cells, sub-confluent monolayers of Vero and C6/36 HT cells were infected with different MOIs of DEN serotype 2 (DEN2) and the amount of NS1 protein released to the culture media was evaluated up to 48 hours post infection for Vero cells and up to 60 hpi for C6/36 HT cells using PlateliaTM Dengue NS1 AG kit
Based on the fact that NS1 protein in infected cell cultures, as well as in humans, is produced and secreted and that the amount of NS1 protein correlates with the viral replication efficiency, in this study we evaluated the use of PlateliaTM Dengue NS1 AG as a surrogate method to monitor semiquantitatively DEN replication in cultured cells
Summary
Dengue is one of the most important arthropod-borne viral diseases in tropical and subtropical areas around the world and represents a serious public health in several countries of America, Asia and Africa. Only in the Americas more than 800,000 cases of dengue fever, the less severe clinical form of dengue infection, and more than 25,000 cases of dengue hemorrhagic fever, the most severe form of dengue syndrome, occurred [1]. During the past years, the incidence of dengue has grown in endemic areas, a specific treatment or vaccines are not yet available. DEN is an enveloped virus of 50 nm in diameter and contains a single strand and positive-polarity RNA as genome of about 10.7 kb [2]. DEN genome encodes for three structural proteins (envelope glycoprotein, E; membrane, M; and capsid, C) and for seven non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5). E protein is the major structural protein exposed on the surface of the particle
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