Molecular Determinants of Human Pathogenic Bunyavirus Virulence and Evolution

人类致病性布尼亚病毒毒力和进化的分子决定因素

• 批准号: 8556042
• 负责人: Hideki Ebihara
• 金额: $ 49.11万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8556042
• 关键词: Acute; Africa; Animals; Antiviral Response; Arthropods; Asia; Biogenesis; Biologic Characteristic; Biological; Birds; Bunyamwera virus; Bunyaviridae; Categories; Cell Line; Cells; Characteristics; China; Chiroptera; Chittoor virus; Clinical; Complex; Crimean Hemorrhagic Fever; Culicidae; Data; Data Analyses; Development; Disease; Disease Outbreaks; Encephalitis; Epidemiology; Event; Evolution; Family; Family member; Fever; Fibroblasts; Future; Gene Mutation; Generations; Genetic; Genetic Variation; Genome; Genotype; Genus Phlebovirus; Goals; Growth; Hantavirus; Hantavirus Pulmonary Syndrome; Human; Human Virus; India; Infection; Interferons; Kenya; Kinetics; Length; Link; Livestock; Maintenance; Mammals; Maps; Measurable; Modeling; Molecular; Molecular Biology; Monkeys; Nairovirus; National Institute of Allergy and Infectious Disease; Nature; North America; Oropouche virus; Orthobunyavirus; Pathogenesis; Plants; Play; Population; Production; Property; Protein Binding; RNA; RNA Viruses; Relative (related person); Research; Research Priority; Rift Valley Fever; Rodent; Role; Serological; Simbu Group Viruses; Simbu virus; Sloths; Somalia; South America; South American; Syndrome; System; Thrombocytopenia; Ticks; Time; Tospovirus; Variant; Viral; Viral Hemorrhagic Fevers; Viral Nonstructural Proteins; Virulence; Virus; Virus Diseases; Work; base; cell type; cytokine; follow-up; genetic analysis; genome sequencing; hemorrhagic fever virus; human disease; influenzavirus; interest; macrophage; member; novel; pathogen; positional cloning; receptor; receptor binding; response; transmission process; vector mosquito; virology; virus genetics; virus host interaction

(1) Mechanisms of virulence acquisition in Ngari virus (NRIV): In 1997-98 a large outbreak of Rift Valley fever (RVF) occurred in Kenya and Somalia. During this outbreak, NRIV was identified as the causative agent of hemorrhagic fever (HF) in a significant proportion of the cases. NRIV was also recognized as a naturally occurring genetic reassortant between Bunyamwera virus (BUNV; L and S segments) and Batai virus (BATV; M segment), both of which belong to the bunyamwera serogroup in the genus Orthobunyavirus. Interestingly, both parental viruses cause febrile illness, but not severe HF in humans. This is a perfect example of the important role that genetic reassortment plays in the evolution of viruses and of the changes in virulence that can result. Therefore, we are using NRIV as a model to understand the molecular mechanisms underlying the emergence of novel pathogenic bunyaviruses in nature. So far, we have determined the full genome sequences of 5 NRIV isolates (genotype: L-BUNV/M-BATV/S-BUNV), including 2 strains from the Kenya/Somalia HF outbreak, as well as 2 BUNV isolates and 5 BATV isolates. Based on analysis of these data we have made several interesting observation related to the emergence of NRIV: 1) that genetic variation among NRIV strains is low, suggesting that a recent single reassortment event may have given rise to all the identified NRIV isolates and 2) that while overall genetic diversity among NRIV strains is low, a significant amount of this total variation is present in strains from a single outbreak, suggesting that multiple introductions into the human population are responsible for the large outbreaks seen with NRIV. In addition to these direct observations, the availability of these sequences also provides us with the possibility to pursue full-length clone generation for these viruses, work that is currently in progress. In future these systems will allow us to construct various reassortant and chimeric viruses and thereby map the viral determinants associated with NRIV virulence and its ability to cause HF. Further, in order to identify measurable phenotypic characteristics that could be related to the acquisition of virulence by NRIV we have been examining growth in various cell lines, including both human and mosquito cells. Interestingly we have determined that while in a highly permissive cell line (Vero) all BATV strains grow well and with similar kinetics, in C6/36 (mosquito cells) only a few BATV strains, including the UgMP-6830 strain, which is the closest relative of the NRIV M-segment, show efficient growth at early time points after infection. This could have implications for successful virus spread in the mosquito vector and potentially limit which BATV strains can generate NRIV-like viruses during reassortment. In addition, we have preliminary data demonstrated enhanced