A study was carried out to determine some of the factors that might distinguish transient from chronic hepadnavirus infection. First, to better characterize chronic infection, Pekin ducks, congenitally infected with the duck hepatitis B virus (DHBV), were used to assess age-dependent variations in viremia, percentage of DHBV-infected hepatocytes, and average levels of DNA replication intermediates in the cytoplasm and of covalently closed circular DNA in the nuclei of infected hepatocytes. Levels of viremia and viral DNA were found to peak at about the time of hatching but persisted at relatively constant levels in chronically infected birds up to 2 years of age. The percentage of infected hepatocytes was also constant, with DHBV replication in virtually 100% of hepatocytes in all birds. Next, we found that adolescent ducks inoculated intravenously with a large dose of DHBV also developed massive infection of hepatocytes with an early but low-level viremia, followed by rapid development of a neutralizing antibody response. No obvious quantitative or qualitative differences between transiently and chronically infected liver tissue were detected in the intracellular markers of viral replication examined. However, in the adolescent duck experiment, DHBV infection was rapidly cleared from the liver even when up to 80% of hepatocytes were initially infected. In all of these ducks, clearance of infection was accompanied by only a mild hepatitis, with no evidence that massive cell death contributed to the clearance. This finding suggested that mechanisms in addition to immune-mediated destruction of hepatocytes might make major contributions to clearance of infections, including physiological turnover of hepatocytes in the presence of a neutralizing antibody response and/or spontaneous loss of the capacity of hepatocytes to support virus replication.

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SUMMARY.We describe a simple, sensrnve affinity technique for the routine measurement of glycosylated plasma proteins in clinical laboratories. The commercially available phenylboronic acid gel used for the chromatography has recently been marketed as a kit for this purpose (Glycogel Test Kit. Pierce Chemical Co). The manufacturers of this kit recommend loading 200 fl.l neat plasma to each I rnl gel column. This high loading is to enable the direct measurement of protein in the bound and unbound fractions at 280 nm. This loading is consistent with 10-15 mg protein being added per ml gel. Our results show that protein levels greater than 2 mg per ml gel overload the column. Therefore we used a modification of the more sensitive Bradford procedure to measure protein. The method discriminates between normals (6'29 ± 1'87%) and diabetic patients (12·62 ± 3'36~~) and has good precision (CV 4-6 %). The results obtained correlate with the colorimetric method using thiobarbituric acid (r C~O' 70) and with glycosylated haemoglobin (r = O' 82).The measurement of glycosylated haemoglobin is gaining increasing use in the management of patients with diabetes." Other proteins are also glycosylated non-enzymically, and it has been shown that both glycosylated albumin and glycosylated plasma proteins respond more quickly than glycosylated haemoglobin to improvement in glycaernic control.f 3The original method for measuring glycosylated albumin.! which involves isolation of the pure protein followed by estimation of the glycosylated fraction, is laborious. Glycosylated plasma proteins can be estimated directly by a colorimetric method" or after protein precipitation." For accurate results, these methods also require the estimation of protein, the removal of glucose," and correction for background colour" on each sample. A specific method for quantitation of the lysine-bound glucose in serum albumin and other glycosylated proteins is also available." This requires isolation of pure protein, followed by hydrolysis and then analysis by HPLC.None of the available methods for measurement of glycosylated plasma proteins is suitable for routine analysis in the clinical laboratory. There have been several recent reports on the use of simple Reprint requests and correspondence to Dr BJ Gould. 16affinity column methods for the measurement of glycosylated haemoglobins. \11-14 A similar chromatographic procedure should senarate glycosylated plasma proteins from their non-glyccsylated counterparts. The get used is a III-aminophenylboronate agarose, which binds selectively to cis-diols.