Breakthroughs to advance the in vitro propagation of human noroviruses

人类诺如病毒体外繁殖取得突破

• 批准号: 7897895
• 负责人: Qiuhong Wang
• 金额: $ 18.75万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-22 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7897895
• 关键词: 3-Dimensional; Adult; Age; Animal Disease Models; Animal Model; Animals; Antibodies; Antibody Formation; Antigen-Presenting Cells; Antigens; Antiviral Agents; Attenuated Vaccines; Bile Acids; Binding; Biopsy; Blood Group Antigens; Calicivirus; Canis familiaris; Categories; Cattle; Cell Culture System; Cell Culture Techniques; Cell Line; Cells; Cessation of life; Child; Classification; Clinical; Coculture Techniques; Collaborations; Colostrum; Complex; Confined Spaces; Culture Media; D Cells; Data; Day center care; Dendritic Cells; Development; Diagnostic Procedure; Disease; Disease Outbreaks; Disease model; Dose; Duodenum; Enteral; Enterocytes; Environment; Epidemiologic Studies; Epidermal Growth Factor; Epithelial Cells; Family Picornaviridae; Family suidae; Fluorescence; Gastroenteritis; Genome; Genomics; Genotype; Gnotobiotic; Goals; Growth Factor; HIV; Hospital Nursing; Hospitalization; Hospitals; Human; Imagery; Immunity; Immunoglobulins; In Vitro; Individual; Infection; Interferon Type I; Interferons; Intestinal Content; Intestines; Knock-out; Knockout Mice; Knowledge; Laboratories; Leukocytes; Life; Mediator of activation protein; Methods; Military Personnel; Molecular; Monitor; Morbidity - disease rate; Mus; National Institute of Allergy and Infectious Disease; Natural Immunity; Newborn Infant; Norovirus; Nucleic Acids; Nursing Homes; Oligonucleotides; Organ; Organ Culture Techniques; Panthera leo; Pathogenesis; Pathway interactions; Patients; Peptides; Pharmaceutical Preparations; Pharmacotherapy; Phenotype; Play; Population; Predisposition; Prevention; Public Health; RNA; RNA replication; Replicon; Reporting; Research; Research Personnel; Resistance; Resources; Role; Rotavirus; STAT protein; STAT1 gene; Sapovirus; School Nursing; Schools; Serial Passage; Serotyping; Shipping; Ships; Signal Pathway; Site; Source; Specificity; Sterility; System; Systemic infection; Techniques; Technology; Testing; Time; Tissues; Transforming Growth Factors; United States National Institutes of Health; Vaccines; Variant; Viral; Viremia; Virus; Virus-like particle; Wild Type Mouse; antigen binding; base; biodefense; cell growth; cell injury; cell type; cytokine; design; economic impact; experience; hepatoma cell; improved; in vitro activity; in vivo; innovation; macrophage; neutralizing antibody; novel strategies; nuclease; pandemic disease; particle; pathogen; prevent; proctolin; protein expression; public health relevance; receptor; research study; resistant strain; translational study; transmission process; viral RNA