growth of BUNV and NRIV strains over BATV strains in primary human macrophages. This finding is currently being followed up and will include the analysis of cytokine production in response to infection with these different viruses, since for other hemorrhagic fever viruses aberrant cytokine responses have been shown to be critical in the development of disease. In addition, the differences observed in virus growth among different cell lines promoted consideration that these viruses might be using distinct receptors. To examine this we have generated soluble version of the receptor-binding subunit of BUNV, BATV and NRIV. However, while we can show that these proteins bind to target cells, in fibroblasts they block infection with all three viruses equally well, indicating that in this cell type receptor usage is overlapping. Studies to examine if this differs in putative primary target cells like macrophages are currently on-going. (2) Molecular determinants of host range in Simbu serogroup viruses: The simbu serogroup also belongs to the genus Orthobunyavirus and can be categorized into those viruses that infect i) humans (Oropouche virus), ii) livestock animals (e.g. Akabane virus) or iii) vertebrate hosts (rodents, birds, monkey etc.) other than humans or livestock (e.g. Mermet virus). Among the simbu-serogroup viruses Oropouche virus (OROV) is unique in causing acute febrile illness in humans. Since its discovery, OROV has caused more than 30 outbreaks with at least 500,000 cases identified between 1960 and 2009 in South America. It is currently unknown why OROV causes disease in human but other simbu viruses do not. In order to identify the viral determinants of host range among simbu serogroup viruses, we will compare the genetic and virological characteristics of OROV and other related viruses, especially those that infect non-livestock animals, since these viruses are genetically closer to OROV than those that infect livestock. To accomplish this, we have determined the full genome sequence of over 25 simbu serogroup viruses. Our analyses have revealed that simbu group viruses that infect vertebrate hosts other than humans or livestock can be divided genetically and geographically into two groups, Old World and New World. In addition, we have identified several South American simbu group viruses that are genetically closely related to the human pathogenic OROV. Interestingly, we found that one of these OROV-like virus, Utinga virus, which is not known to cause illness in humans but was isolated from the same animal reservoir as OROV (sloths), does not encode the non-structural protein NSs, which inhibits the host interferon antiviral response. Based on these results, we have started to analyze the molecular and biological properties of New World simbu group viruses and are attempting to develop reverse genetics systems for OROV to study its molecular biology and pathogenesis. (3) Molecular characterization of uncharacterized orthobunyaviruses and taxonomically ungrouped bunyaviruses potentially causing human disease: In order to better understand the relationships between the molecular biological characteristics of uncharacterized viruses and their zoonotic potential, as well as their evolution, we will conduct an extensive genetic analysis and biological characterization of uncharacterized orthobunyaviruses and taxonomically ungrouped bunyaviruses isolated from Africa, Asia, South and North America. So far, we have determined the genome sequences of around 70 bunyaviruses, including genetically uncharacterized orthobunyaviruses and taxonomically ungrouped bunyaviruses. Our extensive genome sequencing attempts have identified Bhanja virus (BHAV), which was isolated from a tick collected in India, as belonging to a novel species in the genus Phlebovirus. Based on our genetic characterization of the BHAV genome we could determine that BHAV has the highest identity to severe fever with thrombocytopenia syndrome virus (SFTSV), a novel phlebovirus first recognized in 2009 as the cause of a tick-borne hemorrhagic fever-like syndrome in humans in China. Serological analyses also indicate that BHAV is serologically related to SFTSV. These results suggest that BHAV will be classified as a novel species in the genus Phlebovirus and that it constitutes the missing link between the newly identified human pathogen SFTSV and other tick-borne phleboviruses. Sporadic human cases of BHAV and genetic similarities of BHAV to SFTSV suggest pathogenic potential of BHAV for humans. Our sequence data and ongoing molecular characterization of BHAV will facilitate epidemiological and epizoological surveillance for BHAV infections as a potentially under-recognized cause of acute febrile illness.

