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: 7739169
• 负责人: Qiuhong Wang
• 金额: $ 22.5万
• 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 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7739169
• 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）：人类北病毒（Hunovs）是全球胃炎的主要原因，每年造成2300万例胃肠炎病例，仅在美国就产生了实质性的经济影响。缺乏动物疾病模型（直到最近）和用于Hunovs的常规细胞培养系统阻碍了对Nov Replication策略的了解；因此，该类别的B生物防腐病原体不存在疫苗，抗病毒或疗法。肠囊病毒[Novs＆Sapoviruses（SAVS）的突破，使得在体外传播hunovs的这些进步是适当的研究，包括：1）肠道微环境中的介体（肠道含量滤液，胆汁酸，胆汁酸），可促进porcine Save save save save save save save save save; 2）先天免疫在抑制鼠（MNV）感染以及树突状细胞（DCS）和巨噬细胞的证据中的作用，如体内和体外MNV靶标； 3）组织血管组抗原作为影响亨诺夫易感性的人和猪肠细胞的潜在受体； 4）使用具有广泛的HBGA结合的主要Hunov爆发菌株（GII.4）来开发Hunov的Gnotobiotic（GN）猪和小牛疾病模型； 5）初步但未经证实的数据，指控人类三维（3-D）细胞或器官培养物中的Hunov RNA复制。基于这些关键进展，我们的具体目的是：目标1。开发GN Pig和Calf肠道的离体十二指肠和空肠器官培养以传播Hunovs； AIM 2。研究使用3-D猪（IPEC-J2）或人（Hutu80）小肠和其他肠细胞细胞系的使用来传播Hunovs； AIM 3。建立来自GN Pigs/Calves的原发性树突状细胞（DC）/巨噬细胞培养物，并与同源器官，3-D或2-D培养物共同培养它们，或共同培养已建立的DC/巨噬细胞系，含有3-D或2-D培养物，以繁殖Hunov； AIM 4。使用体内动物围栏或体内或3-D培养的蜂巢以及各种常规和非规定的细胞系来传播Hunovs。我们将在4种培养系统中的每一个中测试原始猪和小腿传递的Hunov GII.4应变或新获得的Hunovs（AIMS1-4）。我们假设动物的先前传播选择适合细胞培养的hunov变体，就像我们先前验证的用于体外适应GN猪pass的人轮状病毒一样。模仿肠道微环境的培养基补充剂或状况包括肠道含量滤液，胆汁酸和胰腺素，可增强肠胃病毒在内的肠胃病毒的传播。 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匈奴。霍诺夫人的传播将是推进对11月复制的基础和翻译研究的主要突破，从而为预防和控制和改善全球公共卫生提供新的策略。公共卫生相关性：人类北病毒（Hunovs）是感染各个年龄段的人的新兴病原体。它们是全世界非细菌胃肠炎的主要原因，造成2300万例胃肠炎病例，估计每年有50,000例住院和500例死亡，仅在美国就会造成重大经济影响。全球爆发的数量正在增加，导致易感人群的发病率增加，包括旅行者，巡航和军事船，军事部队，医院和疗养院患者，学校和日托中心的儿童以及其他受限空间中人口高度易感性和变化的人群。他们的低传染剂量，环境抵抗力，应变多样性，无症状的个体脱落和多样化的变速箱车辆具有高度传染性的，促使其分类为B类Biodefense病原体，NIAID，NIH，NIH。缺乏动物疾病模型（直到最近）和匈奴的常规细胞培养系统极大地阻碍了人们对11月复制策略的了解；因此，没有用于预防和控制的疫苗，抗病毒药物或疗法。严重和广泛的爆发通常需要关闭学校，疗养院和医院，这是目前可用于控制的唯一手段。我们将利用我们实验室和其他人的研究中肠胃病毒[Novs和Sapoviruses（SAVS）]的新进展，以开发在实验室细胞培养中传播Hunovs的方法。其中包括使用无毛猪和小牛（唯一用于匈奴的动物疾病模型）传递的hunov gii.4全球占主导地位的菌株，使用培养基补充剂/模仿肠道小钙病毒增长的肠道微环境的培养基补充剂/条件，使用新的肠道和三站培养和共同培养的细胞群体，并具有新的类型这些培养物中的抗病毒免疫。建立用于Hunovs的体外细胞培养系统将提供一个体外平台，以研究Hunov复制机制，筛选抗病毒药物和疗法，以筛选hunovs血清型hunovs并开发减弱的疫苗并评估它们产生保护和保护的中和抗体反应。匈奴人的体外繁殖将为制定新策略和手段提供预防和控制匈奴感染的方法的重大突破，从而改善全球公共卫生。