M. Ruvoletto

Hepatitis C genotypes in patients with dual hepatitis B and C virus infection

In patients with chronic hepatitis B and C virus (HBV, HCV) infection, an inverse relationship in the replicative activity of the two viruses has been reported. In the present study the genotype of HCV was evaluated in 34 consecutive cases found with hepatitis B surface antigen (HBsAg) and anti‐HCV in the serum, in order to identify its possible influence in determining the pattern of HBV/HCV interaction. Nineteen patients were HCV‐RNA positive and could be genotyped: 8 were infected by HCV‐1 (3 by HCV‐1a and 5 by HCV‐1b), 10 by HCV‐2, and only 1 by HCV‐3. Among these, 3 were HBV‐DNA positive, compared to 10 of 15 HCV‐RNA‐negative patients (P = 0.003), and all 3 were coinfected with HCV‐2.

The preS1 domain of hepatitis B virus and IgA cross-react in their binding to the hepatocyte surface

Using a solid-phase assay we have demonstrated specific competition between the preS1 sequence of hepatitis B virus and human IgA in their binding to isolated normal human liver plasma membranes, suggesting molecular mimicry. Monoclonal and polyclonal antibodies raised against virus and IgA epitopes were used to detect and map immunological cross-reactivity to the virus sequence involved in liver cell binding. These findings suggest the existence of a common receptor or of closely related receptors for the attachment of HBV and IgA to human liver cells.

Hepatitis B virus binds to peripheral blood mononuclear cells via the pre S1 protein

The hepatitis B virus has been documented in hepatic and extrahepatic compartments, including bone marrow and peripheral blood cells. The viral protein involved in the attachment to human hepatocytes has been identified within the N-terminus of the pre S1 envelope protein. Using recombinant particles containing the pre S1, pre S2 and S encoded sequences, we studied which virus envelope protein is involved in binding to peripheral blood cells. Mononuclear cells of 20 healthy subjects bound 125I-labelled particles, with a S/N ratio higher than 2.5 (range 2.69-7.77). Binding was abolished by trypsinization. B lymphocytes and monocytes were found to bind viral particles much more efficiently compared to T cells and granulocytes. A monoclonal antibody (MA 18/7), recognizing the (27-49) pre S1 sequence, completely inhibited viral particle attachment to PBMC, while anti-pre S2 (Q 19/10) and anti-S (20/2) monoclonal antibodies had no effect. We conclude that the pre S1 sequence is involved in HBV attachment to PBMC and to hepatocytes. The nature of the cellular attachment site is unknown, but it might be a receptor for physiologic ligands, as occurs with other viruses.

Squamous cell carcinoma antigen-1 (SERPINB3) polymorphism in chronic liver disease

BACKGROUND The serpin squamous cell carcinoma antigen (SCCA, SERPINB3) has been found over-expressed in primary liver cancer and at lower extent in cirrhosis and chronic hepatitis. A novel SCCA-1 variant (SCCA-PD), presenting a single mutation in the reactive centre (Gly351Ala), has been recently identified (rs3180227). AIM To explore SCCA-1 polymorphism in patients with HCV infection as single etiologic factor and different extent of liver disease. METHODS One hundred and fourty-eight patients with chronic HCV infection (45 chronic hepatitis, 53 cirrhosis, 50 HCC) and 50 controls were evaluated. SCCA-1 polymorphism was studied by restriction fragment length polymorphism and confirmed randomly by direct sequencing. Circulating SCCA-IgM complex was determined by ELISA. RESULTS SCCA-PD was detected with higher frequency in cirrhotic patients (45.3%, odds ratio=2.62; 95%CI 1.13-6.10, p=0.038) than in patients with chronic hepatitis or in controls (24.4% and 24%, respectively). Intermediate figures were found in hepatocarcinoma (36.0%). SCCA-IgM in serum was lower in patients carrying SCCA-PD than in wild type patients and the difference was statistically significant in cirrhotic patients (mean+/-S.D.=117.45+/-54.45 U/ml vs. 268.52+/-341.27 U/ml, p=0.026). CONCLUSIONS The newly identified SCCA-PD variant was more frequently found in liver cirrhosis, suggesting that patients carrying this polymorphism are more prone to develop progressive liver fibrosis.

