【摘要】 应用临床阳性样本进行鸭甲型肝炎病毒血清3型（duck hepatitis A virus type 3,DHAV-3）毒株的分离，并利用RT-PCR方法对分离毒株的基因组进行扩增、序列比对分析确定其遗传进化关系。分离毒株分别感染无母源抗体鸭胚，通过测定ELD₅₀比较毒株对鸭胚的致病力；将毒力最强和最弱毒株分别感染5日龄健康雏鸭，记录发病及死亡情况，对雏鸭心脏、肝脏和脾脏组织进行石蜡切片制作，HE染色后观察组织病理变化，分析其对雏鸭的致病性。应用无母源抗体鸭胚自山东菏泽和河北石家庄4个规模化养殖场中成功分离到4株DHAV-3病毒，分别将其命名为HZ-1、HZ-2、HZ-3和HB-1株。序列测定结果显示，分离毒株的全基因组长分别为7 787、7 786、7 785、7 788 bp; G+C含量分别为42.25%、42.15%、42.11%和42.15%。系统进化树分析结果显示，4株DHAV-3病毒与我国山东、安徽、江苏和四川地区毒株的亲缘关系最近，与越南、韩国和黑龙江地区毒株亲缘关系相对较远。鸭胚致病性试验结果显示，HZ-1、HZ-2、HZ-3和HB-1株的半数胚致死量(egg lethal dose, ELD₅₀)分别为10^-7.0/0.2 mL、10^-7.2/0.2 mL、10^-6.3/0.2 mL和10^-7.5/0.2 mL,均属于高致病性毒株。将HB-1株和HZ-1株分别腹腔接种健康雏鸭，观察期内2组雏鸭的病死率均为20%,RT-PCR检测感染率为100%;病理组织学观察显示，肝细胞肿胀、脂肪变性，脾脏出血、坏死，伴有炎症细胞浸润，心脏出血、充血，局部坏死，并伴有炎症细胞浸润。该结果为了解我国DHAV-3田间毒株的分子特征和致病性及开发相关诊断试剂、疫苗和卵黄抗体等生物制品奠定了基础。更多还原

【Abstract】 To understand the molecular characteristics and pathogenicity of duck hepatitis A virus serotype 3（DHAV-3） in China,this study isolated DHAV-3 strains from clinical positive samples.The genomes of the isolated strains were amplified by RT-PCR,and their genetic evolutionary relationships were determined through sequence alignment analysis.The isolated strains were inoculated into duck embryos without maternal antibodies,and their pathogenicily was compared by measuring the ELD₅₀,The most and least virulent strains were then used to infect 5-day-old healthy ducklings to observe morbidity and mortality.Paraffin sections of the heart,liver,and spleen tissues were prepared and stained with hematoxylin and eosin（HE） for histopathological examination to analyze the pathogenicity of the viruses in ducklings.The results showed that four DHAV-3 strains were successfully isolated by duck embryos without maternal antibodies from large-scale farms in Heze,Shandong,and Shijiazhuang,Hebei.These strains were named HZ-1,HZ-2,HZ-3,and HB-1, respectively.Genome sequencing revealed that their full lengths were 7 787,7 786,7 785,and 7 788 bp,with G+C contents of 42.25%,42.15%,42.11 %,and 42.15%,respectively.Phylogenetic analysis indicated that the four DHAV-3 strains were most closely related to isolates from Shandong,Anhui,Jiangsu,and Sichuan,China,while showing relatively distant relationships with strains from Vietnam,South Korea,and Heilongjiang,China.Duck embryo pathogenicity tests demonstrated that the ELD₅₀ values of HZ-1,HZ-2,HZ-3 and HB-1 were 10^-7.0/0.2 mL,10^-7.2/0.2 mL,10^-6.3/0.2 mL and 10^-7.5/0.2 mL,respectively,indicating that all were highly pathogenic strains.After intraperitoneal inoculation of healthy ducklings with HB-1 and HZ-1,both groups exhibited a mortality rate of 20% during the observation period,with a 100% infection rate confirmed by RT-PCR.Histopathological examination revealed hepatocellular swelling and steatosis,splenic hemorrhage and necrosis with inflammatory cell infiltration,as well as cardiac hemorrhage,congestion,focal necrosis,and inflammatory cell infiltration.These findings provide a foundation for understanding the molecular characteristics and pathogenicity of DHAV-3 field strains in China,as well as for the development of related diagnostic reagents,vaccines,yolk antibodies,and other biological products.更多还原