Conformation of the VP2 protein of bluetongue virus (BTV) determines the involvement in virus neutralization of highly conserved epitopes within the BTV serogroup

Seven neutralizing monoclonal antibodies (N-MAbs) were generated to an Australian isolate of bluetongue virus serotype 1 [BTV-1 (Aust)]. At least five of the N-MAbs were specific for epitopes on the outer coat protein VP2 and one was capable of binding SDS-treated protein in a Western blot. Six of the N-MAb panel bound and four of these neutralized BTV-1 (South Africa). None of the N-MAbs neutralized other Australian or South African BTV serotypes. However four of the N-MAb panel bound to a majority and two others bound with varying efficiency, to a more restricted but significant number of heterologous serotypes. Thus epitopes involved in the definition of one BTV serotype may be preserved on other serotypes but not be involved in their neutralization. To investigate the association between these epitopes and factors governing their expression, pools of neutralization-escape variants of BTV-1 (Aust), selected using each of six N-MAbs, were tested in virus neutralization and ELISA binding assays against the N-MAb panel. Each N-MAb displayed a unique reaction pattern with all 25 variants tested. All variants except one showed resistance to neutralization and/or reduced binding with at least three heterologous N-MAbs indicating the N-MAb-defined epitopes were mutually interactive. All 25 variants demonstrated increased resistance to neutralization by a bovine antiserum to BTV-1 (Aust). In total, the results from the variants revealed that the N-MAbs define seven distinct, interdependent neutralization epitopes which form at least part of a major neutralization domain on BTV. A majority of variants bound at least four and up to six N-MAbs, yet still resisted neutralization by them. This observation and the reaction of N-MAbs with BTV-1 (South Africa) and heterologous serotypes suggested that the conformation of the VP2 protein determines whether epitopes, conserved within the BTV serogroup, are involved in neutralization of individual serotypes of the virus.