RESULTS CONCLUSIONS Characterization of virus-like particle of Yellow fever virus in Drosophila expression system Characterization of virus-like particle of Yellow fever virus in Drosophila expression system ABSTRACT Yellow fever virus (YFV) belongs to family Flaviviridae and is important mosquito-borne human pathogens. Virus-like particles(VLPs) of flaviviruses generated from the premembrane (PrM) and envelope (E) genes are a promising vaccine candidate. Here we have established expression of VLP of YFV in Drosophila cell lines. YFV 17D vaccine strain was used in this study. The coding sequences of PrM and E proteins of YFV were cloned into the Drosophila expression vector (pMT/BiP/V5/His-YFV). Drosophila cell line S2 was transfected with this plasmid. PrM & E proteins of YFV expressed in Drosophila cells were recognized by antibodies in human serum derived from yellow fever virus-vaccinated persons in an immunofluorescence assay. The VLP particle of YFV was also detected in electron microscopy. The Drosophila expression system is a more convenient and safer approach to the production of vaccine candidates for YFV. In vivo studies of the VLP of YFV are in progress of determining the immune responses of the yellow fever virus as a vaccine candidate. This study is supported by the research fund (code#2012-NC ) from the Korea Centers for Disease Control & Prevention. REFERENCES 1. Barrett AD, Higgs S. Yellow fever: a disease that has yet to be conquered. Annu Rev Entomol 2007, 52: Barrett AD, Teuwen DE. Yellow fever vaccine-how does it work and why do rare cases of serious adverse events take place? Curr Opin Immunol 2009, 21: Putnak R, Porter K, Schmaljohn C. DNA vaccines for flaviviruses. Adv Virus Res 2003, 61: Guirakhoo F, Zhang ZX, Chambers TJ, Delagrave S, Arroyo J, Barrett AD, et al. Immunogenicity genetic stability, and protective efficacy of a recombinant, chimeric yellow fever-Japanese encephalitis virus (ChimeriVax-JE) as a live, attenuated vaccine candidate against Japanese encephalitis. Virology 1999, 257(2): Guirakhoo F, Weltzin R, Chambers TJ, Zhang ZX, Soike K, Ratterree M, et al. Recombinant chimeric yellow fever-dengue type 2 virus is immunogenic and protective in nonhuman primates. J Virol 2000, 74(12): ● Plasmid pMT/BiP/V5/His-YFV PrM/E containing Yellow fever virus premembrane (PrM) and envelope (E) genes was constructed for Drosophila expression system. ● The virus-like particle (VLP) of YFV was produced in Drosophila S2 cells and was detected in electron microscopy. ● PrM & E proteins of YFV expressed in Drosophila cells were recognized by antibodies in human serum derived from yellow fever virus-vaccinated persons in an immunofluorescence assay. ● Based on these data, in vivo studies of the VLP of YFV are in progress of determining the immune responses of the yellow fever virus as a vaccine candidate Keystone Symposia Oct. 31-Nov. 4, 2013 MATERIALS & METHODS IFA Yellow Fever Virus 17D virus S2 cell Transfection Electron Microscopy 2 days ABC Fig. 1. (A) PCR amplicon from PrM/E gene of YFV. Lane M, 1Kb plus DNA ladder; lane 1, YFV PrM /E. (B) Identification of positive clones using colony PCR method. Lane M, 1Kb Plus DNA ladder; lanes 1-16, colonies of YFV PrM/E. Fig. 2. Sequence analysis of PrM/E genes of YFV. Fig. 3. Electron micrographs of YFV VLPs from Drosophila S2 cells transfected with pMT/BiP /V5/His-YFV PrM/E. YFV VLPs (B, C) were negatively stained and analyzed by TEM. Scale bar is indicated at the bottom. Panel A was from S2 cell control and panel B & C were from transfected cell pellets. Fig. 4. Detection of YFV VLP by indirect immunofluoresecence assay (IFA). Drosophila S2 cells were transfected with pMT/BiP/V5/His-YFV PrM/E plasmid for 48 hrs. YFV VLP in S2 cells were detected with human serum from people vaccinated against YFV 17D vaccine strain. (A) Negative control, (B) YFV VLP. A B