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: 8946509
• 负责人: Hideki Ebihara
• 金额: $ 48.51万
• 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/8946509
• 关键词: Address; Africa; Area; Arthropods; Asia; Biogenesis; Biologic Characteristic; Biological; Biological Assay; Birds; Bolivia; Bunyamwera Group Viruses; Bunyamwera virus; Bunyaviridae; Categories; Cell Line; Characteristics; China; Chiroptera; Chittoor virus; Clinical; Complex; Conserved Sequence; Country; Crimean Hemorrhagic Fever; Culicidae; Data; Data Set; Development; Diagnosis; Disease; Disease Outbreaks; Encephalitis; Epidemiologic Studies; Epidemiology; Evolution; Family; Family member; Fever; Frequencies; Future; Gene Mutation; Genetic; Genetic Drift; Genetic Variation; Genome; Genotype; Genus Phlebovirus; Goals; Growth; Hantavirus; Hantavirus Pulmonary Syndrome; Human; In Vitro; Infection; Japan; Kenya; Knowledge; Large-Scale Sequencing; Link; Maintenance; Mali; Mammals; Methods; Modeling; Molecular; Nairovirus; Names; National Institute of Allergy and Infectious Disease; Nature; North America; Orthobunyavirus; Pathogenesis; Peru; Phlebotomus; Phylogenetic Analysis; Plants; Play; Process; Proteins; Public Health; RNA; RNA Viruses; Reassortant Viruses; Relative (related person); Research; Research Priority; Reverse Transcriptase Polymerase Chain Reaction; Rift Valley Fever; Rift Valley fever virus; Role; Satellite Viruses; Serological; Somalia; South America; South Korea; Syndrome; System; Thrombocytopenia; Ticks; Tospovirus; United States; Universities; Uukuniemi virus; Viral Antigens; Viral Hemorrhagic Fevers; Virulence; Virus; Zambia; base; cell type; cross reactivity; genetic analysis; genome sequencing; human disease; influenzavirus; insight; member; neglect; novel; pathogen; positional cloning; prevent; progenitor; prototype; research study; trait; transmission process; virology; virus host interaction

(1) Mechanisms of virulence acquisition in Ngari virus (NRIV): In 1997-8 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 has been 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. We have recently 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 5 BATV isolates, including the UgMP-6830 strain, which is the closest relative of the NRIV M-segment, and 9 BUNV isolates. Based on the BUNV sequencing data obtained we have identified two distinct virus lineages, one containing viruses related to the prototype strain and another containing strains related to the NRIV progenitor. In addition, as a result of these studies we have identified a novel reassortant of BUNV, the existence of which serves to emphasize that the frequency of such viruses in nature is probably much higher than is currently appreciated. In order to gain insight into factors that facilitate reassortment in nature, we have conducted in vitro reassortment assays between BUNV and BATV, as a model for the emergence of NRIV during co-infection. We found that while nearly all possible reassortants were recovered, and thus are viable, both reassortment frequency and the products of reassortment were influence by the cell type used (mammalian vs arthropod). Combined with phenotypic data from previous experiments in which we analyzes the growth of BUNV and BATV in various relevant cell lines, we are now assessing the characteristics of these reassortants to establish a relationship between these traits and specific gene products/segments. This information will be used to guide our future studies of reassortment using the NRIV reverse genetics system, which we are in the process of establishing, as well as being used as a framework to understand the potential public health impact of other naturally occurring