Characterization of a Sylvatic DENV2 Isolated from a DHF Case

Abstract

Background: Dengue viruses are thought to bemaintained in a sylvatic cycle with the endemic or epidemic viruses arising from ancentral sylvatic strains. In general it is thought that sylvatic dengue viruses are not particularly virulent for humans and may only cause mild disease. In January 2008 we isolated a sylvatic dengue 2 virus from a patient presenting to a fever surveillance clinic. This patient subsequently developed DHF and we present here a description of the case, and early attempts at characterizing the infecting sylvatic virus. Methods: Virus isolation was performed by inoculating C6/36 mosquito cells with 5 ul of serum and incubation of cells at 30 ◦C. One blind passage was performed and when cytopathic effect was observed the isolate was subjected to a Taqman PCR to determine serotype. The nucleotide sequence of the complete E gene was determined by sequencing RT PCR product directly using Big Dye DNA sequencing kit and an ABI 377 sequencer. Dengue virus specific IgM was determined using an IgM Capture ELISA (MAC ELISA) with paired serum specimens tested at 1:100 dilution and dengue IgG was determined using an IgG Capture ELISA (GAC ELISA) in order to determine if the patient had a primary or secondary infection. PRNT50 assays against the sylvatic and currently circulating DENV2 strains were done using serum dilutions in Vero cells in 24 well plates. Western blots were carried out using lysates of infected cells probed with paired sera Results: The phylogenetic analysis of the DENV2 isolated shows that the virus is most closely related to a sylvatic virus P8—1407 isolated in 1970 from a sentinel monkey in Malaysia. This paper will present detailed IgM, IgG, PRNT50 and western blot results for the paired serum against the virus isolated as well as currently circulating DENV2 viruses.