Antigenicity of Japanese encephalitis virus envelope glycoprotein V 3 (E) and its cyanogen bromide cleaved fragments examined by monoclonal antibodies and Western blotting

Abstract

Purified Japanese encephalitis (JE) virus was solubilized under reducing condition by using 2-merceptoethanol (2 ME) or nonreducing condition without 2 ME and its structural proteins were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE), followed by the Western blotting using polyclonal and monoclonal antibodies against JE. The mobilities and reactivities against polyclonal antiserum of V3 (E) and V2 (C) were reduced when virion was disrupted under reducing condition. The 54 K band corresponding to V3 was treated with cyanogen bromide (CNBr) and analyzed by the second SDS-PAGE and Western blotting. By Coomassie Blue staining multiple bands of molecular weight ranging from 54 K to 8 K daltons were revealed for CNBr-treated V3. For the specimens disrupted under reducing condition, uncleaved 54 K and cleaved 8 K, 14 K, 45 K, and 48 K bands were reactive by one of the JE and Murray Valley encephalitis (MVE) crossreactive monoclonal antibodies (NARMA 16), while other monoclones did not show any reactivity. The uncleaved V 3 prepared under nonreducing condition was reactive with several monoclones to almost similar levels. After CNBr treatment, the antigenic epitope(s) for a flavivirus-common monoclone (NARMA 24) and those for NARMA 16 appeared to locate on different fragments, while the epitopes for other monoclones lost their antigenicities. These results indicate the importance of disulfide bond and highly organized structure to maintain some of the epitopes on V 3.