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)阿里病毒(NRIV)的毒力获得机制：1997年8月，肯尼亚和索马里爆发裂谷热(RVF)大流行。在这次疫情爆发期间，NRIV 被确定为很大一部分病例中出血热 (HF) 的病原体。 NRIV 已被认为是布尼亚姆维拉病毒（BUNV；L 和 S 片段）和巴泰病毒（BATV；M 片段）之间天然存在的遗传重配体，这两种病毒均属于正布尼亚病毒属的布尼亚姆维拉血清群。有趣的是，两种亲本病毒都会引起人类发热性疾病，但不会引起严重的心力衰竭。这是基因重排在病毒进化以及由此导致的毒力变化中所发挥的重要作用的完美例子。因此，我们使用 NRIV 作为模型来了解自然界中新型致病性布尼亚病毒出现的分子机制。 我们最近确定了 5 个 NRIV 分离株（基因型：L-BUNV/M-BATV/S-BUNV）的全基因组序列，包括来自肯尼亚/索马里 HF 疫情的 2 个毒株，以及 5 个 BATV 分离株，包括与 NRIV M 段最近的亲属 UgMP-6830 毒株和 9 个 BUNV 分离株。根据获得的 BUNV 测序数据，我们鉴定了两种不同的病毒谱系，一种包含与原型毒株相关的病毒，另一种包含与 NRIV 祖细胞相关的毒株。此外，作为这些研究的结果，我们发现了一种新的 BUNV 重配体，其存在强调了此类病毒在自然界中的出现频率可能比目前认为的要高得多。 为了深入了解自然界中促进重配的因素，我们在 BUNV 和 BATV 之间进行了体外重配测定，作为共感染期间 NRIV 出现的模型。我们发现，虽然几乎所有可能的重配体都被回收，因此是可行的，但重配频率和重配产物均受到所用细胞类型（哺乳动物与节肢动物）的影响。结合之前我们分析 BUNV 和 BATV 在各种相关细胞系中生长的实验的表型数据，我们现在正在评估这些重配体的特征，以建立这些特征与特定基因产物/片段之间的关系。这些信息将用于指导我们未来使用 NRIV 反向遗传学系统进行重配研究（我们正在建立该系统），并用作了解其他自然发生的重配病毒对公共健康的潜在影响的框架，例如我们对正布尼亚病毒属进行大规模系统发育分析时发现的重配病毒。 (2)正布尼亚病毒和未表征的布尼亚病毒的遗传特征和进化模型：布尼亚病毒科是一个异常庞大和多样化的病毒群，仅正布尼亚病毒属就含有150多种命名病毒，其中包括许多人类病原体。由于缺乏足够的遗传数据来促进分子鉴定，正布尼亚病毒历来被分为 18 个不同血清群之一，再次反映了这些病毒之间的高度遗传多样性。然而，这种方法有很大的局限性，因为它通常只关注一种或最多 2 种病毒抗原，使得使用这种方法鉴定重配体的可能性要小得多，特别是在血清群内重配体的情况下。此外，某些病毒之间的血清学交叉反应性可能很高，从而妨碍了基于此类方法的明确鉴定。或者，一些未表征的布尼亚病毒对任何已知组都没有表现出足够的反应性，因此它们的确切性质仍然未知。然而，这些病毒对于我们了解自然界中存在的遗传多样性特别重要，也可能为我们了解该群体的进化提供关键联系。 为了弥补我们知识中的这些空白，并为进化分析生成足够大小和完整性的数据集，我们对各种正布尼亚病毒组进行了大规模测序，包括 Bwamba/Pongola 和 Nyando 组病毒，这些病毒是非洲大部分地区流行但尚未得到充分研究的发热性疾病原因。根据我们的测序工作，我们阐明了这些病毒的分类关系，并确定了两种与 Nyando 病毒组相关的先前未表征的布尼亚病毒（即 Moju dos Campos 和 Kaeng Khoi 病毒）。这些发现表明，该病毒群覆盖的地理区域比以前认识的要大得多（包括亚洲和南美洲），并且可能涉及以前未被认识到对这些病毒群起作用的宿主物种。同样，我们现在正在完成一项研究，旨在检查瓜罗阿病毒的分布情况，瓜罗阿病毒是南美洲一种被忽视的发热性疾病病因，其流行区域似乎正在扩大，包括秘鲁和玻利维亚等国家。根据我们的数据，我们定义了这些病毒的遗传多样性程度，以建立广泛交叉反应的 RT-PCR 引物集，并生成足以对南美洲 GROV 传播进行系统发育地理学建模的数据集，并使我们能够确定活跃传播的区域，作为未来监测工作的一部分，应重点关注这些区域。针对 Bunyamwera 组病毒的其他研究正在进行中。 (3)可能引起人类疾病的蜱传白蛉病毒的分子特征：为了更好地了解未特征病毒的分子生物学特征与其人畜共患潜力之间的关系以及它们的进化，我们正在对与蜱相关的未特征分类学未分组的布尼亚病毒进行广泛的遗传分析和生物学特征，包括从非洲、亚洲、南美洲和北美洲分离的布尼亚病毒。 2005年至2013年期间，严重发烧伴血小板减少综合症病毒（SFTSV）和哈特兰病毒（两种新型蜱传白斑病毒）分别在中国、日本和韩国或美国首次被认为是导致人类严重血小板减少症的原因。尽管这些蜱传白蛉病毒 (TBPV) 与 Bhanja 群病毒 (BHAV) 和 Uukuniemi 群病毒 (UUKV) 一起构成白蛉病毒属的相关群，但由于这些病毒之间的血清学和遗传差异很大，同时对所有 TBPV 进行流行病学研究和诊断一直很困难。因此，根据我们和其他人确定的可用 TBPV 基因组序列，我们开发了一种 RT-PCR 系统，针对 L 基因组片段中的保守序列基序，能够检测所有已知 TBPV 的 L 片段 RNA。这种 RT-PCR 检测使我们能够将一些分类学上未指定的类布尼亚病毒识别为 TBPV。此外，通过与日本北海道大学和山口大学合作，我们利用 RT-PCR 系统在日本、马里、赞比亚和美国发现了新型 TBPV。系统发育分析显示，与蚊子/白蛉传播的病毒（例如裂谷热病毒）相比，与蜱相关的病毒更加多样化，这是以前所认识的，并且代表白蛉病毒属内的主要病毒类型。