DESCRIPTION (provided by applicant): Human noroviruses (HuNoVs) are the leading cause of gastroenteritis worldwide causing 23 million cases of gastroenteritis annually and substantial economic impacts in the US alone. The lack of an animal disease model (until recently) and routine cell culture system for HuNoVs hinders knowledge of NoV replication strategies; consequently, no vaccines, anti-virals or therapies exist for this category B biodefense pathogen. Breakthroughs in enteric calicivirus [NoVs & sapoviruses (SaVs)] research that make it opportune to exploit these advances to propagate HuNoVs in vitro include: 1) Mediators in the intestinal micro-environment (intestinal content filtrates, bile acids) that promote in vitro replication of porcine SaV; 2) Role of innate immunity in inhibiting murine NoV (MNV) infection and evidence for dendritic cells (DCs) and macrophages as in vivo and in vitro MNV targets; 3) Histo-blood group antigens as potential receptors on human and pig enterocytes that influence susceptibility to HuNoV; 4) Use of the dominant HuNoV outbreak strain (GII.4) with broad HBGA binding to develop gnotobiotic (Gn) pig and calf disease models for HuNoV; 5) Preliminary, but unconfirmed data alleging HuNoV RNA replication in human three-dimensional (3-D) cell or organ cultures. Based on these key advances, our specific aims are: Aim 1. Develop ex vivo duodenal and jejunal organ cultures of Gn pig and calf intestine to propagate HuNoVs; Aim 2. Investigate use of 3-D cultures of pig (IPEC-J2) or human (HuTu80) small intestinal and other enterocyte cell lines to propagate HuNoVs; Aim 3. Establish primary dendritic cell (DC)/macrophage cultures from Gn pigs/calves and co-culture these with the homologous organ, 3-D or 2-D cultures, or co-culture the established DC/macrophage cell lines with 3-D or 2-D cultures to propagate HuNoV; Aim 4. Use in vivo animal-passaged or ex vivo organ- or 3-D culture-passaged HuNoVs and various conventional and non-conventional cell lines to propagate HuNoVs. We will test original and Gn pig and calf passaged HuNoV GII.4 strain or newly acquired HuNoVs in each of the 4 culture systems (Aims1- 4). We hypothesize that prior propagation in animals selects for HuNoV variants that adapt to cell culture, like we verified previously for in vitro adaptation of Gn pig-passaged human rotavirus. Medium supplements or conditions mimicking the intestinal microenvironment include intestinal content filtrates, bile acids and pancreatin that enhance propagation of fastidious enteric viruses, including enteric caliciviruses. Other innovative approaches include use of molecular beacons (monitor replication), growth factors (TGF2 and EGF) to promote epithelial cell growth and differentiation, use of HBGA matched organ and 3D cultures to reflect binding by selected HuNoV strains, co-culture strategies to mimic diversity of intestinal cell types including DCs/macrophages, and inhibition of innate immunity to enable in vitro replication of HuNoVs. Propagation of HuNoVs would be a major breakthrough to advance basic and translational studies of NoV replication leading to new strategies for their prevention and control and improved global public health. PUBLIC HEALTH RELEVANCE: Human noroviruses (HuNoVs) are emerging pathogens that infect individuals of all ages. They are the major cause of nonbacterial gastroenteritis worldwide causing 23 million cases of gastroenteritis leading to an estimated 50,000 hospitalizations and 500 deaths annually and substantial economic impacts in the US alone. Global outbreaks are increasing in number leading to increased morbidity in susceptible populations including travelers, those on cruise and military ships, military troops, hospital and nursing home patients, children in schools and day care centers and other settings where there are highly susceptible and changing populations in a confined space. Their low infectious dose, environmental resistance, strain diversity, shedding from asymptomatic individuals and varied transmission vehicles render HuNoVs highly contagious prompting their classification as category B biodefense pathogens by the NIAID, NIH. The lack of an animal disease model (until recently) and routine cell culture system for HuNoVs greatly hinders knowledge of NoV replication strategies; consequently, there are no vaccines, anti-viral drugs or therapies for their prevention and control. Severe and widespread outbreaks often necessitate the closing of schools, nursing homes and hospitals as the only means currently available for their control. We will exploit new advances in enteric calicivirus [NoVs and sapoviruses (SaVs)] research from our lab and others to develop methods to propagate HuNoVs in laboratory cell cultures. These include use of germfree pig and calf (only animal disease models for HuNoV) passaged HuNoV GII.4 strains that are dominant worldwide, use of culture medium supplements/conditions mimicking the intestinal microenvironment in which enteric caliciviruses grow, use of new types of intestinal organ and 3-D cell cultures and co-culture strategies mimicking the diversity of intestinal cell types, and the blocking of innate antiviral immunity in these cultures. Establishment of an in vitro cell culture system for HuNoVs will provide an in vitro platform to investigate the mechanism of HuNoV replication, to screen for anti- viral drugs and therapies, to serotype HuNoVs and to develop attenuated vaccines and evaluate the neutralizing antibody responses they elicit and correlates of protection. In vitro propagation of HuNoVs will provide the major breakthrough needed for development of new strategies and means to prevent and control HuNoV infections and thereby improve public health globally.

