Mérour Emilie, Lamoureux Annie, Biacchesi Stéphane, Brémont Michel
VIM, INRA, Université Paris-Saclay, 78350 Jouy-en-Josas, France.
J Gen Virol. 2016 Apr;97(4):893-900. doi: 10.1099/jgv.0.000411. Epub 2016 Jan 22.
In this study, we aimed to characterize the epitope recognized by the neutralizing 17H23 mAb directed against the E2 glycoprotein of most of salmonid alphavirus (SAV) subtypes and widely used in several laboratories to routinely diagnose SAV. We hypothesized that the 17H23 epitope was located in the major domain B, previously identified in the E2 of mammalian alphaviruses as the domain recognized by most of the E2 neutralizing mAbs. Indeed, the SAV E2 domain B counterpart is contained in the protein domain previously characterized as being recognized by mAb 17H23. Thus, to precisely characterize the 17H23 epitope, we developed an alanine scanning mutagenesis approach coupled with the generation of the respective recombinant SAV (rSAV) by using the available infectious cDNA. Ten mutant rSAVs termed A-J from E2 aa 223-236 were produced and characterized in vitro using indirect immunofluorescence assays on virus-infected cells with mAbs 17H23, 51B8 (another non-neutralizing anti-E2 mAb) and 19F3 directed against the non-structural protein nsp1. Two of the mutant rSAVs (G and H) escaped neutralization by mAb 17H23. In addition, we showed that when juvenile trout were infected by bath immersion with the rSAV mutants, some of them were either totally (D, E and G) or partially (H) attenuated. Together, the data from the in vitro and in vivo experiments indicated that the putative 17H23 amino acid sequence epitope comprised the short amino acid sequence (227)FTSDS(231).
在本研究中，我们旨在鉴定由中和性单克隆抗体17H23识别的表位，该抗体针对大多数鲑鱼α病毒（SAV）亚型的E2糖蛋白，并且在多个实验室中广泛用于常规诊断SAV。我们假设17H23表位位于主要结构域B中，该结构域先前在哺乳动物α病毒的E2中被鉴定为大多数E2中和单克隆抗体识别的结构域。实际上，SAV E2结构域B的对应物包含在先前被鉴定为被单克隆抗体17H23识别的蛋白质结构域中。因此，为了精确鉴定17H23表位，我们开发了一种丙氨酸扫描诱变方法，并通过使用可用的感染性cDNA生成相应的重组SAV（rSAV）。从E2氨基酸223 - 236产生了10个名为A - J的突变rSAV，并在体外使用针对病毒感染细胞的间接免疫荧光试验进行鉴定，所用单克隆抗体有17H23、51B8（另一种非中和性抗E2单克隆抗体）和针对非结构蛋白nsp1的19F3。其中两个突变rSAV（G和H）逃脱了单克隆抗体17H23的中和作用。此外，我们表明，当幼鳟通过浸浴感染rSAV突变体时，其中一些要么完全（D、E和G）要么部分（H）减毒。总之，体外和体内实验的数据表明，推定的17H23氨基酸序列表位包含短氨基酸序列（227）FTSDS（231）。
