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: 8745560
• 负责人: Hideki Ebihara
• 金额: $ 45.43万
• 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/8745560
• 关键词: Africa; Animals; Arthropods; Asia; Base Sequence; Biogenesis; Bioinformatics; Biologic Characteristic; Biological; Biological Assay; Birds; Bite; Bunyamwera virus; Bunyaviridae; Categories; Cell Line; Cells; Characteristics; China; Chiroptera; Chittoor virus; Clinical; Complex; Crimean Hemorrhagic Fever; Culicidae; Data; Detection; Development; Diagnosis; Disease; Disease Outbreaks; Distant; Encephalitis; Epidemiologic Studies; Epidemiology; Evolution; Family; Family member; Fever; Functional RNA; Future; Genbank; Gene Mutation; Genetic; Genetic Drift; Genome; Genotype; Genus Phlebovirus; Goals; Growth; Hantavirus; Hantavirus Pulmonary Syndrome; Human; Infection; Japan; Kenya; Length; Livestock; Maintenance; Mali; Mammalian Cell; Mammals; Maps; Measurable; Methods; Modeling; Molecular; Monkeys; Mutation; Nairovirus; National Institute of Allergy and Infectious Disease; Nature; North America; Nucleotides; One-Step dentin bonding system; Oropouche virus; Orthobunyavirus; Pathogenesis; Patients; Phylogenetic Analysis; Plants; Play; Publishing; RNA; RNA Viruses; Reassortant Viruses; Relative (related person); Research; Research Priority; Reverse Transcriptase Polymerase Chain Reaction; Rift Valley Fever; Rodent; Role; Sampling; Serological; Simbu Group Viruses; Somalia; South America; South Korea; Syndrome; System; Thrombocytopenia; Ticks; Time; Tospovirus; United States; Uukuniemi virus; Viral; Viral Hemorrhagic Fevers; Virulence; Virus; Virus Diseases; base; cell type; design; field study; genetic analysis; genome sequencing; human disease; influenzavirus; insight; interest; member; neutralizing antibody; next generation sequencing; novel; novel virus; pathogen; positional cloning; prototype; research study; screening; transmission process; vector mosquito; 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 4 BUNV isolates, among which we have identified a recent field strain that appears to be much more closely related to NRIV than the prototype lab strain used in most studies to date. Based on analysis of our sequencing data we have made several interesting observation related to the emergence of NRIV, among which is that mutations in the non-coding regions of NRIV are not necessary to facilitate reassortment, something that would have presented a significant hurdle to future studies aimed at generating BUNV/BATV reassortant viruses using reverse genetics. Indeed, with the availability of these complete genome data we are proceeding with the construction of a full-length clone system for these viruses, which will in future 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 previously analysed growth of BUNV and BATV in various relevant cell lines. In particular, we found that in C6/36 (mosquito cells) only a few BATV strains, including the UgMP-6830 strain, 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. We are currently analysing the requirements for reassortment during co-infection in various cell lines, including both C6/36 cells and various mammalian cell types, using a co-infection/reassortment assay that we have established. These experiments will provide us with greater insight into where in the natural infection cycle reassortment occurs and what are the potential products of reassortment. This information will be used to guide our future studies of reassortment using the NRIV reverse genetics system as well as being used as a framework to understand the various other naturally occurring reassortant viruses that we have identified as part of our broad-scale phylogenetic analysis of the Orthobunyavirus genus, which is also currently ongoing. (2) Molecular determinants of host range and antigenic shift in Simbu serogroup viruses: The Simbu serogroup also belongs to the genus Orthobunyavirus and can be categorized into those viruses that infect i) humans (e.g. 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). In order to identify the viral determinants of host range among Simbu serogroup viruses, we have already determined full-length genome sequence of more than 20 Simbu-group viruses/strains, and bioinformatics analyses are ongoing. Oropouche virus (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 humans while other Simbu-group viruses do not. Notably, during the 1999 Oropouche fever outbreak a novel Simbu-group virus, Iquitos virus (IQTV), was isolated from patients with Oropouche fever-like illness. This virus was a novel reassortant possessing L and S genome segments derived from OROV and an M genome segment of a novel Simbu group virus. Importantly, pre-existing neutralizing antibodies in people already infected with OROV do not neutralize and protect from IQTV infection. In order to elucidate the mechanism of antigenic shift associated with the emergence of novel genetic reassortants we have been carrying out genome sequencing on 39 OROV strains and 12 strains of IQTV. Our analyses have revealed that i) IQTV M segments are genetically close to those of OROV, compared with M segment sequences of other Simbu group viruses, but distant enough to belong to a distinct species, and ii) all OROV S genome segment sequences published to date (or registered in Genbank) are missing 200 nucleotides compared with the full-genome sequence that we completed using a combination of next generation sequencing and careful sanger-based sequencing. We are now developing a reverse genetics system for OROV based on our sequence results, which will represent a critical step towards characterization of the molecular determinants of host range and antigenic shift in Simbu serogroup viruses. (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 will conduct an extensive genetic analysis and biological characterization of uncharacterized taxonomically ungrouped bunyaviruses isolated from Africa, Asia, South and North America. During 2005-2013, Severe Fever with Thrombocytopenia Syndrome virus (SFTSV) and Heartland virus, 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 Bhanja group viruses (BHAVs) and Uukuniemi group viruses (UUKVs), the epidemiological study and diagnosis of all TBPVs simultaneously has been difficult due to serological and genetic divergence among these viruses. Therefore, we expanded our extensive genome sequencing attempts to the various tick-borne phleboviruses and established a simple and fast detection method for multiple TBPVs by using one-step RT-PCR with a single pair of degenerate primers designed based on conserved virus genome sequences. Moreover, we identified novel TBPVs from uncharacterized bunyavirus samples and ticks corrected in Mali, strongly demonstrating that our simple RT-PCR system is of sufficient sensitivity and broad-reactivity to detect a range of TBPVs, including previously uncharacterized viruses that are divergent from known TBPVs. Our system will help field studies to identify both known and novel TBPVs, and can be used for the screening of uncharacterized febrile illnesses associated with tick bites.

