Three Distinct Southern Blot Hybridization Patterns of HBV-DNA in the Sera of HBV Carriers.

Abstract

The molecular species of hepatitis B virus (HBV)-DNA of 44 sera taken from 25 HBV carriers were examined by Southern blot hybridization with a biotin-labeled HBV-DNA probe and classified into three patterns. Type I consisted of two distinct bands of 4.0 kb and 3.2 kb. Type II consisted of the 4.0 kb and 3.2 kb bands and a smear between these two bands. Type III showed a broad band below 4.0 kb. With endogenous HBV-DNA polymerase reaction, the smear disappeared and 4.0 kb and 3.2 kb bands appeared. After EcoRI digestion, the 4.0 kb band disappeared to form a single band of 3.2 kb. These results suggest that the 4.0 kb and 3.2 kb forms are a relaxed circular, fully double-stranded DNA and a linear, fully double-stranded DNA, respectively, and that the smear between 4.0 kb and 3.2 kb is formed from the sum of relaxed circular, partially double-stranded DNAs with various length of plus strands. Comparison between histological diagnosis and Southern blot hybridization patterns of 25 HBV carriers indicates that these three patterns are closely related to the degree of hepatitis. Southern blot hybridization; HBV-DNA; HBV carriers Hepatitis B virus (HBV), or Dane particle, has a lipid envelope embedded with surface antigen proteins. Inside the lipid envelope there is a core particle, in which double-stranded HBV-DNA exists. HBV also contains a partially single-stranded circular DNA, which consists of a complete minus strand of approximately 3,200 bases and a plus strand of 1,700-2,800 bases; HBV is, therefore, heterogeneous in terms of the length of the plus strand (Gerber and Thung 1985;Krogsgaard 1988).HBV itself does not have cytopathic effect on hepatocytes, and liver cell

Relationship between Molecular State of Serum HBV-DNA and Clinical Features of Hepatitis B Virus Carriers.

“…The same three hybridization patterns as those reported previously (Yamada et al 1993) were reproducibly identified in this study. Type I consisted of two disinct bands of 4.0 kb and 3.2 kb.…”

“…HBV DNA spot hybridization Spot hybridization of serum HBV-DNA was performed using a cloned HBV-DNA labeled with biotin as described previously (Yamada et al 1993). …”

“…Southern blot analysis of HBV DNA Extraction of HBV-DNA from serum and Southern blot analysis were performed as described previously (Yamada et al 1993). Briefly, the DNAs obtained from sera were electrophoresed through a 1% agarose gel and transferred to nitrocellulose filter (Advantec Toyo Corp., Tokyo).…”

“…The filter was dried for 3 hr at 80°C and then was prehybridized for 4 hr at 42°C in 50% formamide, 5X SSC (1X SSC is 150 mM NaCl,15 mM sodium citrate), 5X Denhardt's solution (1X Denhardt's solution is 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone and 0.02% Ficoll 400), 25 mM sodium phosphate (pH 6.5), and 500 pg/ml sonicated salmon sperm DNA (Pharmacia, Uppsala, Sweden). The prehybridization solution was removed and then the filter was hybridized for 18 hr at 42°C in 45% formamide, 5X SSC, 1X Denhardt's solution, 20 mM sodium phosphate (pH 6.5), 5% dextran sulfate, 200 pg/ml sonicated salmon sperm DNA, and a cloned HBV-DNA labeled with biotin (Yamada et al 1993). The filter was then washed twice in 2X SSC with 0.1% sodium dodecyl sulphate (SDS), twice in 0.2X SSC with 0.1% SDS for 3 min at room temperature, twice in 0.16X SSC with 0.1% SDS for 15 min at 50°C, and once in 2X SSC.…”

“…These molecular states represented the length of the plus strand of HBV-DNA. The patients who had HBV with incomplete, short plus stranded DNA appeared to have chronic liver disease with active inflammation (Yamada et al 1993), although the number of patients examined was insufficient to draw a positive conclusion.…”