鸭1型甲肝病毒致病型变异的分子机理研究

Duck hepatitis A virus type 1 (DHAV-1) is one of the most important infectious agents which pose a heavy threat to duck industry. Many clinical cases caused by DHAV-1 were characterized with yellowish or Hemorrhagic points in pancreas in duckling since 2011. This newly emerged clinical appearance in duckling caused by DHAV-1, termed as pancreotropic DHAV-1, is a novel pathotype which distinct from the hepatitis appearance caused by classical DHAV-1 (termed as hepatotropic DHAV-1). To investigate the discrepancy and molecular basis of the two pathotype virus, we selected two DHAV-1 strains with different pathotype to conduct a series of experiments on animals and monolayer cell. After the inoculation of the pancreotropic DHAV-1 and hepatotropic DHAV-1, we compared the proliferation properties, tissue and primary cell damage ability and mRNA expression of different innate immune genes in monolayer cells and ducklings. By using reverse genetics technology, we rescued a batch of recombinant viruses with different gene region of the two viruses exchanged between each other. We detected the proliferation properties, tissue and primary cell damage ability and the innate immunity response in duckling,and then mapped the tissue tropism dominants in the genome of DHAV-1. Based on the study of viral gene exchange,tissue tropism and interaction with innate immune system of host, we will further disclosed the discrepancies between the two different pathotype viruses, and elucidate molecular basis of pathogenic phenotypic alteration. These outcomes will lay the foundation for development of gene-deletion vaccine and genetic engineering attenuated vaccine specific for pancreotropic DHAV-1. The research about the pathotype variation of DHAV-1 will have great theoretical significance and potential applications.

目前，鸭1型甲肝病毒（DHAV-1）仍为危害我国养鸭业的重要病毒之一，且于2011年出现了不同于典型肝炎（肝炎型）而以胰腺发黄为特征病变的新的致病型（胰腺炎型）。本项目选取两种致病型DHAV-1的代表毒株，比较其在细胞及雏鸭体内的增殖特性、对细胞和组织损伤能力、诱导雏鸭先天性免疫应答等情况；同时利用反向遗传操作技术构建两种致病型DHAV-1基因组不同基因区段互换的重组嵌合病毒，测定这些不同重组嵌合病毒的增殖特性、引起组织损伤能力和所致特征病变、诱导雏鸭先天性免疫相关因子的表达及其组织嗜性相关基因分析。从病毒基因突变、组织亲嗜性及与宿主免疫系统的相互作用等层面探明两种不同致病型DHAV-1的差异，明确DHAV-1致病型变异的分子机理，为基因缺失胰腺炎型DHAV-1弱毒疫苗、基因工程疫苗等研发奠定基础。因此，本项目研究不仅具有重要的学术价值和科学意义，而且还具有潜在的应用前景。

鸭1型甲肝病毒（DHAV-1）仍为危害我国养鸭业的重要病毒之一，近年来在鸭群中又出现了一种由DHAV-1感染引起的，不同于经典的肝炎型DHAV-1的疾病，该病主要以胰腺发黄、出血为特征病变，称为胰腺炎型DHAV-1。本项目选取两种致病型的代表毒株，从组织、细胞、基因三个层面探明两种不同致病型的差异及引起致病型差异的分子基础。结果表明，不同致病型的DHAV-1病毒FZ86株和MPZJ1206株感染肝细胞引起的细胞整体病变、超微结构病变、细胞凋亡等均存在显著差异，经典肝炎型毒株感染后可诱导肝细胞出现显著的细胞病变，细胞超微结构出现大量异质性变化，凋亡小体蓄积明显。雏半番鸭感染胰腺型DHAV-1后病变以胰腺出血、发黄为特征，胰腺上皮细胞大面积坏死，细胞呈颗粒状变性和坏死。不同致病型的DHAV-1病毒FZ86株和MPZJ1206株结构蛋白基因互换后，可影响病毒在宿主体内的增殖活性及致病性。以上研究结果表明肝炎型和胰腺炎型DHAV-1感染雏鸭具有明显的组织嗜性差异，分别导致肝炎型和胰腺型的临床表现，而病毒VP基因的差异是导致两种DHAV-1对雏鸭的感染的组织嗜性及病理损失表现不同的特点的原因之一，该研究成果为今后开展该病毒的基因缺失弱毒疫苗、基因工程疫苗等的研发奠定基础。