(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 暴发的 2 个毒株，以及 5 个 BATV 分离株，包括 UgMP-6830 毒株，它是 NRIV M 段的最近亲，以及 4 个 BUNV 分离株，其中我们鉴定了一个最近的野外毒株，它似乎与我们的关系更为密切。到 NRIV 比迄今为止大多数研究中使用的原型实验室菌株要高。基于对我们测序数据的分析，我们对 NRIV 的出现进行了一些有趣的观察，其中包括 NRIV 非编码区的突变对于促进重配来说并不是必需的，这可能会给未来旨在利用反向遗传学生成 BUNV/BATV 重配病毒的研究带来重大障碍。事实上，随着这些完整基因组数据的可用性，我们正在着手构建这些病毒的全长克隆系统，这将使我们将来能够构建各种重配和嵌合病毒，从而绘制与 NRIV 毒力及其引起心衰能力相关的病毒决定因素。 此外，为了确定可能与 NRIV 毒力获得相关的可测量表型特征，我们之前分析了 BUNV 和 BATV 在各种相关细胞系中的生长。特别是，我们发现在 C6/36（蚊子细胞）中，只有少数 BATV 菌株（包括 UgMP-6830 菌株）在感染后的早期时间点表现出有效生长。这可能对病毒在蚊子媒介中的成功传播产生影响，并可能限制 BATV 菌株在重配过程中产生 NRIV 样病毒。我们目前正在使用我们建立的共感染/重配测定分析各种细胞系（包括 C6/36 细胞和各种哺乳动物细胞类型）共感染期间重配的要求。这些实验将使我们更深入地了解自然感染周期重配发生的位置以及重配的潜在产物是什么。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，并用作了解各种其他自然发生的重配病毒的框架，我们已将这些病毒确定为正布尼亚病毒属大规模系统发育分析的一部分，该分析目前也在进行中。 (2) Simbu 血清群病毒宿主范围和抗原转变的分子决定因素：Simbu 血清群也属于 Orthobunyavirus 属，可分为感染 i) 人类（例如 Oropouche 病毒）、ii）家畜动物（例如 Akabane 病毒）或 iii）脊椎动物宿主（啮齿动物、鸟类、猴子等）的病毒 比人类或牲畜（例如 Mermet 病毒）。为了确定 Simbu 血清群病毒宿主范围的病毒决定因素，我们已经确定了 20 多种 Simbu 群病毒/毒株的全长基因组序列，生物信息学分析正在进行中。 1960 年至 2009 年间，奥罗普切病毒 (OROV) 在南美洲引发了 30 多次疫情，至少发现了 50 万例病例。目前尚不清楚为什么 OROV 会引起人类疾病，而其他辛布族病毒却不会。值得注意的是，在 1999 年奥罗普切热爆发期间，从奥罗普切热类疾病患者中分离出了一种新型辛布族病毒——伊基托斯病毒 (IQTV)。该病毒是一种新型重配病毒，具有源自 OROV 的 L 和 S 基因组片段以及新型辛布族病毒的 M 基因组片段。重要的是，已经感染 OROV 的人体内预先存在的中和抗体不能中和和保护 IQTV 感染。为了阐明与新型基因重配出现相关的抗原转移机制，我们对 39 个 OROV 毒株和 12 个 IQTV 毒株进行了基因组测序。我们的分析表明，i) 与其他辛布族病毒的 M 片段序列相比，IQTV M 片段在遗传上与 OROV 的片段相似，但距离足够远，足以属于不同的物种；ii) 迄今为止发表的（或在 Genbank 中注册的）所有 OROV S 基因组片段序列与我们使用下一代测序和仔细桑格测序相结合完成的全基因组序列相比，缺失了 200 个核苷酸。 测序。我们现在正在根据我们的序列结果开发 OROV 的反向遗传学系统，这将代表朝着表征 Simbu 血清群病毒宿主范围和抗原转变的分子决定因素迈出关键一步。 (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，我们将对从非洲、亚洲、南美洲和北美洲分离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。 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（一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，同时对所有 TBPV 进行流行病学研究和诊断一直很困难。因此，我们将广泛的基因组测序尝试扩展到各种蜱传白蛉病毒，并通过使用一步RT-PCR和基于保守病毒基因组序列设计的一对简并引物，建立了一种简单快速的多种TBPV检测方法。此外，我们从马里未定性的布尼亚病毒样本和蜱中鉴定出新型 TBPV，这有力地证明了我们的简单 RT-PCR 系统具有足够的灵敏度和广泛的反应性，可以检测一系列 TBPV，包括与已知 TBPV 不同的先前未定性的病毒。我们的系统将帮助现场研究识别已知和新型的 TBPV，并可用于筛查与蜱虫叮咬相关的未表征的发热性疾病。