Out of the reservoir: identification and characterization of viral and host factors governing the pathobiology of zoonotic cowpox viruses

这是一项延续的建议，我们在第一个供资期的成果基础上继续努力。虽然我们首次能够从啮齿动物宿主——普通田鼠中分离并详细描述牛痘病毒（CPXV），但我们仍在继续进行实地研究。我们的目标是更好地了解CPXV在水库内维持和传播的生态学，通过优化的采样方案筛选进一步的分离株，并通过确定特定栖息地的血清流行率。第一个田鼠分离物可以与从意外/溢出宿主（大鼠、猫、牛）获得的CPXV序列进行比较。我们推测，病毒编码蛋白的改变允许CPXV在储存库中维持，并导致CPXV成功溢出到意外宿主中。我们将在这里研究是否以及如何确定CPXV因子有助于维持自然田鼠储存库和溢出到不同的意外宿主。重要的是，我们将确定这些因素如何与毒性联系起来。实验将首先建立基于全长克隆的重组和嵌合CPXV，并通过细胞生物学、生化和免疫学分析来确定蛋白质的功能。除此之外，我们将对潜在的物种特异性CPXV免疫逃避蛋白（例如，7tGP， CrmE和D7L蛋白）以及CPXV对田鼠物种Mx蛋白的可能干扰进行详细的实验。我们将在已建立的合作基础上与SPP1596的其他小组合作，主要是R.G. Ulrich（田鼠的筛选包括流行研究），M. Marz （RNAseq分析和CPXV基因组的自动注释），K. kamerer - schadt（生态学和建模），G. Kochs（田鼠Mx蛋白的作用）和C. Drosten（人畜共患啮齿动物传播病原体的总体背景）。

This is a continuation proposal in which we build on results from the first funding period. Although we were able, for the first time, to isolate and characterize in detail a cowpox virus (CPXV) from the rodent reservoir, the common vole, we are continuing field studies. Our goal is to gain a better understanding of the ecology of CPXV maintenance and transmission within the reservoir, by screening for further isolates with an optimized sampling scheme and by determining seroprevalences in defined habitats. The first vole isolate allowed comparison with available CPXV sequences from accidental/spill-over hosts (rats, cats, cattle). Alterations in virus-encoded proteins were identified that we surmise allow CPXV maintenance in the reservoir and others that are responsible for successful spill-over into accidental hosts. We will here investigate if and how the identified CPXV factors contribute to maintenance in the natural vole reservoir and to spill-overs into different accidental hosts. Importantly, we shall determine how those factors are linked to virulence. The experiments will be done by first creating recombinant and chimeric CPXV that are based on full-length clones and by performing cell biological, biochemical and immunological assays to determine protein function. Among others, we will perform detailed experiments on potentially species-specific CPXV immune evasion proteins (e.g., 7tGP, CrmE and D7L proteins) as well on the possible interference of CPXV with Mx proteins in vole species. We shall cooperate with other groups of SPP1596 based on established cooperations, primarily R.G. Ulrich (screening of voles including prevalence studies), M. Marz (RNAseq analysis and automated annotation of CPXV genomes), K. Kramer-Schadt (ecology and modeling), G. Kochs (role of the vole Mx proteins) and C. Drosten (overall context of zoonotic rodent borne pathogens).