新生期树鼩接种人乙型肝炎病毒的长期实验观察

目的 观察新生期树鼩接种HBV后体内HBV感染标志物的长期动态.方法 6只树鼩于新生期接种人HBV DNA阳性血清,每4-6周抽血1次和每6～12周做肝活体组织检查1次,应用巢式聚合酶链反应(nPCR)、荧光定量聚合酶链反应(FQ-PCR)、Southern blot、酶联免疫吸附试验和免疫组织化学染色等方法动态观察血清和肝组织中HBV感染标志物的消长,用电镜寻找肝组织内的HBV颗粒和用光镜观察肝组织病理变化.结果 新生期树鼩接种后48周,3只动物(1、2和6号)血清和肝组织标本经多对引物进行的nPCR,均稳定显示HBV DNA阳性,肝组织HBVcccDNA阳性;FQ-PCR显示血清和肝组织HBV DNA的拷贝数分别为103-104/ml和每微克肝组织总DNA 107～108拷贝;Southern blot检测显示肝组织存在HBV复制中间体HBV cccDNA和HBV单链DNA;酶联免疫吸附试验检测显示血清HBsAg持续阳性;免疫组织化学染色可见数量逐步增多的HBsAg阳性肝细胞.其中的1号动物至接种后2年每微克肝组织总DNA仍可测得107～108拷贝的HBV DNA,电镜下可见疑似HBV颗粒.另3只动物除nPCR显示肝组织HBV DNA阳性条带和FQ-PCR显示低拷贝数(每微克肝组织总DNA103拷贝)HBV DNA外,其余的HBV感染标志物均为阴性或一过性阳性.结论 新生树鼩能够长期感染HBV,并且HBV能够在树鼩体内稳定复制和长期存在.

Long-term observation of hepatitis B virus (HBV) replication in new-born tree shrews inoculated with HBV

Objective To observe the hepatitis B virus (HBV) replication in the tree shrews that were inoculated with HBV at neonatal period.Methods Six new-born tree shrews were inoculated with human HBV positive serum.Blood samples and liver biopsies were colleted at different time points after inoculation.The HBV infection markers were tested by nested polymerase chain reaction (nPCR),fluorescence quantitative polymerase chain reaction (FQ-PCR),Southern blot,ELISA and immunohistoehemistry staining.The liver tissues were observed under electron and light microscope.Results 48 weeks after inoculation,HBV DNA and HBV cccDNA were detected in the serum and liver samples of three animals (number 1,2 and 6) by nPCR.The copy-numbers of HBV DNA detected by FQ-PCR in their serum and liver samples were 103 and 104/ml respectively,and the total DNA in 1 μg liver tissue was 107-108.Southern blot indicated that HBV replication intermediates such as HBV cccDNA and HBV ssDNA was detectable in liver tissues.HBsAg was detected by ELISA,and immunohistochemical staining showed a gradual increase of HBsAg-positive liver cells.High copy number of HBV DNA and suspected HBV EM particles could be detected in the liver samples from one of the three animals that have survived more than 2 years after inoculation.The other three animals showed low HBV DNA copy number,and the rest of the signs of HBV infection were negative or transiently positive.Conclusions Neonatal tree shrews can be infected with human HBV.HBV can replicate inside the liver cells of tree shrew.