Retreatment of chronic hepatitis C (CHC) with sequential interferon-ribavirin combination (IFN-RIBA) therapy

Most patients with CHC have transient response (Relapsers-R) or no response (NR) when treated with IFN. To assess efficacy of IFN-RIBA in these patients, 50 R and 50 NR after IFN therapy were randomized to be retreated with IFN alone or with IFN-RIBA. IFN was given to all cases at 6MU tiw and at 2 mo patients were randomized to continue with IFN alone or to add RIBA 1800-1200 mglday) for 6 mo. ALT and HCV-RNA (PCR) End Theraov Resoonse and Sustained (12 mo) Resoonse are shown in the table. Previous NR (ALT) ETR(RNA) (ALT) SR (RNA) IFN monotherapy (24) 5(21 %I 3(12%) 0 0 IFN-RIBA (241 12(50%) 4(16%) 1(4%1 0 Previnus R IFN monotherapy (26) 1 16(61X) 10(38%) 1 6(23%) 4(15%) IFN.RIBA (28) 1 22(85%1 18(69%) 1 14(54%) 12(46%) IFN-RIBA significantly improved rate of SR in R (P=O.O3) but not in NR, in which only ALT ETR was improved. IFN-RIBA vs IFN improved SR by 27% with HCV-1 and by 4% with HCV-213. SR rates were 27% and 40% respectively with IFN alone and IFN.RIBA in R after 3MU x 6 mo IFN while the corresponding figures for R after higher/longer IFN therapy were 7% and 57%. Addition of RIBA to IFN improved SR only in patients with normal ALT at randomization. In conclusion, IFN-RIBA combination therapy was superior to IFN monotherapy in retreating previous IFN relapsers, particularly those with HCV-1 and those who had already being treated with high dose IFN during the I” cycle. Our schedule of IFN.RIBA was not effective in previous IFN non responders. A NOVEL HEPATITIS E (HEY) ISOLATED FROM A PATIENT WITH ACUTE nA-C HEPATITIS IN ITALY L. Roman& E. Tanzi. A. Zanetti. G. Sl *Viral Discovery Group, Abbott Laboratories, USA Objective: To assess the aetiological role of HEV in acute nA-C hepatitis and to characterize a viral isolate from a patient with no history of travel to areas where HEV is endemic. P&t&s and methods: We studied 216 patients with nA-C hepatitis (negativity for IgM anti-HAV, HBsAg, IgM anti-HBc, anti-HCV, HCV-RNA and exclusion of autoimmunity, alcohol or hepatotoxic drugs). All sera were tested by EIA for both IgG (Abbott Labs) and IgM (in house assay) anti-HEV. Sera and stools (when available) collected during the acute phase were examined by nested RT-PCR using primers derived from the ORFl region. Oligonucleotide primers based on the 5’-end of the ORFl of HEV-US1 were used to identified an isolate from a patient with no known risk factors. Results: 22/216 (10.2%) patients were found HEV-RNA and IgM positive during acute phase (ALT mean peak 2768 IUil, range 396-12290). The acute disease had a benign course with ALT normalization in 3-5 weeks in 20 (91%) patients. One patient with a history of chronic hepatitis C died from fulminant hepatitis and 1 patient (co-infected with HAV) had a clinically severe course (ALT 12290 IU/l, AST 17870 IUil) with ALT normalization within 8 weeks. 17 (77.3%) acute hepatitis E infections occurred in patients who travelled in endemic areas and 5 (22.7%) in patients with no history of travel. Pairwaise alignments of the nucleotide sequence from a HEV F’CR positive patient with no history of travel indicate that the isolate is significantly divergent from the two original isolates of HEV Burma and Mexico (79.9 and 80.7% respectively) and also significantly divergent from the new US isolates (85.8-88.6%). Phylogenetic analysis indicates that the Italian isolate represents a fourth branch distinct from those represented by the Burmese, Mexican and US isolates. Conclusions: HEV is responsible of about 10% of acute nA-C hepatitis in Italy. The identification of a new HEV variant may be important in understanding the epidemiology of HEV infection in our country.

Serum HBV-DNA in anti-HBe positive patients detected by filter and liquid phase hybridization assays

Serum HBV-DNA is considered the best parameter for monitoring HBV replication in the liver. The filter hybridization assay (spot test) with 32P-labelled HBV-DNA has been the technique more frequently used to date. A simple solution hybridization assay, in which 125I HBV-DNA is used as labelled probe, has been recently standardized. We have compared the performances of these two assays for the detection of HBV-DNA. The results were similar with the two methods: an agreement was found in 39/44 (89%) samples. Three sera were positive only by the spot assay-and two only by the liquid phase assay. However, in these cases, HBV-DNA levels were near the sensitivity limits of the assay. Therefore, the filter and the liquid phase assays can be considered to be suitable methods to monitor HBV replication, a fundamental index for the clinical assessment and prognosis of patients with HBsAg positive chronic hepatitis.