7600 series

The biochemical analyzer was provided by the Second Affiliated Hospital of Shandong University of Traditional Chinese Medicine, and the model is Hitachi 7600 Series, the automatic biochemical analyzer (made in Japan). The transmission electron microscope was provided by the electron microscope room of Medical College of Shandong University, model: JME-d-1200EX (made in Japan), resolution: 0.1–10 nm, and acceleration voltage: 60–100 kV. The ultrathin slicer was provided by the electron microscope room of Medical College of Shandong University, and the model is Ultracut E, Cambridge, UK.

Liver biochemical parameters were determined by a biochemistry analyzer (7600 Series; Hitachi, Tokyo, Japan). Platelet was measured by Sysmex XN-2000 (Kobe, Japan). Serum HBsAg, anti-HBs, hepatitis B e antigen (HBeAg), hepatitis B e antibody (anti-HBe), and hepatitis B core antibody (anti-HBc) were detected using an enzyme-linked immunosorbent assay kit (ARCHITECT i2000 SR; Abbott Architect, USA). Serum HBsAg were retested (Roche Cobas e602; Roche, Switzerland) when it exceeded the upper linearity limit (250 IU/mL). HBV DNA was quantified by using a real-time PCR assay (DAAN Diagnostics, Guangzhou, China). Detection limits of HBsAg, anti-HBs, HBeAg, anti-HBe, anti-HBc, and HBV DNA were 0.05 IU/mL, 10 IU/L, 1 S/CO, 1 S/CO, 1 S/CO, 500 IU/mL, respectively. The upper limit of normal (ULN) of ALT has been defined as 40 U/L for women and 50 U/L for men. The ULN of bilirubin has been defined as 21 μmol/L for women and 26 μmol/L for men. The normal range of albumin was 40-55 g/L. In addition, hepatitis B core-related antigen (HBcrAg) was not detected due to the lack of serum samples.

Mouse sera from both C57/BL6 and BALB/c were collected at the 1d, 3d, 7d, 10d, 2w, 3w, 4w, 5w, 6w, 7w, 8w, 9w, 10w… after being injected with different concentrations of pAAV-HBV1.2 plasmid DNA. Alanine aminotransferase (ALT) levels of the BALB/c mice injected with 1 μg, 5 μg, 10 μg, or 100 μg pAAV-HBV1.2 plasmid DNA were measured on full automated biochemistry analyzer (7600 Series; Hitachi, Tokyo, Japan) by using ALT reagent (Denka Seiken, Tokyo, Japan).
The HBsAg, HBeAg, HBcAb and HBsAb were determined using commercial enzyme-linked immunosorbent assay (ELISA) kits (Kehua, Shanghai, China). Ten-fold diluted serum samples were used for detection.

Fasting blood serum samples were used for laboratory tests. Serologies for hepatitis B surface antigen were detected with an automated blood analyzer (Advia-Bayer, Leverkusen, Germany). Routine blood (measured by XT-2000i, Sysmex, Kobe, Japan) and biochemistry variables (measured by 7600 Series, Hitachi, Tokyo, Japan) were assessed, including PLT count, serum ALT, AST, GGT, ALB, total bilirubin (TBIL), alkaline phosphatase (ALP), and globulin concentrations. Using the formulas shown in Table 1, the API (10 (link)), AAR (10 (link),11 (link)), APRI (10 (link),11 (link)), GPRI (11 (link)), S index (12 (link)), FIB-4 (13 (link)), and Fibro-Q (14 (link)) were calculated, all of these being considered noninvasive models for evaluating the degree of liver fibrosis.

Serum anti-HEV immunoglobulin IgM and IgG were tested using enzyme-linked immunosorbent assay (ELISA) (MP Biomedicals, Singapore) according to the manufacturers’ instructions. Liver functions were tested using fully-auto-biochemistry-analysis instruments (7600 Series; Hitachi, Japan). Serum HBV markers were detected using ELISA methods (ARCHITECT i2000 SR; Abbott, Germany). HBV viral load quantification was detected using real-time PCR (ABI 7500; Applied Biosystems Inc, United States). Routine blood tests were performed using automated blood cell analyzers (XT-2000i; Sysmex, Japan).