title: Factors influencing dengue virus isolation by C6/36 cell culture and mosquito inoculation of nested PCR-positive clinical samples.
abstract: Dengue viral isolation is necessary for definitive diagnosis, pathogenesis and evolutionary research, vaccine candidates, and diagnostic materials. Using standardized techniques, we analyzed isolation rates of 1,544 randomly selected polymerase chain reaction (PCR)-positive samples, representing all four dengue serotypes, from patients with serologically confirmed dengue infections and evaluated whether clinical and laboratory results could be predictive of isolation using standard and mosquito isolation techniques. Viruses were isolated from 62.5% of the samples by direct application to C6/36 cells and increased to 79.4% when amplifying C6/36 negative samples by intrathorasic inoculation in Toxyrhynchites splendens mosquitoes. High viremia, measured by reverse transcriptase (RT)-PCR, was a strong predictor for viral isolation by either method. Isolation was most successful in samples collected early in the disease, had low antibody levels, temperatures greater than 38°C, and had a final clinical diagnosis of dengue fever. Dengue serotypes also played a role in the success of viral isolation.
authors.affiliation: Department of Virology, United States Army Medical Component, Armed Forces Research, Institute of Medical Sciences, Bangkok, Thailand. richard.jarman@afrims.org
authors.forename: Richard G
authors.initials: RG
authors.lastname: Jarman
authors.forename: Ananda
authors.initials: A
authors.lastname: Nisalak
authors.forename: Kathryn B
authors.initials: KB
authors.lastname: Anderson
authors.forename: Chonticha
authors.initials: C
authors.lastname: Klungthong
authors.forename: Butsaya
authors.initials: B
authors.lastname: Thaisomboonsuk
authors.forename: Winai
authors.initials: W
authors.lastname: Kaneechit
authors.forename: Siripen
authors.initials: S
authors.lastname: Kalayanarooj
authors.forename: Robert V
authors.initials: RV
authors.lastname: Gibbons
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D009033:Culicidae / Q000821:virology*; D003716:Dengue Virus / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D004664:Encephalitis Virus, Japanese / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay; D007074:Immunoglobulin G / Q000276:immunology; D007075:Immunoglobulin M / Q000276:immunology; D016133:Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013696:Temperature
chemical_list: D007074:Immunoglobulin G; D007075:Immunoglobulin M