描述（由申请人提供）：人类诺如病毒 (HuNoV) 是全世界胃肠炎的主要原因，每年造成 2300 万例胃肠炎病例，仅在美国就造成了巨大的经济影响。 HuNoV 动物疾病模型（直到最近）和常规细胞培养系统的缺乏阻碍了对 NoV 复制策略的了解；因此，对于这种 B 类生物防御病原体，尚无疫苗、抗病毒药物或疗法。肠道杯状病毒 [NoV 和沙波病毒 (SaV)] 研究的突破使得有机会利用这些进展在体外繁殖 HuNoV，包括： 1) 肠道微环境中的介质（肠内容物滤液、胆汁酸）促进体外复制猪SaV； 2）先天免疫在抑制小鼠NoV（MNV）感染中的作用以及树突状细胞（DC）和巨噬细胞作为体内外MNV靶标的证据； 3) 组织血型抗原作为人和猪肠细胞上的潜在受体，影响对 HuNoV 的易感性； 4) 使用具有广泛 HBGA 结合的显性 HuNoV 爆发菌株 (GII.4) 来开发 HuNoV 的限菌 (Gn) 猪和牛犊疾病模型； 5) 初步但未经证实的数据表明 HuNoV RNA 在人类三维 (3-D) 细胞或器官培养物中复制。基于这些关键进展，我们的具体目标是： 目标 1. 开发 Gn 猪和小牛肠的离体十二指肠和空肠器官培养物以繁殖 HuNoV；目标 2. 研究使用猪 (IPEC-J2) 或人 (HuTu80) 小肠和其他肠上皮细胞系的 3-D 培养物来繁殖 HuNoV；目标 3. 建立来自 Gn 猪/牛的原代树突细胞 (DC)/巨噬细胞培养物，并将其与同源器官、3-D 或 2-D 培养物共培养，或将建立的 DC/巨噬细胞系与 3 -D或2-D培养物以传播HuNoV；目标 4. 使用体内动物传代或离体器官或 3D 培养物传代的 HuNoV 以及各种常规和非常规细胞系来繁殖 HuNoV。我们将在 4 个培养系统 (Aims1-4) 中测试原始和 Gn 猪和小牛传代的 HuNoV GII.4 菌株或新获得的 HuNoV。我们假设动物中先前的繁殖选择了适应细胞培养的 HuNoV 变体，就像我们之前验证的 Gn 猪传代人轮状病毒的体外适应一样。培养基补充剂或模拟肠道微环境的条件包括肠内容物滤液、胆汁酸和胰酶，它们可增强挑剔的肠道病毒（包括肠道杯状病毒）的繁殖。其他创新方法包括使用分子信标（监测复制）、生长因子（TGF2 和 EGF）来促进上皮细胞生长和分化、使用 HBGA 匹配器官和 3D 培养物来反映所选 HuNoV 毒株的结合、共培养策略来模拟包括 DC/巨噬细胞在内的肠道细胞类型的多样性，以及抑制先天免疫以实现 HuNoV 的体外复制。 HuNoV 的传播将是推进 NoV 复制的基础和转化研究的重大突破，从而制定预防和控制新策略并改善全球公共卫生。公共卫生相关性：人类诺如病毒 (HuNoV) 是感染所有年龄段个体的新兴病原体。它们是全球非细菌性胃肠炎的主要原因，导致 2300 万例胃肠炎病例，每年导致估计 50,000 人住院治疗和 500 人死亡，仅在美国就造成了巨大的经济影响。全球疫情爆发数量不断增加，导致易感人群发病率上升，包括旅行者、游轮和军舰上的人、军队、医院和疗养院患者、学校和日托中心以及其他高度易感和人口变化的环境中的儿童在一个密闭的空间里。 HuNoV 的低传染剂量、环境抵抗力、菌株多样性、无症状个体的脱落和不同的传播媒介使 HuNoV 具有高度传染性，促使其被 NIAID、NIH 归类为 B 类生物防御病原体。 HuNoV 动物疾病模型（直到最近）和常规细胞培养系统的缺乏极大地阻碍了对 NoV 复制策略的了解；因此，没有疫苗、抗病毒药物或疗法来预防和控制它们。严重而广泛的疫情爆发往往需要关闭学校、疗养院和医院，这是目前控制疫情的唯一手段。我们将利用我们实验室和其他实验室在肠道杯状病毒 [NoV 和 Sapoviruses (SaV)] 研究方面的新进展，开发在实验室细胞培养物中繁殖 HuNoV 的方法。其中包括使用无菌猪和小牛（仅限 HuNoV 动物疾病模型）传代的 HuNoV GII.4 菌株（在世界范围内占主导地位）、使用培养基补充剂/条件模仿肠道杯状病毒生长的肠道微环境、使用新型肠道微生物器官和 3D 细胞培养以及共培养策略模仿肠道细胞类型的多样性，并阻断这些培养物中的先天抗病毒免疫。 HuNoV 体外细胞培养系统的建立将为研究 HuNoV 复制机制、筛选抗病毒药物和疗法、对 HuNoV 进行血清分型、开发减毒疫苗并评估其引起的中和抗体反应提供一个体外平台。以及保护的相关性。 HuNoV 的体外繁殖将为制定预防和控制 HuNoV 感染的新策略和手段提供所需的重大突破，从而改善全球公共卫生。

项目成果

Failure of propagation of human norovirus in intestinal epithelial cells with microvilli grown in three-dimensional cultures.

• DOI: 10.1007/s00705-013-1806-4
• 发表时间: 2014-02
• 期刊: ARCHIVES OF VIROLOGY
• 影响因子: 2.7
• 作者: Takanashi, Sayaka;Saif, Linda J.;Hughes, John H.;Meulia, Tea;Jung, Kwonil;Scheuer, Kelly A.;Wang, Qiuhong
• 通讯作者: Wang, Qiuhong