Molecular Biologic and Molecular Epidemiologic Study of Group A Human Rotavirus

1.通过免疫人轮状病毒，建立了62株分泌中和人轮状病毒中和单抗的杂交瘤细胞株。根据这些单抗对不同HRV毒株的中和和酶联免疫吸附试验的反应模式，它们被分为血清型特异性单抗和交叉反应单抗。免疫沉淀法和基因重组株的蛋白质特异性分析表明，虽然大多数血清型特异性抗原主要存在于VP7中，部分(仅部分2型特异性抗原)存在于VP3中，但交叉反应抗原主要位于VP3中，部分位于VP7中。2.利用这些单抗，我们筛选了KU株(血清1型HRV)的抗原性突变体，该突变体能抵抗所用单抗的中和作用。迄今进行的去VP7单抗与抗体选择的抗原突变体之间的交叉中和试验确定了一种交叉反应a…更多和五个不同的血清型特异性中和表位，这些表位在操作上彼此重叠，构成VP7中的一个抗原位点。通过对野生型和突变型KU株VP7基因的序列分析，确定了导致突变株抗原性改变的VP7氨基酸替换。所有抗原突变体中的氨基酸替换都发生在两个可变区之一：氨基酸87到101和氨基酸208到221。迄今进行的VP3单抗与相应抗原突变体的交叉中和试验表明，VP3中至少有三个不同的交叉反应中和表位。抗原性突变体的VP3基因测序也表明VP3有三个不同的中和表位：突变体在305、433或392位(或在三维结构上似乎接近392的439位)保持了单一的氨基酸替换。一个包含第一个中和表位序列的合成肽(氨基酸296到313)与其相应的单抗发生反应，表明该区域包含一个“连续的”抗原决定簇。较少

1. By immunizing human rotavirus(HRV), we established 62 hybridomas that secrete monoclonal antibodies (Mabs) neutralizing HRV. Based on the reactivity patterns of these Mabs against various HRV strains in neutralization and enzyme-linked immunosorbent assays, they were classified into either serotype-specific or cross-reactive Mabs. Protein specificity of these mabs determined by both immunoprecipitation analysis and the use of genetically reassorted HRV strains revealed that while the majority of serotype-specific antigens resided for the most part in VP7 and partly (only a part of serotype 2-specific antigen) in VP3, cross-reactive antigens were located mostly in VP3 and partly in VP7. 2. By using these Mabs we selected antigenic mutants of KU strain (serotype 1 HRV) that resisted neutralization by the Mabs used for their selection. Cross-neutralization tests between the VP7-derected mabs and the antibody-selected antigenic mutants carried out to date identified one cross-reactive a … More nd five distinct serotype-specific neutralization epitopes which operationally overlapped one another and constituted a single antigenic site in VP7. The amino acid substitution in VP7 that are responsible for the antigenic alterations in the mutants were identified from sequencing of VP7 genes of the wild and mutant KU strains. All the amino acid substitutions in the antigenic mutants occurred in one of the two variable regions: amino acid 87 to 101 and 208 to 221.Cross-neutralization tests between the VP3-directed Mabs and the corresponding antigenic mutants carried out to date identified at least three distinct cross-reactive neutralization epitopes in VP3. Sequencing of VP3 genes of the antigenic mutants also indicated three distinct neutralization epitopes in VP3: the mutants sustained a single amino acid substitution at position 305, 433 or 392 (or 439 which seemed to be located in close proximity to 392 in three dimensional structure). A synthetic peptide (amino acids 296 to 313) which included the sequence of the first neutralization epitope reacted with its corresponding Mab, suggesting that the region contains a "sequential" antigenic determinant. Less