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Plaque assays for titrating dengue virus (DENV) are time-consuming and not suitable for strains that do not plaque. Fluorescence-activated cell sorting (FACS) has been used to detect DENV-infected cells. Here we describe a FACS-based assay for titrating DENV. We determined that at 24 h postinfection, the number of infected cells detected by FACS represented the first round of infection and therefore could be used as a readout of the number of infectious particles in the inoculum. When the titers of different laboratory and clinical strains of DENV were compared using FACS, plaque, and endpoint dilution assays, for most strains the FACS titers were comparable to titers obtained by plaque or endpoint dilution assays. The FACS assay is an improvement over the plaque assay because the infection period is reduced from 5 to 7 days to 24 h and the assay can be used to titrate clinical isolates that frequently do not form clear plaques on cell monolayers. The novel FACS-based methods described here will facilitate laboratory studies of dengue.Dengue is a mosquito-borne viral disease of global public health significance. Throughout the world there are more than 2.5 billion people at risk of dengue virus (DENV) infection. Each year there are an estimated 100 million cases of dengue viral infection worldwide, with 250,000 people developing the more severe dengue hemorrhagic fever/dengue shock syndrome, which is often fatal (5). DENV is a positive-polarity RNA virus in the family Flaviviridae, and the DENV complex consists of four distinct serotypes designated DENV1 through DENV4 (10).Laboratory studies of dengue virus are difficult because the virus does not grow to high titers in cell culture and assays for titrating virus and measuring virus neutralization are timeconsuming. These problems are exacerbated when working with primary clinical isolates of virus. Standard methods for titrating DENV and measuring the ability of antisera to neutralize the virus are based on plaque assays which require 5 to 7 days to complete. Some isolates, especially among primary clinical isolates, do not form clear plaques on cell monolayers. Better methods for titrating the virus and measuring the ability of antisera to neutralize dengue need to be developed. Within the past 10 years, fluorescence-activated cell sorter (FACS)-based methods have been developed to follow infection and determine titers of viruses such as human immunodeficiency virus, herpesvirus, measles virus, influenza virus, Epstein-Barr virus, and rabies virus (1,4,12,13,16). FACS has also been used to detect DENV in clinical samples and to measure the ability of the virus to infect a variety of cells (2,3,7,8,11,17). Here we report on a FACS-based assay for titrating DENVs and for characterizing the ability of antisera to neutralize the virus. MATERIALS AND METHODSCells, viruses, and antisera. Aedes albopictus C6/36 mosquito cells were obtained from the Centers for Disease Control and Prevention, Fort Collins. Vero 76 (African Green monkey kidney) cells were purchased fro...

Sindbis virus infection of mice has provided valuable insight into viral and host factors that contribute to virus-induced neurologic disease. In an effort to further define the viral genetic elements that contribute to adult mouse neurovirulence, the neurovirulent Sindbis virus strain AR86 was compared to the closely related (22 single amino acid coding changes and the presence or absence of an 18-amino-acid sequence in nsP3 [positions 386 to 403]) but avirulent Girdwood strain. Initial studies using chimeric viruses demonstrated that genetic elements within the nonstructural and structural coding regions contributed to AR86 neurovirulence. Detailed mapping studies identified three major determinants in the nonstructural region, at nsP1 538 (Ile to Thr; avirulent to virulent), an 18-amino-acid deletion in nsP3 (positions 386 to 403), and nsP3 537 (opal to Cys; avirulent to virulent), as well as a single determinant in the structural genes at E2 243 (Leu to Ser; avirulent to virulent), which were essential for AR86 adult mouse neurovirulence. Replacing these codons in AR86 with those found in Girdwood resulted in the attenuation of AR86, while the four corresponding AR86 changes in the Girdwood genetic background increased virulence to the level of wild-type AR86. The attenuating mutations did not adversely affect viral replication in vitro, and the attenuated viruses established infection in the brain and spinal cord as efficiently as the virulent viruses. However, the virus containing the four virulence determinants grew to higher levels in the spinal cord at late times postinfection, suggesting that the virus containing the four attenuating determinants either failed to spread or was cleared more efficiently than the wild-type virus.