Efficient infection of primary tupaia hepatocytes with purified human and woolly monkey hepatitis B virus

Abstract

The Asian tree shrew, Tupaia belangeri, has been proposed as a novel animal model for studying hepatitis B virus (HBV) infection. Here, we describe a protocol for efficient and reproducible infection of primary tupaia hepatocytes with HBV. We report that human serum interferes with HBV binding to the hepatocytes, thus limiting the maximum multiplicity of infection. Purification of HBV virions by gradient sedimentation greatly enhances virus binding and infectivity. Covalently closed circular DNA was clearly detectable by Southern blot analysis and newly synthesized single-stranded HBV DNA was visible 2 weeks postinoculation. Primary tupaia hepatocytes are also susceptible to infection with the recently discovered woolly monkey hepatitis B virus (WMHBV) but not to woodchuck hepatitis virus infection. Compared to HBV, WMHBV replicated at a higher rate with single-stranded DNA detectable within the first week postinoculation. Primary tupaia hepatocytes should represent a useful system for studying early steps of HBV and WMHBV infection.

FIG. 1

HBV binding to PTH. Cells were incubated for 6 h with increasing volumes of HBV DNA-positive serum (lanes 2 to 5) or a mixture of HBV-positive serum (10 μl) and HBV-negative human serum (200 μl) (lane 6). HBV binding was assessed by Southern blot analysis. Numbers on top of the figure indicate the volume of the respective serum samples. M, 3.2-kbp linear HBV DNA; n.i., noninfected control; RC, relaxed circular viral DNA; p.i., postincubation.

FIG. 2

Effect of virus purification on viral attachment to PTH. (A) Purification of HBV particles by Nycodenz gradient centrifugation. (Top) Coomassie blue staining of fractionated serum proteins. (Middle) Detection of HBsAg by Western blot analysis. (Bottom) Dot blot analysis of viral DNA. Fractions 6, 7, and 8 contain purified virions. (B) Binding assay with purified (right) versus nonpurified (left) viral particles. Viral DNA was detected by Southern blot analysis. For nomenclature, see the legend to Fig. 1.

FIG. 3

Infection studies with purified HBV. (A) Infectivity of purified and nonpurified virus. Viral DNA was detected by Southern blot analysis on day 6 postinfection. Note that cccDNA is clearly detectable only in PTH inoculated with purified virus. (B) Inoculation of primary rat hepatocytes (lane 1) with gradient-purified HBV compared to PTH (lane 2). Note that cccDNA is formed in PTH but not in primary rat hepatocytes. The circular nature of the fast-migrating HBV DNA species was confirmed by digesting the DNA with NcoI, which cuts the HBV genome once (lane 3). (C) Time course of HBV infection in PTH. Cells were incubated with purified HBV virions and harvested on days 1, 7, and 16 postinfection as indicated at the top of the figure. Viral replicative intermediates were analyzed by Southern blot analysis. Note that cccDNA was detectable on day 7, and ssDNA was detectable on day 16 post infection. Se, purified HBV DNA from serum; den., denatured serum DNA; Pl, 3.2-kbp circular plasmid DNA; M, 3.2-kbp linear HBV DNA; RC, relaxed circular viral DNA; DL, double-stranded linear DNA.

FIG. 4

Infection of PTH with WMHBV and WHV. (A) WMHBV-positive serum was processed by gradient centrifugation as described for HBV. Cells were harvested on days 1, 7, and 13 postinfection. Viral DNA was analyzed by Southern blotting. Note that cccDNA and ssDNA are generated in WMHBV-infected cells. (B) Control infection with WHV. PTH were incubated with gradient-purified WHV and harvested on day 6 postinfection. Note that WHV relaxed circular DNA but no cccDNA is visible on the Southern blot. For abbreviations, see the legend to Fig. 3.

FIG. 5

Viral replication and transcription in HBV- and WMHBV-infected PTH. (A) Southern blot analysis of infected PTH on day 6 postinfection (top); dot blot analysis of HBV and WMHBV serum (2 μl each; bottom). Note that only WMHBV produced ssDNA at this time point. For abbreviations, see the legend to Fig. 3. (B) Northern blot analysis of infected PTH. Lysates from cells infected with fractions 6 to 8 were pooled. pgRNA, pregenomic viral RNA. sgRNA, subgenomic viral RNA coding for surface proteins.