The animals used in this study were apparently healthy Manchurian chipmunks (Tamias sibiricus asiaticus), weighing approximately 80gm, captured from fields in the Kyunggido and Kangwondo provinces of Korea. Sera were obtained from all animals by cardiac puncture and extracted liver tissues were fixed in 10% neutral formalin.

Each serum was tested for HBsAg, anti-HBs, and anti-HBc by radioimmunoassay using commercial kits (Abbott laboratories, North Chicago, III).

To test for the presence of particles similar to HBV in chipmunk sera, sera were pelletted and examined by electron microscope. Each serum sample was centrifuged at 10,000 rpm for 10 min to remove precipitated protein and other debris; 200 μl of each supernatant diluted with 800 μl of buffer was layered over an 11 ml gradient of 10–20% (Wt/Vol) sucrose containing 10mM Tris-HCl (pH 7.4), 0.1M NaCl, and 5mM EDTA.

After centrifugation for 13 hr at 35,000 rpm in a Spinco SW 40.1 rotor at 20°C, the supernatant was thoroughly removed by aspiration, and the pellet was suspended in 100 μl of the buffer. The suspension was spotted on a carbon-coated grid and the material was stained with 1% phosphotungstic acid and examined by electron microscope.

Pooled sera from Manchurian chipmunks containing HBsAg reactivities and virus-like particles and pooled human sera positive for HBsAg and HBeAg were used for an extensive virion DNA polymerase reaction in the presence of [α-³²P] dCTP to radiolabel the viral DNA by the method described by Landers et al⁹⁾. Serum samples from chipmunks and humans were clarified by centrifugation at 10,000 rpm for 10 min; 4ml of each supernatant was layered over 8 ml of 30% sucrose (wt/vol) in TNEMBSA (10mM Tris-HCl, 0.1M NaCl, 0.001M EDTA, 0.1% 2-mercaptoethanol, and Img of bovine serum albumin per ml, pH7.5) and pelleted in a SW 40.1 rotor for 5 hr at 39,000 rpm and 4°C. The pellet was redissolved in 200μl of 0.01M Tris-HCl (pH 7.5), 0.1M NaCl, 0.5% Nonidet P-40, and 0.1% 2-mercaptoethanol.

The 50μl of Tris-phosphate mixture (2.0M Tris-HCl [pH 7.5], 0.08M MgCl₂, 0.24M NH₄Cl, 1mM each dGTP) dTTP and dATP, 2.5μM [α-³²P] dCTP [800 ci/mmol] was added and the reaction mixture was placed at 37°C for 20 min. Unlabeled dCTP was then added to a final concentration of 0.2mM and the reaction mixture was incubated for 3hr at 37°C.

The reaction mixture was digested with proteinase K in 0.01M Tris-HCl (pH 7.5)-0.01M EDTA-0.15% sodium dodecyl sulfate for 2 hr at 56°C. The reaction mixture was extracted three times with phenol equilibrated with TNEME. A one-tenth volume of 2M sodium acetate (pH 5.2) and 2 volumes of 100% ethanol were added to precipitate the DNA. After 2hr at −20°C, the ethanol precipitate was pelleted in a Microfuge for 5 min and resuspended in 10mM NaCl-10mM Tris (pH7.5)-1 mM EDTA.

Agarose gel electrophoresis was conducted in a horizontal apparatus using gels of 1% agarose. Gels were run for 12hr at 30V. The size markers were Hind III fragments of bacteriophage lambda DNA. The electrophoresis buffer consisted of 89mM Tris-89mM boric acid-2.5mM EDTA. The gel was subjected to autoradiography at −70°C for 120 hr using a Dupont Cronex lightning-plus intensifying screen and Kodak XR-5 film.

Liver tissue fixed in 10% neutral formalin was embeded in paraffin, sectioned at 5 μm and stained with hematoxylin and eosin.

Each of fifteen Manchurian chipmunks without serologic markers of HBV was inoculated with 100μl of pooled HBsAg-reactive sera from chipmunks. The animals were sacrificed two months later and serologic tests for HBV markers and histologic exams of liver tissues were performed.

Of 192 tested animals, HBsAg reactivities were found in the sera of 8 animals and anti-HBs in 11 animals by radioimmunoassay. Anti-HBc was not detected in any case (Table 1). No HBsAg-positive serum produced more than 10-fold amount of ¹²⁵I-labeled anti-HBs binding than that of the HBsAg-negative control serum.

All the HBsAg-positive sera contained the particles shown in Fig. 1. The most abundant form in all of the HBsAg-positive sera was a small spherical particle 20–22nm (mean 21nm) in diameter. The HBsAg-positive sera also contained doublelayered spherical particles 39–42nm (mean 41nm) in diameter with an outer shell and an inner spherical core, similar to the Dane particles of HBV. However, filamentous form of the particle was not observed in any of the HBsAg-positive sera. No such particles were found in HBsAg-negative sera including anti-HBs-positive sera.

The DNA polymerase activity in HBV repairs a single-stranded region in the viral DNA, and the reaction catalyzed by this enzyme can be used to radiolabel the viral DNA and make it fully double stranded. Similarly prepared preparations of particles from HBsAg-positive Manchurian chipmunk and human sera were used for an extensive virion DNA polymerase reaction in the presence of [α-³²P] dCTP to radiolabel the DNA and close the single-stranded gap. The DNA was then extracted from each reaction mixture and analyzed by agarose gel electrophoresis and autoradiography.

The results in Fig. 2 show that after an extensive DNA polymerase reaction the radiolabeled DNA from Manchurian chipmunks and human serum particles formed moderately sharp bands with similar electrophoretic mobilities. This form of HBV DNA is fully double-stranded circular molecules⁹⁾. These results indicate that particles in the HBsAg-positive chipmunk sera contain DNA with a similar size to that of HBV DNA and a virion DNA polymerase that can repair the single-stranded region.

Histologic examination of liver tissues from chipmunks containing HBsAg reactivities and virus-like particles in their sera reavealed no evidence of cell necrosis, infiltration of inflammatory cells or fibrosis (Fig. 3). Chipmunks without HBsAg reactivities showed no evidence of liver diseases, either.

No HBsAg-reactivity was detected in sera obtained from 15 Manchurian chipmunks two months after inoculation with HBsAg-positive sera. However, histologic exams of liver tissues revealed pathologic findings of acute hepatitis in 4 animals, chronic persistent hepatitis in 2, and chronic active hepatitis in 5 including 2 cases showing various appearances of dysplastic hepatocytes with hyperchromatic giant nuclei and ground-glassy ballooned cytoplasms (Fig. 4).

This frequent occurence of significant hepatitis in the inoculated animals is in sharp contrast to the silent histologic findings of the other animals examined immediately after capture without inoculation.