DENV1-4, mixture of dengue virus types 1, 2, 3, and 4. and WNV infections to assess the sensitivity and specificity of the MAC-ELISA using VLPs or SMB antigens. In addition, serum specimens from patients infected with either Powassan virus or La Crosse encephalitis virus were used to evaluate the cross-reactivity of seven mosquito-borne viral antigens. The results of the present studies showed higher sensitivity when using SLEV and WNV VLPs and higher specificity when using SLEV, WNV, and the mixture of DENV-1 to -4 VLPs in the MAC-ELISA than when using corresponding SMB antigens. Receiver operating characteristic (ROC) curve analysis, a plot of the sensitivity versus false positive rate (100 ? specificity), was applied to discriminate the accuracy of tests comparing the use of VLPs and SMB antigen. The measurement of assay performance by the ROC analysis indicated that there were statistically Montelukast significant differences in assay performance between DENV and WNV VLPs and the respective SMB antigens. Additionally, VLPs had a lower cutoff positive/negative ratio than corresponding SMB antigens when employed for the confirmation of current infections. The VLPs also performed better than SMB antigens in the MAC-ELISA, as indicated by a higher positive prediction value and positive likelihood ratio test. Cell lines continuously secreting these VLPs are therefore a significantly improved source of serodiagnostic antigens compared to the traditional sources of virus-infected tissue culture or suckling mouse brain. Members of the genus have an 11-kb, single-stranded, positive-sense RNA genome which translates and encodes capsid (C), premembrane/membrane (prM/M), and envelope (E) structural proteins and seven nonstructural proteins. During natural flavivirus infection, noninfectious virus-like particles (VLPs) are produced in addition to infectious, mature virions (25). Flavivirus VLPs have structural and physiochemical properties similar Rabbit Polyclonal to EDG1 to mature virus particles. VLPs have been Montelukast characterized for several flaviviruses, including tick-borne encephalitis virus (27), Japanese Montelukast encephalitis virus (JEV) (2, 14, 17), West Nile virus (WNV) (5), St. Louis encephalitis virus (SLEV) (23), dengue virus type 2 (DENV-2) (3) and DENV-1, -3, and -4 (23), and Murray Valley encephalitis virus (18). We have previously described WNV, JEV, SLEV, and DENV-1 to -4 plasmid constructs that direct the expression of prM/M and E proteins and secretion of VLPs into Montelukast the tissue culture media of plasmid-transformed cells. Plasmid DNA containing a eukaryotic transcriptional unit consisting of the human cytomegalovirus immediate early gene promoter, Kozak consensus ribosomal binding sequence, the signal sequence derived from the carboxy terminus of the C protein of JEV, and the prM/M and E gene regions is sufficient for production of VLPs. The transformation of tissue culture cells with plasmid DNA is therefore advantageous for antigen production, since these cells secrete viral prM and E proteins in VLPs having proper conformation and presentation of epitopes similar to those of virion particles. Dengue fever and/or dengue hemorrhagic fever (DHF), caused by four serotypes of DENV, is the most important arbovirus disease in terms of morbidity and mortality. Annually, it is estimated that 50 million to 100 million people may be infected with DENV worldwide, with more than 2.5 billion people living in areas where dengue is endemic and at risk of infection. DENV is spread by the Montelukast bite of infected mosquitoes, with more than half of individuals infected being asymptomatic or having an undifferentiated fever (1, 7). In addition to the relatively mild form, dengue fever, an increase in the incidence of the more serious diseases DHF and dengue shock syndrome has been observed over the last 50 years, with an estimated 250,000 to 500,000 cases of DHF and 24,000 deaths reported annually in recent years (9). The dengue serogroup consists of four antigenically related but distinct serotypes. Cross-reactive antibodies which react to similar epitopes presented on other flaviviruses, particularly for viruses within the same serogroup, are produced during flavivirus infection. For DENV, infection results in the production of neutralizing antibodies and lifelong immunity to the homologous serotype. In an early study, Albert Sabin demonstrated that the volunteers challenged with a second DENV serotype were fully cross-protected for only 2 months and partially protected for up to 9 months after infection with the first serotype but were not protected thereafter (26). Subsequent secondary infection by one or more of the three heterologous serotypes is generally accepted as a major risk factor for DHF and/or dengue shock syndrome due to antibody-dependent enhancement (ADE) (1, 10, 11, 12). Additionally, this ADE phenomenon has been observed within members of the JEV serocomplex under experimental conditions (19). Isolation and characterization of virus, detection of genomic sequence, detection of virus-specific antigen(s), and detection.