Optimization On The Model Of New-Born Tree Shrew Infected With Human HBV

Objective:To further optimize the new-born tree shrew infection of hepatitis B virus (HBV) model on the aspects such as source of infection, route of inoculation, testing method and other support measures.Methods:Part I. Optimizing the sources of infection. The main sources of infections included: Single or mixed sera from hepatitis B patients, serum from confirmed HBV-infected tree shrew, cells culture medium of HepG2.2.15 that secretes HBV particles, plasmid pT-HBV1.3 that contains HBV genome, and purified HBV-infection serum. Part II. Optimizing the route of inoculation. The main routes of inoculation included: Subcutaneous injection, intraperitoneal injection, subcutaneous plus intraperitoneal injection, intravenous plus intraperitoneal injection, percutaneous direct liver injection, and laparotomy indirect injection through spleen to liver. Part III. Optimizing the testing method for detection of HBV infected tree shrew in vivo. The man testing methods included: Applying fluorescence quantitative polymerase chain reaction (FQ-PCR) to detect HBV DNA in serum and liver samples, ELISA and time-resolved fluoroimmunoassay (TRFIA) to detect HBV immunological markers in serum, dot blot to detect HBV DNA in liver samples, electron microscopy to detect HBV particles in serum and liver samples, immunohistochemical staining to detect HBsAg/HBcAg in liver tissue, and optical microscopy to observe histopathology changes in liver tissue. Part IV. Exploring other support measures for increasing infection efficacy. The main measures included: Reproduction of the confirmed HBV-infected female tree shrew, inoculating HBV serum to female tree shrews during their late pregnancy, applying immunosuppressive agent to animals during the period of HBV inoculation.Results:Part I. Among the various sources of infection, single serum from a patient and serum from infected tree shrew showed a better effect: among the six tree shrews that showed persistent infection (carried HBV infection signs for 68-200 weeks), five were inoculated with single HBV patient serum (with or without purification), and one was inoculated with infected tree shrew's serum. Tree shrews inoculated with other sources showed no evidence as chronic HBV carrier. Part II. Among the six tree shrews with persistent infection, four were inoculated by subcutaneous injection, and two were inoculated by subcutaneous plus intraperitoneal injection. Other infection routes showed no clear effect on persistent infection. Part III. FQ-PCR results showed the copy numbers of HBV DNA in the serum sample from the six chronic infected animals were 103-104/ml since 12 or 30 weeks after inoculation, while their copy numbers of HBV DNA in liver samples were 104-106/μg total liver DNA at the 12th-week after inoculation, and increased to 107-108 afterwards. The results from ELISA/TRFIA on serum HBsAg showed, among the six animals with chronic infection, four were HBsAg persistently positive and maintained at high level, and the other two showed intermittent weak positive. Dot blot on the liver samples from four chronically infected animals all showed HBV DNA positive. Electron microscopy on samples from three chronic infected animals, all showed large and small spherical particles and tubular particles in their serum, and two showed suspect HBV Dane spherical particles with diameter around 40nm, located in cytoplasm of lever cells and arranged as grid-like. Immunohistochemistry on liver tissues from three chronic infected animals showed visible HBsAg and HBcAg positive hepatocytes. Light microscope observation on liver tissues of the infected animals showed only lightly pathological changes such as mild liver cell proliferation and edema. Part IV. Four pups born from a persistent infected female tree shrew were all died at babyhood, in which the HBV DNA was not detected in the liver tissues by FQ-PCR. Among the tree shrews born from the female tree shrews inoculated HBV at their late pregnancy, some showed medium level of HBV DNA copy numbers (103～105/μg total liver DNA) in liver sample since 6- or 12-weeks after inoculation, some showed low level HBsAg in serum, but none showed evidently chronic infection by 48 weeks after inoculation. Tree shrew were inoculated neonatally and receive immunosuppressant (CsA) at the same time showed HBV DNA negative or low copy numbers (103~104) in liver tissues, a few of them showed casually serum HBsAg week-positive at the 12th-week after inoculation. Among the tree shrews that were inoculated HBV and received immunosuppressant at their adulthood, only a small part showed low copy numbers (103~104) of HBV DNA in liver, while the most still showed HBsAb in their serum quite soon after inoculation. All the six tree shrew with persistent infection in this study were inoculated within 3 days of age.Conclusions:1. Serum from single HBV patient and infected tree shrew are the better sources for infecting neonatal tree shrew. 2. Subcutaneous injection and subcutaneous plus intraperitoneal injection are the better routes for inoculating neonatal tree shrew. 3. FQ-PCR diagnostic kit for HBV DNA and ELISA/TRFIA diagnostic kit for serum HBV immunological markers are suitable for detecting tree shrew samples, and the results are stable. Dot blot, electron microscopy and immunohistochemical staining are suitable methods for verifying HBV infection of tree shrew in vivo.