Establishment Of A Rat Model Of Liver Fibrosis Induced By RAAV8-1.3HBV

Objective:Chronic hepatitis B (CHB) is a widespread infectious disease, As of now, about 400 million people worldwide are chronically infected with hepatitis B virus (HBV).10%-30% of those patients are at increased risk of developing a progressive liver fibrosis, cirrhosis and HCC, especially in the Asia-Pacific region. Annually nearly 1 million people die from HBV. It has become clear that continuous HBV replication can revert liver fibrosis or even cirrhosis. In natural condition, the host range of HBV is limited to humans and primates, which are not commonly used in scientific research. The lack of convenient animal model that closely mimics HBV infection in patients has greatly hampered the progress of HBV research. HBV-transgenic mice are the most widely used model. That mice have the viral genome integrated into the chromosome and express HBV DNA export viral particles into the serum, but the integrated HBV genome persists in every mouse cell and they are immunologically tolerant to viral antigens. At present, the two most common methods used for experimental liver fibrosis are the administration of carbon tetrachloride (CC14) and the bile duct ligation (BDL). Unfortunately, those models can hardly mimic specific human HBV conditions that result in liver fibrosis.In clinical practice, it. Since it exists many objective limitations to research HBV related liver fibrosis especially the mild one as a result of requirements of liver biopsy, a applicable animal models of HBV related liver fibrosis may fill this void.In the present study,we describe a rat model by injecting a hepatotropic recombinant adeno associated virus carries the HBV genome(rAAV8-1.3HBV). The rats were bled to monitor hepatitis B antigens and HBV-DNA copy number, and meanwhile liver biopsy was performed to examine the stage of liver fibrosis. To our knowledge,this is the first rat model with humanized, persistent HBV replication that allows the evaluation of immune response, inflammation and liver fibrosis.Methods:For establishment of the rat model, Neonatal (PO) Sprague-Dawley rats (n=13,6 male,7 female) received a single intraperitoneal injection of 1×1011 vg of a recombinant virus carries 1.3 copies of the HBV genome and is packaged in AAV8 capsid(rAAV8-1.3HBV).5 neonatal rats were injected an equivalent volume of phosphate buffered saline (PBS) as negative control. To identify the value of the model, all rats were bled retro-orbitally to monitor hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) by ELISA and DNA samples were assayed with real-time quantitative PCR. Serum alanine aminotransferase (ALT) were measured also. twelve weeks after injection, all rats were sacrificed and samples of liver tissue were collected and stained with HE and Masson’s to degree the inflammation activity and liver fibrosis. Immunohistochemical (IHC) staining was carried out as described previously using commercially available antibodies against HBcAg to assess HBV replication, TGF-β1 and a-SMA to assess the stage of fibrosis.Results:All rats were HBsAg and HBeAg positive at 2 weeks post-injection. In male rats, HBsAg and HBeAg levels increased to a mean concentration of 3820±828 IU/mL and 133±46 NCU/mL, respectively. Male hosts displayed HBV antigens activity higher than that observed in female hosts. Then, HBsAg and HBeAg levels declined gradually thereafter.12 weeks after injection, HBsAg in all rats remained positive while HBeAg changed from positive to negative in most of rats(n=11,84.6%). The HBV DNA levels, ranging from 2.95×106 to 8.27×104 copy/mL (mean titers were 8.27×106 and 5.71×105 copy/mL from male and female, respectively) at 2weeks, slowly declined since then. The mean titers became 8.59×103 and 8.56×103 from male and female at 12 weeks post-injection. Paraffin sections of liver tissue were stained with HE/Masson. Results showed significant swelling of liver cell, focal necrosis and eosinophilic bodies associated with an inflammatory reaction. The inflammed portal field is somewhat expanded with fibrosis but has intact limiting plate. HBcAg was positive in all HBV rats. We also observed a high IHC expression of TGF-β1 which may predict a early stages of liver fibrosis.Conclusions:The HBV-related liver fibrosis rat model induced by rAAV-1.3HBV injection is the first rat model with humanized HBV replication that allows the evaluation of the immune response, inflammation and liver fibrosis.