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（HuNoVs）是全球胃肠炎的主要原因，每年仅在美国就造成2300万例胃肠炎和重大经济影响。缺乏动物疾病模型（直到最近）和常规HuNoVs细胞培养系统阻碍了对NoV复制策略的了解；因此，没有针对这种B类生物防御病原体的疫苗、抗病毒药物或治疗方法。肠道杯状病毒（NoVs & sapovirus, SaV）研究的突破，使得利用这些进展在体外繁殖HuNoVs成为可能，包括：1)肠道微环境中的介质（肠内容物滤液、胆汁酸）促进猪SaV的体外复制；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和体外MNV靶点的证据；3)组织血型抗原作为人类和猪肠细胞的潜在受体影响对HuNoV的敏感性；4)利用具有广泛HBGA结合的甲型h1n1流感爆发优势毒株（GII.4）建立猪和犊牛甲型h1n1流感的非生物（Gn）疾病模型；5)初步但未经证实的数据声称人类三维（3-D）细胞或器官培养中有HuNoV RNA复制。基于这些关键进展，我们的具体目标是：目标1。在猪和犊牛肠道中培养体外十二指肠和空肠器官以繁殖HuNoVs；目标2。研究利用猪（IPEC-J2）或人（HuTu80）小肠和其他肠细胞系的三维培养来繁殖HuNoVs；目标3。建立原代树突状细胞(DC)/巨噬细胞培养物，并将其与同源器官，3d或2d培养物共培养，或将已建立的DC/巨噬细胞系与3d或2d培养物共培养以繁殖HuNoV；目标4。使用体内动物传代或离体器官传代或3-D培养传代的人造血干细胞和各种常规和非常规细胞系来繁殖人造血干细胞。我们将在4个培养系统（Aims1- 4）中分别测试原代和新代猪和小牛的HuNoV GII.4菌株或新获得的HuNoV。我们假设先前在动物中的繁殖选择了适应细胞培养的HuNoV变体，就像我们之前在猪传代的人类轮状病毒的体外适应中验证的那样。培养基补充物或模拟肠道微环境的条件包括肠道内容物滤液、胆汁酸和胰酶，它们能增强包括肠杯状病毒在内的精细肠道病毒的繁殖。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）来促进上皮细胞的生长和分化，使用HBGA匹配的器官和3D培养来反映选定HuNoV菌株的结合，共同培养策略来模拟肠道细胞类型的多样性，包括dc /巨噬细胞，以及抑制先天免疫来实现HuNoV的体外复制。人类病毒的繁殖将是推进NoV复制基础研究和转化研究的重大突破，从而为其预防和控制以及改善全球公共卫生制定新的战略。公共卫生相关性：人类诺如病毒（HuNoVs）是一种新出现的病原体，可感染所有年龄的个体。它们是全球非细菌性肠胃炎的主要原因，每年造成2300万例肠胃炎，估计有5万人住院治疗，500人死亡，仅在美国就造成了巨大的经济影响。全球疫情的数量正在增加，导致易感人群的发病率增加，包括旅行者、游轮和军舰上的人、军队、医院和疗养院的病人、学校和日托中心的儿童以及在密闭空间内有高度易感和不断变化的人群的其他环境中的儿童。它们的低感染剂量、环境抗性、菌株多样性、无症状个体的脱落和多种传播媒介使HuNoVs具有高度传染性，促使其被NIAID和NIH列为B类生物防御病原体。缺乏动物疾病模型（直到最近）和常规细胞培养系统的HuNoVs极大地阻碍了NoV复制策略的知识；因此，没有疫苗、抗病毒药物或治疗方法来预防和控制它们。严重和广泛的疫情往往需要关闭学校、疗养院和医院，这是目前唯一可用的控制手段。我们将利用我们实验室和其他实验室在肠杯状病毒（NoVs和sav）研究方面的新进展，开发在实验室细胞培养中繁殖HuNoVs的方法。这些方法包括使用世界范围内占主导地位的无菌猪和小牛（仅用于HuNoV的动物疾病模型）传代HuNoV GII.4菌株，使用模拟肠道口状病毒生长的肠道微环境的培养基补充剂/条件，使用新型肠道器官和3-D细胞培养以及模拟肠道细胞类型多样性的共培养策略，以及阻断这些培养物中的先天抗病毒免疫。建立人体免疫抑制病毒体外细胞培养系统将为研究人体免疫抑制病毒复制机制、筛选抗病毒药物和治疗方法、对人体免疫抑制病毒进行血清分型、开发减毒疫苗、评估其引起的中和抗体反应和相关保护提供一个体外平台。人乳头状瘤病毒体外繁殖将为制定预防和控制人乳头状瘤病毒感染的新战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.

在三维培养物中生长的微绒毛的肠上皮细胞中人类诺如病毒传播的失败。

• 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