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 2 A 组轮状病毒 (RVA) 仍然是幼年动物急性胃肠炎和死亡的重要原因 3、儿童。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 我们之前进行了比较序列分析，确定了两个关键氨基酸取代 6 与细胞培养适应和减毒相关：大多数人和猪 RVA 品系 VP4 中的 D385N 7 仅猪 OSU 品系 VP4 中的 D393H。虽然这些或类似的突变之前被认为对于 8细胞培养适应/减毒，其功能尚未得到实验证实。使用我们的猪肠 9 肠类（PIE）系统，我们已经证明与宿主聚糖相互作用的对比模式决定 图10 不同RVA毒株的复制效能。 OSU 复制受到独特且显着的下调 11 去除了末端唾液酸，而后者显着增强了新型猪 G9P[13] 菌株的复制。 12 OSU 是历史上占主导地位的 RVA 变体，其特征是体内和体外复制强劲，并且始终如一 13 几十年来一直与仔猪腹泻病相关，而 G9P[13] 和 G9P[19] 最近才在全球范围内出现 猪和人类中 14 种新出现的 RVA 变异。后一种菌株可能已经进化获得了一些生物特征 15 个优势允许它们在不同的主机中有效传播和复制。强毒 OSU 和 G9P[13] 菌株具有 VP4疏水环的氨基酸位置385和393有16个独特的氨基酸取代，这可能是 与宿主聚糖的 17 种可变相互作用。此外，我们最近的研究表明，宿主（PIE）转录组 18 与 OSU 和 G9P[13] 感染相关的反应显着不同，改变了聚糖表达、胆固醇 19 以菌株特异性方式进行代谢和先天免疫信号传导。这些发现表明，进化 20 RVA 对宿主和宿主聚糖的适应是允许其跨物种（包括 21 人畜共患病）传播。 22 按照之前优化的方案，我们的实验室已成功将 11 个有毒 RVA OSU 片段克隆到 23 pT7质粒并拯救了感染性病毒。我们现在建议利用这个反向遗传学系统进行深入探索 24 RVA 毒力、细胞附着和复制的分子机制。实现这一目标的具体目的 25 项总结如下： 26 目标 1：评估 D385N 和 D393H 取代（单独或与 OSU 中的其他突变组合）是否 27 VP4) 引入 OSU RGS (ic-virOSU) 将赋予子代病毒 (ic-attOSU) 减毒表型。目的 28 2：评估 G9P[13] 样突变（S385N 和 D393N）是否会赋予后代 G9P[13] 样聚糖特异性 29 病毒（ic-G9OSU）。目标 3：对野生型 (ic-virOSU) 和突变型 (OSU- 30 att 和 OSU-G9) OSU 变体。我们的 PIE 和 Gn 猪模型以及新建立的 RGS 将允许研究 31 已识别的 VP4 突变如何改变 RVA 毒力以及与宿主聚糖的选择性相互作用。 32