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Abstract
The envelope (E) glycoprotein of JEV is the major antigen to elicit neutralizing antibody (NAb) against JEV infection. In order to develop a rapid and safe neutralization assay system for evaluation of the JEV vaccine strains, we constructed JEV-pseudotyped viruses with JEV env genes (Nakayama-NIH, Beijing-1). The titers of JEV-pseudotyped viruses with NK and BJ strains were 4.0×104 IFU/ml and 1.3×105 IFU/ml in Vero cell cultures, respectively. We have analyzed the neutralization activity of immunized mouse sera with JEV-NK and JEV-BJ pseudotyped viruses. The neutralizing antibody titers of NK and BJ (50% reduction of virus) were about 1:10,000 at each immunized sera. Compared with conventional plaque reduction neutralization test (PRNT), the method using JEV-pseudotyped virus has desirable advantages such as more rapid, easier, and non-biohazardous. This neutralization assay system might be useful to evaluate NAb activity against JEV vaccine strains or vaccine candidates.
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Figure 1.
Expression of JEV-envelope (E) protein. Cell lysate and culture media of JEV-pseudotyped viruses were subjected to SDS-PAGE. JEV-E proteins were detected by Western blotting using sera from mouse immunized with JEV (Nakayama-NIH strain). The bands show the JEV-E protein in cell lysates and culture media. Nontransfected TELCeB6 cells were used as a negative control.
Figure 2.
Cells in the JEV reduction by neutralization using sera from mouse immunized with JEV in Vero cells. (A) 100% of neutralizing activity; (B) No neutralizing activity.
Figure 3.
Neutralization assay with JEV-pseudotyped viruses. Two different JEV-pseudotyped viruses (NK and BJ strain) were incubated with either normal serum (Δ), or two sera from mouse immunized with JEV-NK strain ( and JEV-BJ strain (❍). (A) Neutralization assay of used JEV-NK pseudotyped virus. (B) JEV-BJ pseudotyped virus.
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