鸭坦布苏病毒准种遗传多样性及宿主适应的分子机制研究

Since April 2010, a severe outbreak of duck viral infection, with egg drop, feed uptake decline and ovary-oviduct disease,has spread around the major duck-producing regions in China. A new flavivirus closely related to Tembusu virus was isolated in different areas,and a similar disease was reproduced in healthy egg-producing ducks infected with the isolated virus.As a newly emerging virus, it caused a great loss for Chinese duck industry.Tembusu and related viruses have been isolated from mosquitos in Mylaysia and Thailand. Neutralizing antibodies were detected in chickens,Bornean orandutans and humans. The public health threats of this virus should be assessed for the zoonotic potential of flaviviruses. In this study, the inter-strain genomic diversity and the quasispecies composition of the isolates collected from endemic areas will be deeply sequenced using the next-generation sequencing. The relationship between quasispecies diversity and host adaptability will be investigated by sequential in vivo infection experiments with ducklings.The genotypes with selection advantage will be identified and purified. To get insights into the host switching of the Tembusu virus, the genetic evolution of the virus and host adptation will be measured via in vivo passage. Cloned virus will be sequentially and alternatively passed 20 times in ducklings and mosquitos. Resulting viruses will be genetically characterized. The nucleotide mutation rates of virus in disparate hosts will be monitored. Combined the experimental results and bioinformatic analysis, the key mutated genes will be selected and its role for the host adaptation will be confirmed with reverse genetic manipulation on the viral genome. These results will further reveal the virological and host factrors contributing to the maintainance and evolution of duck Tembusu virus in nature.

鸭坦布苏病毒感染自2010年在我国发生以来已蔓延至我国各主要养鸭地区，给养殖业造成巨大的经济损失。病毒主要侵害鸭生殖和中枢神经系统，造成严重的产蛋下降和脑炎，亦可造成鹅、鸡和麻雀感染。坦布苏病毒早期主要存在于马来西亚和泰国的蚊虫中，偶尔有猩猩和人检测抗体阳性，此次我国发生大规模家禽感染表明该病毒具有极强的宿主适应性和致病力。作为一种新发传染病，了解影响其发生和发展的病原学和环境因素，对开展疾病的风险预测、预防和控制具有重要科学意义。本项目拟对我国鸭坦布苏病毒代表性分离毒基因组进行深度测序分析，了解病毒准种的遗传多样性和鸭群感染的主要基因型；通过动物继代感染试验确定宿主选择压力对病毒遗传变异的影响，并进一步采用反向遗传定向突变和动物感染试验验证病毒关键基因突变与宿主适应的相关性，从病原学角度揭示坦布苏病毒分子演化和病毒跨宿主感染的分子机制，为深入开展疾病防控研究及公共卫生风险评估奠定基础。

鸭坦布苏病毒感染子2010年首先在我国爆发以来，已蔓延至全国主要水禽养殖地区并严重威胁着水禽产业发展。本项目通过对不同l毒株基因分析和血清学调查表明坦布苏病毒感染持续存在于商品水禽中，但我国家禽致病性坦布苏病毒新分离株尚未发生显著的遗传学变异。利用高通量测序技术对坦布苏病毒原始毒株以及高代次细胞适应毒株的全基因组进行了深度测序并对其单核苷酸变异多态性进行分析结果表明， 随着病毒在细胞培养中的传代，高代次适应毒株的SNP位点数目显著增加，变异频率也显著升高。准种多样性指数统计学分析结果显示，高代次毒株的香农熵（Sn）与辛普森指数（Sp）均显著高于原始毒株，表明鸭坦布苏病毒在细胞上传代适应过程中，其准种多样性不断增加。. 以JXSP 株为对象，通过连续传代使其能稳定适应BHK-21细胞，制备了矿物油乳剂灭活疫苗，动物免疫试验结果表表明雏鸭首次免疫后对强毒感染的保护率可达87%，组织脏器载毒量显著低于未免疫鸭；二次免疫则能完全抵抗病毒感染；产蛋鸭经二次免疫后可有效抵抗强毒的攻击，二免后抗体能持续4个月以上。另一方面，随着病毒传代代次增加，病毒对试验鸭的致病性明显下降。动物感染试验结果表明，高代次毒株JXSP2-310已充分致弱，仅在感染雏鸭的脾脏及胸腺组织中检测到少量病毒，易感雏鸭体内连续传代无毒力返强现象。以105 PFU/只的剂量免疫蛋鸭10天后攻击强毒，保护率可达100%，可以作为弱毒疫苗的候选株。基因组序列分析显示，与强毒株JXSP2-4相比，高代次弱毒株JXSP2-310全基因组共产生了41个核苷酸位点的变异，导致19个氨基酸突变，其中E蛋白的氨基酸变异率最高。为进一步揭示病毒基因突变与毒力变化的相关性，本研究采用反向遗传操作技术构建的JXSP2-4感染性cDNA克隆，将弱毒株JXSP2-310蛋白编码基因置换到JXSP2-4相应区域，拯救获得嵌合毒株。细胞感染试验表明，嵌合毒株在BHK-21细胞上的繁殖能力显著高于亲本拯救毒株。动物感染试验表明，感染雏鸭血液、脑及心脏组织中病毒含量显著低于亲本拯救毒株组。结果显示，鸭坦布苏病毒E和NS5蛋白基因的氨基酸变异是影响病毒繁殖能力和病毒毒力的关键因素。本项目的研究结果为进一步开展病毒感染致病机制和免疫防治奠定了坚实的基础。