Long-Tren Observation On New-Born Tree Shrew Inoculated With Hepatitis B Virus

Objective By applying a series of molecular biological and immunological assay, to observe the hepatitis B virus (HBV) infection markers in serum and liver samples of the tree shrews that were inoculated with HBV at neonatal period, and then to explore and confirm the possibility of tree shrew infected with HBV chronically.Methods Part I. Firstly, for optimizing the procedure of nested polymerase chain reaction (nPCR), serum samples of tree shrew were prepared by boiling or by alkaline lysis. Then nPCR results from the samples treated by different ways were compared on abundance of product, sensitivity of detection and reproducibility of result. Secondly, for optimizing primers for nPCR, four sets of primers were designed from the conservative sections of HBV genome, two sets for HBV S gene and two sets for HBV C gene respectively. Then the amplifying efficiency of the primer sets was compared from each other. Part II. Six tree shrews were inoculated with HBV at neonatal period. Then their serum and liver samples were detected by a series of methods as following: Fluorescence quantitative polymerase chain reaction (FQ-PCR) and nPCR were applied to detect the levels of HBV DNA in serum and liver samples. FQ-PCR and Southern blot were applied to detect HBV cccDNA in liver samples. ELISA and immunohistochemistry were applied to observe HBV infection markers in serum and liver samples. Electron and optical microscopy were used to observe HBV particles and histopathology changes in the liver tissue.Results Part I. The treatment of serum samples with alkaline lysis for the detection of HBV DNA by nPCR was more sensitive than treated by boiling. Two sets of nPCR primer were selected for detecting HBV S and HBV C gene respectively. Part II. The serum samples of animal number 1, 2 and 6 collected from 20th-week to 48th-week after inoculation from showed stably positive bands by nPCR, with the two primers sets for HBV S and HBV C gene respectively, while the remaining three animals showed negative results all the time. By the same nPCR primers mentioned above, however, the liver tissue samples from all of the six animals continuously showed positive results from 12th- to 48th-weeks after inoculation. The results of sequencing on nPCR products showed 96.0% and 98.0% homologous to the corresponding fragment in HBV C and S gene respectively. FQ-PCR showed the copy numbers of HBV DNA in the serum sample from animal 1, 2 and 6 were 103~104/ml from 20th- to 48th-weeks after inoculation, while the remaining three animals were negative all the time. The copy numbers of HBV DNA in the liver sample of animal 1, 2 and 6 were 107~108/μg total liver DNA from 24th- to 48th-weeks after inoculation, while the remaining three animals maintained at the level of 103~104/μg total liver DNA. HBV cccDNA was detected by nPCR in the liver samples of animal 1, 2 and 6, and the sequencing on nPCR products showed 100% homology comparing to the corresponding fragment in HBV genome. HBV cccDNA and HBV ssDNA, two forms of intermediate during HBV replication, were detected in the liver samples of animal 1, 2 and 6 by Southern blot, and these HBV intermediates had an increasing trend among the liver samples collected at 12th-, 24th- and 36th-week after inoculation. ELISA results showed all of the serum samples collected at 12th-, 24th-, 36th- and 48th-week from animal 1, 2 and 6 were HBsAg positive. While only one of the three remaining animals showed serum HBsAg positive at 24th-week and transformed to negative afterwards, and the other two animals were serum HBsAg negative all the time. Immunohistochemistry detected HBsAg in liver tissues of animal 1, 2 and 6, and the number of HBsAg-positive liver cells had an increasing trend with time. The remaining three animals showed no sign of HBsAg in their liver tissues all the time. By electron microscopy, suspect HBV spherical particles with diameter around 40nm were observed in the liver tissues of animal 1, while minor histopathology changes in liver tissues of the infected animals were observed by optical microscopy.Conclusions For nPCR detection, the method of alkaline lysis is the better choice for serum samples preparation. The two sets of nPCR primers that were selected respectively for HBV C and S gene are applicable to detect HBV DNA in serum and liver samples of tree shrew. Neonatal tree shrew can be infected with human HBV, and HBV can survive and replicate inside the liver cells of tree shrew.