reassortant viruses, such as that identified as part of our broad-scale phylogenetic analysis of the Orthobunyavirus genus. (2) Genetic characterization and evolutionary modelling for orthobunyaviruses and uncharacterized bunyaviruses: The Bunyaviridae is an unusually large and diverse virus group, with the genus Orthobunyavirus alone containing more than 150 named viruses, among which are numerous human pathogens. In the absence of a sufficient body of genetic data to facilitate molecular identification, orthobunyaviruses have historically been classified into one of 18 distinct serogroups, again reflecting the high degree of genetic diversity between these viruses. However, this approach has significant limitations in that it usually focuses on only one or at most 2 viral antigens, making it much less likely that reassortants will be identified using this approach, particularly in the case of reassortants within a serogroup. In addition, serological cross-reactivity can be high among certain viruses, preventing a definitive identification based on such methods. Alternatively, some uncharacterized bunyaviruses do not show sufficient reactivity to any known group and as a result their exact nature remains unknown. However, these viruses are particularly important to our understanding of the genetic diversity that exists in nature and may also provide critical links in our understanding of the evolution of this group. In order to address these gaps in our knowledge and generate datasets of sufficient size and completeness for evolutionary analysis, we have undertaken large-scale sequencing of various orthobunyavirus groups, including the Bwamba/Pongola and Nyando group viruses, which are prevalent but understudied causes of febrile illness throughout much of Africa. Based on our sequencing efforts we have clarified the taxonomic relationships of these viruses, as well as identifying two previously uncharacterized bunyaviruses that are related to the Nyando virus group (i.e. Moju dos Campos and Kaeng Khoi viruses). These findings showed that this virus groups covers a much larger geographical area than was previously appreciated (including both Asia and South America) and may involve host species that had not previously been recognized to play a role for these virus groups. Similarly, we are now completing a study aimed at examining the distribution of Guaroa virus, a neglected cause of febrile illness in South America that seems to be undergoing expansion of its endemic area to include countries such as Peru and Bolivia. Based on our data we have defined the degree of genetic diversity of these viruses to establish broadly cross-reactive RT-PCR primer sets and generated datasets sufficient to allow phylogeographic modelling of GROV spread within South America and allowing us to identify regions of active spread that should be focused on as a part of future surveillance efforts. Additional studies focusing on the Bunyamwera group viruses are on-going. (3) Molecular characterization of tick-borne phleboviruses 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 are conducting an extensive genetic analysis and biological characterization of uncharacterized taxonomically ungrouped bunyaviruses associated with ticks, including those isolated from Africa, Asia, South and North America. During 2005-2013, Severe Fever with Thrombocytopenia Syndrome virus (SFTSV) and Heartland virus, two novel tick-borne phleboviruses, were first recognized as the causes of severe illness with thrombocytopenia among humans in China, Japan and South Korea, or the United States, respectively. Although these tick-borne phleboviruses (TBPVs) comprise a related group in the