鸭坦布苏病毒毒力致弱的分子基础

The newly emerged infectious disease caused by duck Tembusu virus (DTMUV) had resulted in a huge economic loss to the duck industry in China since 2010. As a member of the genus Flavivirus in the family Flaviviridae, DTMUV may be a potential threat to human health. To date, the pathogenesis of DTMUV is largely unknown and no vaccine against DTMUV infection is available. An attenuated virus (FX2010-180P) was developed by serial passage of FX2010 strain, a pathogenic DTMUV isolate, in chicken embryo fibroblasts (CEFs). FX2010-180P was attenuated significantly in ducks, and the replication in mice was also restricted obviously. Comparing with FX2010, there were 19 amino acid changes in the FX2010-180P . Which mutation play a key role in attenuation? Which mutation determines the neurovirulence or neuroinvasiveness of DTMUV? And which mutation is involved in cell tropism or attachment/entry? To answer those questions, a series of the recombinant chimeric viruses based on FX2010 and FX2010-180P will be rescued through a reverse genetics approach . The virulence of these rescued virus will be evaluatd in ducks and suckling mice. The results will help us to understand the molecular basis of DTMUV virulence and provide theoretical basis for DTMUV pathogenesis.

2010年我国新发的鸭坦布苏病毒病给养鸭业造成了巨大的经济损失。作为黄病毒属成员，该病毒也可能对人类健康存在潜在威胁。目前，没有疫苗用于该病的预防，鸭坦布苏病毒的致病机制等重大科学问题也不清楚。我们把鸭坦布苏病毒强毒株（FX2010）在CEF上连续传代培养180代获得了一株弱毒株（FX2010-180P）。该弱毒株对鸭的致病力显著减弱，在鼠体内的复制能力明显降低。与FX2010相比，FX2010-180P有19个氨基酸突变。那么，哪些突变对病毒毒力致弱起到了关键作用？哪些与病毒（嗜）神经毒力相关？哪些与病毒吸附或进入宿主呼吸道相关？这些是本项目要解答的科学问题。本项目利用强毒株和弱毒株的反向遗传操作系统，把强、弱毒的基因相互替换或相互点突变，构建新的重组病毒，通过病毒对鸭和乳鼠的致病力评价，全面解析鸭坦布苏病毒毒力致弱的分子基础，为进一步探讨鸭坦布苏病毒的致病机制奠定基础。

本研究在成功建立的鸭坦布苏病毒强毒株FX2010和致弱毒株FX2010-180P反向遗传操作系统基础上，成功拯救了一系列鸭坦布苏病毒嵌合病毒和点突变病毒，动物实验证明了鸭坦布苏病毒FX2010 E蛋白结构域的120为氨基酸由D(天冬氨酸)变为N(天冬酰胺)时，仅能在脾脏中低水平复制，丧失了在鸭之间传播的能力，表现出与鸭坦布苏病毒弱毒疫苗株FX-180P相似的生物学特性。表明E蛋白结构域Ⅱ中的120位氨基酸由D变为N是导致坦布苏病毒毒力减弱的关键氨基酸，突变该位点能够显著降低病毒在鸭子体内的复制能力和在鸭群间的水平传播能力。通过对鸭坦布苏病毒FX2010株E蛋白的序列比对和糖基化位点的预测和分析，鸭坦布苏病毒FX2010与GenBank中发布的鸭坦布苏病毒序列的E120位点全部是D，说明鸭坦布苏病毒在自然界中倾向于表现是D，而鸭坦布苏病毒FX2010在E154位置存在1个糖基化位点，而将E120位点（D突变为N）的突变增加了潜在的N糖基化位点，该增加的糖基化位点可能与其致病性有关。本研究首次从分子水平上深入阐明影响鸭坦布苏病毒毒力的分子基础，证明E120位点（D突变为N）是鸭坦布苏病毒毒力减弱的关键位点，为进一步深入研究鸭坦布苏病毒和其他黄病毒属病毒的致病机制奠定了基础。