Rotavirus Reverse Genetics System to Study Viral Pathogenesis and Receptor Interactions

轮状病毒反向遗传学系统研究病毒发病机制和受体相互作用

• 批准号: 10739026
• 负责人: Linda J. Saif
• 金额: $ 19.69万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-10 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10739026
• 关键词: Acute; Address; Amino Acid Substitution; Attenuated; Binding; Biological; Blood Group Antigens; Cell Adhesion; Cell Culture Techniques; Cell-Matrix Junction; Cells; Cessation of life; Child; Cholesterol; Clinical; Down-Regulation; Epithelial Cells; Excision; Family suidae; Gastroenteritis; Gene Exchanges; Gene Expression Profile; Genes; Genetic Transcription; Gnotobiotic; Goals; Heterogeneity; Human; Hydrophobicity; Immune signaling; In Vitro; Individual; Infection; Intestines; Knowledge; Metabolism; Modeling; Modification; Molecular; Mutation; Natural Immunity; Neuraminidase; Pathogenesis; Phenotype; Physiological; Plasmids; Polysaccharides; Positioning Attribute; Proliferating; Protocols documentation; RNA; Rotavirus; Sequence Analysis; Series; Severity of illness; Sialic Acids; Sialyltransferases; Signal Pathway; Small Intestines; Source; Specificity; System; Testing; Therapeutic; Transfection; Translational Research; Variant; Viral Pathogenesis; Viral Physiology; Virulence; Virulent; Virus; Virus Receptors; Zoonoses; attenuation; comparative; design; diarrheal disease; genome analysis; improved; in vivo; innovation; intestinal epithelium; juvenile animal; mortality; mutant; novel; porcine model; receptor; recombinant virus; response; reverse genetics; tool; transcriptome; transmission process; vaccination strategy; vector

1 Abstract 2 Rotaviruses (RVs) of group A (RVAs) remain an important cause of acute gastroenteritis and mortality in young animals 3 and children. Despite their significance, mechanisms of RVA cell entry, replication and attenuation remain poorly 4 understood indicating a critical need to address these knowledge gaps and design universal control approaches. 5 We have previously conducted comparative sequence analysis that identified two key amino acid substitutions 6 associated with cell culture adaptation and attenuation: D385N in the VP4 of most human and porcine RVA strains and 7 D393H in porcine OSU strain VP4 only. While these or similar mutations were previously suggested to be important for 8 cell culture adaptation/attenuation, their function has not been confirmed experimentally. Using our porcine intestinal 9 enteroid (PIE) system, we have demonstrated that contrasting modes of interactions with the host glycans determine 10 replication efficacy of different RVA strains. OSU replication was uniquely and significantly down-regulated by the 11 removal of terminal sialic acids, while the latter significantly enhanced the replication of a novel porcine G9P[13] strain. 12 OSU is a historic dominant RVA variant characterized by robust in vivo and in vitro replication and consistently 13 associated with diarrheal disease in piglets for several decades, while G9P[13] and G9P[19] are recent, globally 14 emerging RVA variants in swine and humans. It is possible that the latter strains have evolved to acquire some biological 15 advantage allowing for their efficient spread and replication in different hosts. Virulent OSU and G9P[13] strains have 16 unique aa substitutions in the aa positions 385 and 393 of the VP4 hydrophobic loop, which could be a reason for 17 variable interactions with host glycans. Additionally, our recent study demonstrated that the host (PIE) transcriptome 18 response associated with OSU and G9P[13] infections differed drastically altering glycan expression, cholesterol 19 metabolism and innate immune signaling in a strain-specific manner. These findings suggest that evolutionary 20 adaptation of RVAs to the host and host glycans is a critical mechanism allowing for their interspecies (including 21 zoonotic) transmission. 22 Following a previously optimized protocol, our lab has successfully cloned 11 segments of virulent RVA OSU into the 23 pT7 plasmid and rescued infectious virus. We now propose to use this reverse genetics system for in-depth exploration 24 of the molecular mechanisms of RVA virulence, cell attachment and replication. The specific aims to achieve this goal 25 are summarized below: 26 Aim 1: Evaluate if the D385N and D393H substitutions (individually or in combination with other mutations in the OSU 27 VP4) introduced into OSU RGS (ic-virOSU) will confer an attenuated phenotype to the progeny virus (ic-attOSU). Aim 28 2: Evaluate if G9P[13]-like mutations (S385N and D393N) will confer the G9P[13]-like glycan specificity to the progeny 29 virus (ic-G9OSU). Aim 3: To conduct comparative transcriptome analysis of the wild-type (ic-virOSU) and mutant (OSU- 30 att and OSU-G9) OSU variants. Our PIE and Gn pig models as well as the newly established RGS will allow to study 31 how the identified VP4 mutations alter RVA virulence and the selective interactions with host glycans. 32

