E种肠道病毒HY12小鼠感染模型与致病机理及病毒准种异质性

Bovine enterovirus is the etiological agent related to a newly identified epidemic disease of cattle with a high morbidity and mortality in many regions in our country, and caused a huge economical loss to cattle industry. Previously, we reported the isolation and characterization of the first enterovirus E strain (HY12) in our country from a unknown disease in a cattle farm with a severe respiratory and enteric disease, and high morbidity and mortality. Preliminary studies in our laboratory showed that HY12 is highly pathogenic, causing cytopathic effects in cell culture as early as 6 h PI with an unusual high titration of TCID50 about 10^-13/ml ; Phylogenetic analysis showed that HY12 isolate belongs to neither serotype/genotype 1 nor serotype/genotype 2; it belongs to a new serotype/genotype, named serotype/genotype 3 within enterovirus E. HY12 was also revealed to contain unique mutations in its encoding capsid proteins in relation to the known enteroviruses and exhibit a relatively regular, higher frequency of amino acids mutation in VP1 capsid protein during the passages. Understanding the mechanism underlying the unique biological characters of HY12 and its pathogenesis and eliciting immunity will provides the fundamental for preventing and controlling this newly emerging viral infection. In this project, we will employ the HY12 to establish a viral infection mouse model and then used this animal model system to study following aspects: 1) the host and tissue tropisms for HY12 by using immunohistochemistry and molecular biology approaches to determine the HY12 virus antigen distribution, thus the tropism for different tissue. 2): Use virology, molecular biology and immunology approaches to determine virus infection route, infection dose (infectivity), and virus shedding pattern for HY12. 3) employ histology, pathology, immunology and IHC approaches to determine the pathological lesions, microscopic lesions and antibody change pattern in the virus infected mouse model, thus elucidating the mechanism of HY12 viral pathogenesis and its eliciting immunity. 4) use virology, molecular biology bioinformatics approaches to determine the biological properties, diversity, and complexity of HY12 quasispecies, quasispecies evolution and the underlying mechanism with varied pressures (immune pressure, etc.). Results from this project will contributes significantly to the understanding HY12 virus infection and elucidating the mechanism of viral pathogenesis and the immunity, thus providing the fundamentals for the prevention and control of this newly emerging epidemic disease.

牛肠道病毒感染是国内新发的一种严重危害养牛业的疫病，其病原体为小RNA病毒科肠道病毒属成员。HY12是从牛群暴发的临床上以咳嗽、呼吸困难和严重腹泻、发病率和致死率高达50%以上的新发疫病分离的病原体，也是国内分离的首株E种肠道病毒。HY12的TCID50异常高，引起细胞病变时间短，病毒编码VP1序列在传代过程出现规律的突变。有关病毒的致病机理等问题不清楚，缺乏研究。本研究以HY12为研究对象，通过建立小鼠感染模型，确定HY12病毒感染途径、剂量、致病性及对组织亲嗜性，进而为揭示病毒的致病机理及免疫机理提供动物模型。应用病毒学、分子生物学和信息生物学等手段，确定HY12病毒准种的生物学特性、复杂性和多样性及准种进化与分子机制。该研究结果不仅揭示出E种肠道病毒的致病机理，而且对于揭示与阐明病毒(准种)遗传变异、进化规律及其机制，病毒对宿主亲嗜性及致病性差异和对病毒性疫病的防控等发挥重要作用。

肠道病毒HY12毒株是本团队从国内牛群暴发的临床表现为严重腹泻、呼吸困难、发病率和致死率高达50%以上的新发疫病中分离的病原体，也是国内分离获得的首株E种肠道病毒。有关该病毒的毒力、致病机理及遗传进化等众多问题不清楚，缺乏研究。本研究以分离的国内首株E种肠道病毒HY12毒株为研究对象，建立了HY12病毒感染的小鼠模型。通过不同途径与不同剂量感染不同品系的小鼠，确定出ICR乳鼠为HY12病毒最易感小鼠品系。HY12病毒通过滴鼻、消化道灌服、腹腔注射、皮下注射和肌肉注射途径接种乳鼠，发现不同途径接种病毒均可导致小鼠感染。HY12病毒感染乳鼠的最低感染剂量为2×106 TCID50。感染后，病毒在小鼠体内带毒时间至少为18天。病理组织学观察发现，感染小鼠的小肠、肺脏、肝脏、脑、肾脏均出现程度不同的细胞病变及炎性细胞浸润。免疫组织化学检测结果揭示， HY12病毒具有广泛的组织亲嗜性，但主要分布在小肠、肺脏和脑组织。建立的小鼠模型为HY12病毒的致病机制及病毒引发的宿主免疫应答等方面研究打下了基础。为探究HY12毒株的遗传变异特点，本研究将HY12病毒在抗体存在或无抗体存在条件下，连续传代，通过扩增不同代次HY12毒株的基因序列，确定病毒的遗传变异规律。序列测定与分析发现，VP1和VP2基因分别存在着不同程度的准种多样性变化，且抗体的选择压力显著降低HY12病毒准种的多样性，同时导致某些准种演变为新的优势准种。在无抗体压力存在下，不同代次的HY12病毒其VP1基因序列与HY12病毒的主序列不完全相同，为不同的克隆类型，从而确定了HY12 准种存在明显的多样性和复杂性。更重要的发现是，与第1 代的HY12 病毒相比，第40 代次后HY12病毒VP1 基因272 位核苷酸发生非同义的点突变（272 G>A），导致其91 位氨基酸由精氨酸突变为组氨酸（R91H）。这些规律性的非同义突变是否影响病毒的生物学特性、病毒的毒力与致病性等，将是急需阐明的科学问题。深入挖掘HY12病毒准种及其突变、进化与机理，将为阐明病毒遗传变异及致病机理提供了理论依据。