Vaccines and molecular tools for the control of the emerging bunyavirus, severe fever with thrombocytopenia syndrome phlebovirus (SFTSV)

新形成的布尼亚病毒目的病毒经常由节肢动物传播，许多成员能够引起大量的人、动物和疾病。世界卫生组织已将几种布尼亚病毒确定为关键的新兴病原体，这一题为“加快疫苗开发的健康途径”的呼吁也强调了人们对这类病毒的兴趣。该项目专注于布尼亚病毒属现病毒科的严重发热并伴有血小板减少综合征的静脉病毒(SFTS静脉病毒；SFTSV)，该病毒还包括亚洲和美国的重要新病原体(哈特兰病毒，HRTV)。到目前为止，中国报告了2500多例新城疫，韩国和日本分别报告了172例和96例，病死率从8.2%到32.6%不等。所有流行国家的年度报告发病率都在上升，例如中国，2010年报告了53例，而2013年为676例。为了应对新出现的布尼亚病毒带来的威胁，MRC格拉斯哥大学病毒研究中心(CVR)的科学家们开发了一种“反向遗传学”系统，利用该系统可以操纵SFTSV，并允许合理设计合理减毒的病毒候选疫苗。这种反向遗传学系统使得能够从编码病毒基因组RNA的cDNA拷贝的质粒中快速恢复具有复制能力的布尼亚病毒。这项技术有助于绘制以体液和细胞免疫反应为目标的表位图，以及识别和操纵病毒编码的毒力因子。许多布尼亚病毒的NSS蛋白被认为是毒力因子，因为它们有能力对抗哺乳动物的先天免疫反应。因此，这些基于反向遗传学的策略提供了一种现实的手段，为新出现的布尼亚病毒病开发合理的候选疫苗，以及开发有助于进一步阐明感染和病毒发病机制的免疫反应的病毒。我们现在已经产生了一种重组的NSS-Deletant SFTSV(称为SFTSVdelNSs)，它不能对抗感染时安装的I型干扰素反应。这有助于研究先天免疫反应在控制SFTSV中的作用，以及在缺乏毒力因子NSS的情况下体内的组织趋向性和致病机制，从而评估此类潜在减毒病毒作为疫苗候选。基于反向遗传学的方法生产重组布尼亚病毒的优势超出了生产减毒病毒候选疫苗的范围。该系统还允许开发和生产病毒样颗粒(VLP)。重组病毒和VLP都可以测试它们在免疫动物和保护性候选疫苗中诱导免疫反应的能力。这项建议将联合CVR和北京生命科学研究院(BIOLS)的研究人员，阐明SFTSV感染的宿主免疫应答和致病机制，重组病毒候选疫苗的开发和进一步研究SFTSV的工具。目的1.阐明SFTSV NSS在体内组织趋向性和致病机制中的作用。我们还将评估重组SFTSV(缺乏NSS；SFTSVdelNSS)作为疫苗候选者与VLP的比较。目的2.生产针对SFTSV的新型中和抗体，用于基础研究和潜在的治疗药物。我们将使用一种表达报告基因的新型SFTS病毒来研究抗体反应，以研究患者血清和受感染动物的免疫反应。这将使我们能够定义中和抗体反应的广度，并绘制病毒粒子上的抗原决定簇。将通过突变分析来研究SFTSV Gn结构蛋白(已知的抗体反应目标)的抗原变异和逃避中和。该项目还将包括为合作伙伴BIOLS提供反向遗传学技术方面的培训。

Viruses in the newly formed order Bunyavirales are frequently arthropod-transmitted with many members capable of causing substantial human, animal and disease. The WHO has identified several bunyaviruses as key emerging pathogens, and this call entitled "One health approaches to accelerate vaccine development" also emphasizes interest in this group of viruses. This project focuses on severe fever with thrombocytopenia syndrome phlebovirus (SFTS phlebovirus; SFTSV) of the Phenuiviridae family of bunyaviruses which also includes significant emerging pathogens in both Asia and the USA (Heartland virus, HRTV). To date, over 2500 SFTSV cases have been reported in China, 172 cases in South Korea and 96 cases in Japan, with case fatality rates ranging from 8.2% to 32.6%. The annual incidence of reporting is increasing in all endemic countries, e.g. in China where 53 cases were reported in 2010, compared to 676 in 2013. To address the threat posed by novel, emerging bunyaviruses, scientists at the MRC-University of Glasgow Centre for Virus Research (CVR) have developed a "reverse genetics" system with which SFTSV can be manipulated, and which permit the rational design of rationally attenuated virus vaccine candidates. Such reverse genetics systems enable the rapid recovery of replication competent bunyaviruses from plasmids encoding cDNA copies of the viral genomic RNA. This technology facilitates the mapping of epitopes targeted by the humoral and cellular immune response as well as the identification and manipulation of virally-encoded virulence factors. The NSs proteins of many bunyaviruses are recognised as virulence factors due to their ability to counteract the mammalian innate immune responses. Hence, these reverse genetics-based strategies offer a realistic means to develop rational vaccine candidates for emerging bunyaviral diseases, as well as the development of viruses that help to further elucidate the immune responses mounted to infection and viral pathogenesis. We have now generated a recombinant NSs-deletant SFTSV (called SFTSVdelNSs) which is unable to antagonise type I interferon responses mounted in response to infection. This facilitates the studies into the role of innate immune responses in controlling SFTSV, as well as in vivo tissue tropism and pathogenesis in absence of the virulence factor NSs, leading to the evaluation of such potentially attenuated viruses as vaccine candidates. The strengths of reverse genetics-based approaches to producing recombinant bunyaviruses extends beyond the production of attenuated virus vaccine candidates. The system also allows the development and production of virus-like particles (VLPs). Both recombinant viruses and VLPs can be tested for their ability to induce immune responses in immunised animals as well as protective vaccine candidates. This proposal will bring together CVR and Beijing Institutes of Life Science (BIOLS) researchers to elucidate host immune responses to SFTSV infection and pathogenesis, the development of recombinant virus vaccine candidates and tools to further study SFTSV.Aim 1. To elucidate the contribution of SFTSV NSs to in vivo tissue tropism and pathogenesis. We will also assess a recombinant SFTSV (lacking NSs; SFTSVdelNSs) as vaccine candidate in comparison to VLPs.Aim 2. To produce novel neutralising antibodies against SFTSV for basic research and as potential therapeutic agents. We will investigate antibody responses using a novel SFTS viruses expressing reporter genes to study immune responses in both patient sera and infected animals. This will allow us to define the breadth of the neutralising antibody responses and to map antigenic determinants on the virion. Antigenic variation and escape from neutralisation of the SFTSV Gn structural protein (known target for antibody responses) will be investigated through mutational analysis. The project will also include training in reverse genetics technology for the partner BIOLS.