genus Phlebovirus, along with the Bhanja group viruses (BHAVs) and Uukuniemi group viruses (UUKVs), the epidemiological study and diagnosis of all TBPVs simultaneously has been difficult due to the high degree of serological and genetic divergence among these viruses. Therefore, based on available TBPV genome sequences, determined by us and others, we have developed an RT-PCR system targeting a conserved sequence motif in the L genome segment that is able to detect the L segment RNA of all known TBPVs. This RT-PCR assay allowed us to identify some taxonomically unassigned bunya-like viruses as TBPVs. Furthermore, by collaborating with Hokkaido University and Yamaguchi University in Japan, we have discovered novel TBPVs in Japan, Mali, Zambia, and the United States using our RT-PCR system. Phylogenetic analysis revealed that viruses associated with ticks are more divergent that was previously appreciated and represent the dominant virus type within the genus Phlebovirus, as compared with mosquito/phlebotomus-borne viruses (e.g., Rift Valley fever virus).

（1）NGARI病毒（NRIV）的毒力获取机制：在1997 - 8年，肯尼亚和索马里发生了大量裂谷谷热（RVF）爆发。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被认为是Bunyamwera病毒（BUNV； L和S段）和Batai病毒（BATV; M段）之间的一种天然存在的遗传性重构，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。 我们最近确定了5个NRIV分离株（基因型：L-BUNV/M-BATV/S-BUNV）的完整基因组序列，包括来自肯尼亚/索马里HF暴发的2种菌株以及5个BATV分离株，包括UGMP-6830菌株，这是NRIV M-6830菌株的最接近的NRIV M-M-M-sement sement exment sement sement sement sement sement sement sement sement sement sement和9 bunv bunv隔离株。根据获得的BUNV测序数据，我们已经确定了两个不同的病毒谱系，一种含有与原型菌株相关的病毒，另一种包含与NRIV祖细胞相关的菌株。此外，由于这些研究，我们已经确定了BUNV的新型bunv，其存在强调，这种病毒在自然界中的频率可能比目前所欣赏的要高得多。 为了深入了解促进自然界重新分类的因素，我们在BUNV和BATV之间进行了体外的重新分类测定，这是在共同感染过程中NRIV出现的模型。我们发现，虽然恢复了几乎所有可能的重组，因此是可行的，但重新分配频率和重新分类的产物都受到所使用的细胞类型（哺乳动物与节肢动物）的影响。结合以前实验的表型数据，我们在各种相关细胞系中分析了BUNV和BATV的生长，我们现在正在评估这些重新评估的特征，以在这些性状和特定基因产品/段之间建立关系。这些信息将用于指导我们未来对使用NRIV反向遗传系统重新分类的研究，我们正在建立该系统，并用作理解其他自然发生的重新分类病毒的潜在公共卫生影响，例如被确定为我们的宽尺性分析的一部分的公共健康影响。 （2）正质阳离子病毒和未表征的Bunyaviruse的遗传表征和进化模型：Bunyaviridae是一种异常大而多样的病毒群，单独包含超过150个命名的病毒，其中有许多人类病原体。在没有足够的遗传数据体系来促进分子鉴定的情况下，历史上已经将正骨尚病毒分类为18种不同的血清群之一，再次反映了这些病毒之间的高度遗传多样性。但是，这种方法具有重大的局限性，因为它通常仅关注一种或最多的2种病毒抗原，因此使用这种方法鉴定出重新成分的可能性要小得多，尤其是在血清群中重新分类的情况下。此外，在某些病毒中，血清学交叉反应性可能很高，从而阻止了基于这种方法的确定识别。或者，某些未表征的Bunyavirus对任何已知组都没有足够的反应性，因此它们的确切性质仍然未知。但是，这些病毒对于我们对自然界中存在的遗传多样性的理解尤为重要，并且还可能在我们对该群体演变的理解中提供关键联系。 为了在我们的知识中解决这些差距，并生成了足够大小和完整性的数据集以进行进化分析，我们已经对各种正骨基质病毒组进行了大规模测序，包括Bwamba/Pongola和Nyando Group病毒病毒，这些病毒普遍存在，但这些病毒普遍存在，但本次经中遗忘的是整个非洲大部分地区的大量疾病。基于我们的测序工作，我们阐明了这些病毒的分类关系，并确定了与Nyando病毒组相关的两个先前未表征的BUNYAVIRES（即Moju Dos Campos和Kaeng Khoi Viruse）。这些发现表明，该病毒组涵盖的地理区域比以前所欣赏的更大（包括亚洲和南美），并且可能涉及以前尚未被认为对这些病毒组发挥作用的宿主物种。同样，我们现在正在完成一项研究，旨在检查瓜鲁阿病毒的分布，瓜鲁亚病毒的分布是南美的一个被忽视的高温疾病原因，似乎正在其流行地区扩大，以包括秘鲁和玻利维亚等国家。基于我们的数据，我们定义了这些病毒的遗传多样性程度，以建立足够的足够的交叉反应性RT-PCR引物集和生成的数据集，以允许南美Grov扩散的植物地理学建模，并允许我们确定应将其作为未来监视工作的一部分的主动扩散区域。针对Bunyamwera组病毒的其他研究正在进行中。 (3) Molecular characterization of tick-borne phleboviruses 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 are conducting an extensive genetic analysis and biological characterization of uncharacterized taxonomically ungrouped bunyaviruses associated with ticks, including those isolated from Africa, Asia, South and North America.在2005年至2013年期间，血小板减少综合征病毒（SFTSV）和Heartland病毒严重发烧，两种新型的Tick传播鼻息病毒首先被认为是在中国，日本和韩国或韩国或美国的人类中患有血小板减少症的严重疾病的原因。尽管这些tick传播的静脉病毒（TBPV）包括属phlebovirus属的一个相关组，以及Bhanja组病毒（BHAVS）和UUKUNIEMI组病毒（UUKVS）（UUKVS），在所有TBPV的流行病学研究和诊断中都归功于这些疾病的所有概念，并且在这些概述中都是备受统治程度。因此，基于由我们和其他人确定的可用TBPV基因组序列，我们开发了一个针对L基因组段中保守序列基序的RT-PCR系统，该系统能够检测所有已知TBPV的L段RNA。这种RT-PCR分析使我们能够将一些类似于分类的Bunya样病毒识别为TBPV。此外，通过与日本的北海道大学和山古奇大学合作，我们使用我们的RT-PCR系统在日本，马里，赞比亚和美国发现了小说的TBPV。系统发育分析表明，与寄生虫/芬氏病毒/省植物病毒相比，与tick相关的病毒更具不同的不同，代表了phlebovirus属内的主要病毒类型（例如，裂谷谷发烧病毒）。