（1）NGARI病毒（NRIV）中毒力获取的机制：在1997 - 98年，肯尼亚和索马里发生了很大的裂谷发烧（RVF）。在此暴发期间，在相当一部分病例中，NRIV被确定为出血热（HF）的病因。 NRIV也被公认为是Bunyamwera病毒（BUNV； L和S段）和Batai病毒（BATV; M段）之间自然存在的遗传替代品，它们均属于Orthobunyavirus属的Bunyamwera血清群。有趣的是，两种父母病毒会引起高温疾病，但在人类中并不严重。这是一个完美的例子，说明遗传重新分类在病毒的进化以及可能导致的毒力变化中发挥作用的重要作用。因此，我们将NRIV用作模型来理解自然界中新型致病性Bunyavires出现的分子机制。 到目前为止，我们已经确定了5个NRIV分离株的完整基因组序列（基因型：L-bunv/m-batv/s-bunv），包括来自肯尼亚/索马里亚HF暴发的2种菌株，以及2个BUNV分离株和5个BATV分离株。基于对这些数据的分析，我们对NRIV的出现进行了几个有趣的观察：1）NRIV菌株之间的遗传变异很低，这表明最近的单个保修事件可能导致了所有已确定的NRIV隔离株，并且2）2）虽然NRIV中的总体多样性的整体概率是较低的，但该概率的范围是较低的，这表明人类的范围很低，这是对人类的重要量，这一点是较低的，这是一个较低的范围，这是对人类的重要量，这是对人类的重要量，这是对人类的重要量，这是对人类的重要量，这是对人类的重要量，这是对人类的范围，这是对人类的重要量，这是对人类的范围，这是对人类的总体差异，这是对人类的影响，这是对人的总体差异，而这一范围却是较低的范围。 NRIV看到的大爆发。除了这些直接观察之外，这些序列的可用性还为我们提供了为这些病毒（目前正在进行的工作）追求全长克隆生成的可能性。将来，这些系统将使我们能够构建各种重新排化和嵌合病毒，从而绘制与NRIV毒力相关的病毒决定因素及其引起HF的能力。 此外，为了确定可能与NRIV获取毒力有关的可测量表型特征，我们一直在研究包括人和蚊子在内的各种细胞系的生长。有趣的是，我们已经确定，尽管在高度允许的细胞系（VERO）中，所有BATV菌株都可以很好地生长，并且具有相似的动力学，但在C6/36（蚊子细胞）中，只有少数BATV菌株，包括UGMP-6830菌株，包括UGMP-6830菌株，这是NRIV M分段的最接近的M-s元素，在感染后早期显示出有效的增长。这可能对成功的病毒在蚊子载体中的传播产生影响，并且在重新分类过程中BATV菌株可能会产生类似NRIV的病毒。此外，我们的初步数据表明，在原代人巨噬细胞中，BUNV和NRIV菌株的生长增强了。目前正在跟踪这一发现，并将包括针对这些不同病毒感染的细胞因子产生分析，因为对于其他出血性发烧病毒而言，已经证明细胞因子反应异常，这对于疾病的发展至关重要。另外，不同细胞系中病毒生长观察到的差异促进了考虑这些病毒可能使用不同受体的考虑。为了研究这一点，我们已经生成了BUNV，BATV和NRIV的受体结合亚基的可溶性版本。但是，尽管我们可以证明这些蛋白质与靶细胞结合，但在成纤维细胞中它们可以很好地阻断所有三种病毒的感染，这表明在这种细胞类型的受体使用中，使用是重叠的。研究目前正在进行研究的研究中，巨噬细胞（如巨噬细胞）是否有所不同。 (2) Molecular determinants of host range in Simbu serogroup viruses: The simbu serogroup also belongs to the genus Orthobunyavirus and can be categorized into those viruses that infect i) humans (Oropouche virus), ii) livestock animals (e.g. Akabane virus) or iii) vertebrate hosts (rodents, birds, monkey etc.) other than humans or livestock （例如Mermet病毒）。