Use of Centrifugal Filter Devices to Concentrate Dengue Virus in Mosquito per os Infection Experiments
Since the introduction of West Nile virus into the United States in 1999, there has been a greater awareness of arboviruses, consequently, diagnostic testing for West Nile virus and other arboviruses has increased both in U.S. and international public health laboratories. The Centers for Disease Control and Prevention/Division of Vector-Borne Infectious Diseases/Arbovirus Diagnostic and Reference Laboratory produces and provides the serodiagnostic reagents which are not available commercially. Reagents needed to conduct the enzyme-linked immunoassay (ELISA) include a virus-specific non-infectious antigen. Antigens for Japanese encephalitis and the four dengue virus serotypes have been developed from COS-1 transformed cells that secrete non-infectious, virus-like particles into the cell culture supernatant. Four methods for concentrating the supernatant are discussed here. The methods are ultracentrifugation, polyethylene glycol precipitation, and two ultrafiltration methods: the Stirred Cell (Millipore Corporation, Billerica, MA) and the Pellicon 2 (Millipore Corporation, Billerica, MA). Ultracentrifugation and the Pellicon 2 ultrafiltration system produced antigen at a sufficient concentration for use in the ELISA. Large volumes were concentrated in a shorter time in the Pellicon 2 ultrafiltration system. An additional filtration step was necessary to produce antigen of sufficient concentration for use in the microsphere-based immunoassay, which requires antigen concentrated an additional 10 times.