一种新出现的人-猪杂合型正呼肠孤病毒感染性克隆及σ1s功能研究

Reoviruses are members of the Reoviridae family, which includes several genera that cause disease in humans and animals. Mammalian orthoreoviruses (MRVs) are highly tractable experimental models for studies of double-stranded (ds) RNA virus replication and pathogenesis.It is well known that reovirus genomes are prone to various types of genome alterations including intragenic rearrangement and reassortment under laboratory and natural conditions.Recently, mammalian reovirus (MRV) genome reassortment leads to enhanced virulence, even the events that cause human encephalitis have been reported, which will undoubtedly aggravate the public health significance of reovirus.At the same time, due to the complexity of MRV genome hinder the establishment of reverse genetics system, and this critical technological gap is perhaps the single most important limitation to studies of these viruses. Until now, a strategy to rescue infectious virus from cloned cDNA has not been available for any member of mammalian orthoreoviruses in China. Recently，we report the discovery and characterization of a novel mink reovirus MRV-HB-A strain, which seems to be closely related to human isolates and porcine strains indicated a high probability that MRV-HB-A results from a reassortment of human and swine strains. σ1s is a nonstructural protein and is a key determinant of the capacity of reoviruses to induce a G2/M cell cycle arrest in infected cells, suggesting that it plays an important role during viral pathogenesis. To determine the function of σ1s in the pathogenesis of reovirus, on the one hand, we are planning to use plasmid-based reverse genetics to engineer a recombinant reovirus deficient in σ1s expression and compared the growth,replication and dissemination of wild-type and σ1s-null viruses in vitro and in vivo. On the other hand, the yeast two-hybrid system will be used to identify interactions of σ1s with host proteins,σ1s with the binding properties of the host protein will be analyze by co-immunoprecipitation combined with confocal laser technology. We will also investigate whether the host protein influences reovirus replication and pathogenicity by the over-expression and knockdown technology.

近年来,哺乳动物呼肠孤病毒（MRV）基因组重配后毒力增强,甚至引起人脑炎的事件时有报道,这无疑提升了呼肠孤病毒的公共卫生意义。同时,由于MRV基因组组成的复杂性妨碍了其反向遗传系统的建立，也明显制约了该科病毒的研究进程。迄今，国内尚无MRV感染性克隆及成功拯救病毒的报道。本研究前期分离到一株水貂呼肠孤病毒（HB-A），分析发现，该毒株可能是人源病毒与猪源病毒重配后产生的杂合新病毒。为了深入研究其感染机制和相关基因功能，一方面利用反向遗传学技术，构建并拯救σ1s突变型重组病毒，比较野生型与σ1s突变型病毒在感染、复制、移行和分布上的差异，初步明确σ1s对水貂呼肠孤病毒感染力的影响；另一方面利用双杂交系统筛选与σ1s互作的宿主蛋白，再通过免疫共沉淀结合激光共聚焦技术，明确σ1s与宿主蛋白的结合特性，进而采用过表达和Knockdown技术揭示σ1s调控MRV感染、复制以及凋亡的分子机制。

2014 年山东某貂厂发生以腹泻症状为主的疾病，取肠道内容物接种于BHK-21和Vero 细胞，经盲传，细胞出现了变圆、脱落等病变。经电镜观察，观察到无囊膜、二十面体对称、双层衣壳和直径约 75 nm 的病毒粒子。RT-PCR和BLAST 结果表明，病毒粒子为哺乳动物正呼肠孤病毒，命名为 MRV3 SD-14 株。测定了 SD-14 株的病毒滴度、血凝性以及致病性，SD-14 株在 BHK-21 和 Vero 细胞上的TCID50为105.625TCID50/mL 和 105TCID50/mL；SD-14 株在4℃、室温以及 37℃条件下，对人O型血红细胞以及鸡、豚鼠、牛和兔的红细胞均不发生凝集反应；将SD-14株经口和脑内接种乳鼠，对乳鼠不致病，对其生长没有明显影响。设计特异引物，经RT-PCR 获得了SD-14株全基因组序列，序列分析结果表明，SD-14株为血清3型呼肠孤病毒，其序列与人源 MRV2Tou05、猪源的GD-1株、貂源的HB-A株具有高度的序列相似性。综合序列的相似性以及根据每个片段所构建的进化树，推测SD-14 株为人源、猪源以及貂源呼肠孤病毒混合感染产生的新的重组病毒。SD-14株是首次从水貂中分离获得的血清 3 型呼肠孤病毒。此外，序列分析显示，SD-14 株的 S1片段第246个密码子为终止密码子，可能造成σ1蛋白翻译提前终止，从而对病毒的生物学特性产生影响。因此在构建HB-A全基因组cDNA感染性克隆时，又构建了σ1s 蛋白缺失突变型呼肠孤病毒cDNA体系，进行一系列条件优化后， rHB-A拯救成功，但是病毒滴度较低，仅能通过间接免疫荧光检测到少量病毒蛋白的表达。 . 基于呼肠孤病毒保守L1基因序列，建立了探针法检测该病原的荧光定量RT-PCR方法，该方法灵敏性高。本研究采用病毒中和试验调查了山东、河北以及吉林水貂群中呼肠孤病毒的血清抗体水平，以期从抗体水平了解呼肠孤病毒感染的流行状况。结果表明该病毒在上述地区水貂群中具有较高的感染率。为该病的防控提供可靠的数据。 . 同时基于病毒感染细胞后引起的蛋白质组学进行研究，通路分析及western blot 初步证明IFITM参与病毒感染与复制过程中，对病毒发挥抑制效果，具体机制待进一步探索。

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