在Simbu-Serogroup病毒中，Oropouche病毒（OROV）在引起人类急性发热疾病方面是独一无二的。自发现发现以来，OROV引起了30多次疫情，在1960年至2009年在南美发现了至少500,000例案件。目前尚不清楚OROV为什么在人类中引起疾病​​，但其他SIMBU病毒却没有。为了鉴定SIMBU血清群病毒中宿主范围的病毒决定因素，我们将比较OROV和其他相关病毒的遗传和病毒学特征，尤其是那些感染非种植动物的病毒的病毒，因为这些病毒在遗传上比感染牲畜的病毒更接近OROV。为此，我们确定了超过25个SIMBU血清群病毒的完整基因组序列。我们的分析表明，感染脊椎动物或牲畜以外的其他脊椎动物的SIMBU组病毒可以在遗传和地理上分为两组，分为旧世界和新世界。此外，我们已经确定了几种与人类致病性OROV密切相关的南美SIMBU组病毒。有趣的是，我们发现这些OROV样病毒之一Utinga病毒在人类中尚未引起疾病，但与OROV（懒惰）（懒惰者）分离出来，并未编码非结构性蛋白质NSS，这抑制了宿主宿主抗病毒抗病毒抗病毒反应。基于这些结果，我们已经开始分析新世界SIMBU组病毒的分子和生物学特性，并正在尝试开发OROV研究其分子生物学和发病机理的反向遗传系统。 (3) Molecular characterization of uncharacterized orthobunyaviruses and taxonomically ungrouped bunyaviruses potentially causing human disease: In order to better understand the relationships between the molecular biological characteristics of uncharacterized viruses and their zoonotic potential, as well as their evolution, we will conduct an extensive genetic analysis and biological characterization of uncharacterized orthobunyaviruses and taxonomically ungrouped与非洲，亚洲，南美和北美分离的BUNYAVIRUS。到目前为止，我们已经确定了大约70个Bunyavires的基因组序列，包括遗传上未表征的矫形病毒和分类学未分组的Bunyaviruses。我们广泛的基因组测序尝试已经确定了Bhanja病毒（BHAV），该病毒是从印度收集的壁虱中分离出来的，属于phlebovirus属的新物种。根据我们对BHAV基因组的遗传特征，我们可以确定BHAV对血小板减少综合征病毒（SFTSV）的严重发烧具有最高的认同，这是一种新型的呼吸病毒，于2009年首次被认为是中国人类tick虫出血性出血热综合症的原因。血清学分析还表明，BHAV在血清学上与SFTSV相关。这些结果表明，BHAV将被归类为Phlebovirus属中的一种新物种，并且它构成了新鉴定出的人类病原体SFTSV和其他Tick传播的phleboviruses之间缺失的联系。 BHAV的BHAV和BHAV遗传相似性的零星人类对SFTSV的遗传相似性表明BHAV对人类的致病潜力。我们的序列数据和BHAV的持续分子表征将促进对BHAV感染的流行病学和表演性监测，这可能是急性发热疾病的潜在认可的原因。