1摘要 A组（RVA）的2个轮状病毒（RVS）仍然是年轻动物急性胃炎和死亡率的重要原因 3和孩子。尽管具有重要意义，但RVA细胞进入，复制和衰减的机制仍然很差 4理解表明，迫切需要解决这些知识差距并设计通用控制方法。 5我们先前已经进行了比较序列分析，该分析鉴定了两个关键的氨基酸取代 6与细胞培养适应和衰减相关：大多数人和猪RVA菌株的VP4中的D385N以及 仅在猪OSU菌株VP4中7 D393H。虽然这些或类似的突变以前被认为对 8细胞培养适应/衰减，其功能尚未通过实验确认。使用我们的猪肠 9 Enteroid（PIE）系统，我们已经证明了与宿主聚糖的相互作用模式确定 10不同RVA菌株的复制功效。 OSU复制是独特而显着下调的 11去除末端唾液酸，而后者显着增强了新型猪G9P菌株的复制。 12 OSU是一种历史悠久的RVA变体，其特征是体内和体外复制的稳健性和一贯的特征 13与小猪中的腹泻病有关数十年，而G9P [13]和G9P [19]是最近的，全球 14种新兴的RVA变体中的猪和人类变体。后一种菌株可能已经发展为获得一些生物学 15优势允许它们在不同主机中的有效分布和复制。强烈的OSU和G9P [13]菌株具有 VP4疏水循环的AA位置385和393中的16个独特的AA取代，这可能是一个原因 17个与宿主聚糖的变量相互作用。此外，我们最近的研究表明宿主（PIE）转录组 18与OSU和G9P [13]感染相关的反应发生了巨大改变，胆固醇的聚糖表达发生了巨大改变 19代谢和先天免疫信号以特异性方式传导。这些发现表明进化 20对宿主和宿主聚糖的改编是允许其间隔的关键机制（包括 21人畜共动性）传播。 22遵循先前优化的协议，我们的实验室成功将11个有毒的RVA OSU克隆到了 23 PT7质粒并营救了传染病。现在，我们建议使用这种反向遗传学系统进行深入探索 RVA毒力，细胞附着和复制的分子机制24。具体目的是实现这一目标 25总结如下： 26 AIM 1：评估D385N和D393H是否取代（单独或与OSU中的其他突变组合 27 VP4）引入的OSU RGS（IC-Virosu）将赋予后代病毒（IC-Attosu）的衰减表型。目的 28 2：评估G9P [13]类似突变（S385N和D393N）是否会授予G9P [13]类似于后代的聚糖特异性 29病毒（IC-G9OSU）。目标3：进行野生型（IC-Virosu）和突变体（OSU-）的比较转录组分析 30 ATT和OSU-G9）OSU变体。我们的派和GN猪模型以及新建立的RGS将允许学习 31鉴定的VP4突变如何改变RVA毒力和与宿主聚糖的选择性相互作用。 32