A Molecularly Cloned, Live-Attenuated Japanese Encephalitis Vaccine SA14-14-2 Virus: A Conserved Single Amino Acid in the ij Hairpin of the Viral E Glycoprotein Determines Neurovirulence in Mice
Figure 6
E-244: A major determinant of viral infectivity in mouse and human neuronal cells in vitro.
(A-C) Viral growth. NSC-34 (A), SH-SY5Y (B), or BHK-21 (C) cells were infected at an MOI of 0.1 with the parent or one of the following four E-244 mutant viruses: G244E, G244D, G244R, and G244K. At the indicated time points, culture supernatants were harvested for virus titration on BHK-21 cells. (D-F) Viral particle infectivity. NSC-34 (D), SH-SY5Y (E), or BHK-21 (F) cells were infected at an MOI of 1 with each of the five viruses, as indicated. At 12–15 hpi, the number of infected cells was measured by flow cytometry using mouse α-JEV antiserum. The results are the average of two independent experiments, presented as -fold changes in infectivity relative to the parent (infectivity value of 1). (G-I) Viral RNA infectivity/replicability. NSC-34 (G), SH-SY5Y (H), or BHK-21 (I) cells were transfected with RNAs transcribed from the parent or each mutant cDNA, as indicated. At 4 dpt, RNA infectivity was quantified by infectious center assay, coupled with staining of cell monolayers using an α-JEV antiserum. (J-L) Virus yield. At 20 h after RNA transfection, culture supernatants from RNA-transfected NSC-34 (J), SH-SY5Y (K), or BHK-21 (L) cells were harvested for virus titration on BHK-21 cells.