Hepatitis B surface antigen variants in vaccinees, blood donors and an interferon-treated patient.

Variants of hepatitis B virus (HBV), with amino acid substitutions in the major antigenic "a" determinant of hepatitis B surface antigen (HBsAg), have been described mainly in vaccinated children. In the present study in addition to vaccinated children, we have investigated Chinese blood donors positive for anti-HBc alone, and a patient with continuing liver disease after interferon-induced seroconversion to anti-HBs. Variants were detected in two of four children with break-through infections. One child had a double mutation (P142S and G145R) and the other a G145A substitution. Three of seven anti-HBc positive Chinese blood donors had a T131I substitution, whilst the interferon-treated patient had a treble amino acid substitution (P142S, G145R and N146D). The present results indicate that HBsAg variants may exist in individuals other than vaccinated children.

Intrahepatic transfusion-transmitted virus detected by in situ hybridization in patients with liver diseases.

Transfusion-transmitted virus (TTV) has been identified from patients with post-transfusion hepatitis of unknown aetiology, but the clinical relevance remains unclear. The aim of this study was to evaluate TTV in liver. We studied 15 patients with hepatitis non-A-E and 44 with hepatitis B virus (HBV). The nested polymerase chain reaction (PCR) with primers corresponding to the conserved region of the published TTV genome was employed to amplify TTV fragments in serum, and in situ hybridization was used to detect TTV in biopsied liver specimens. TTV DNA was detected in serum from six (40%) of 15 patients with hepatitis of unknown aetiology and from 16 (36.4%) of 44 patients with chronic hepatitis B, respectively. The intrahepatic viral fragment was detected in 17 (77.3%) of 22 patients with TTV in serum. There was no statistical difference in the prevalence of TTV infection between the two groups (hepatitis non-A-E 40% vs HBV 25%, P > 0.75). When patients in both groups, with and without TTV, were compared, no differences were found in serum alanine aminotransferase (ALT) levels (hepatitis non-A-E: 131.5 +/- 66.6 vs 244.2 +/- 257.4, P=0.955; HBV: 240.1 +/- 418.9 vs 214.6 +/- 276.7 U l(-1), P=0.761) or histological activity index (grade) score (hepatitis non-A-E: 6.4 +/- 5.5 vs 5.6 +/- 5.9, P=0.689; HBV: 5.6 +/- 3.7 vs 5.5 +/- 3.7, P=0.345). HBV DNA levels in patients with and without TTV co-infection did not differ significantly (300 +/- 776.4 microg ml(-1) vs 97.1 +/- 160.5 microg ml(-1), P=0.980). Hence, TTV does exist in liver, but plays no role in hepatitis or aggravation of liver damage when co-infected with HBV.

A prospective study of transfusion-transmitted virus transmission by blood transfusion.

Recently, a new single-stranded DNA virus (TT virus, TTV) has been isolated and related to post-transfusion hepatitis. The aim of this study was to investigate the prevalence of TTV in blood donors and blood recipients, and the incidence of TTV transmission by blood transfusion. TTV DNA and serum markers of hepatitis B virus (HBV) and hepatitis C virus (HCV), were examined in 130 blood recipients, and the presence of TTV was studied in their 340 corresponding blood donors. The prevalence of TTV infection was 10.6% (36/340) in donors and 8.5% (11/130) in blood recipients, before transfusion. Eighteen subjects (15.1%) were found to be TTV positive, after transfusion, in the 119 blood recipients without TTV before transfusion; at least one of the corresponding donors was TTV positive. There were 46 subjects with post-transfusion hepatitis virus infection, 45 with HCV infection (including seven co-infected with TTV) and two with HBV infection (including one co-infected with HCV and one co-infected with TTV). The recipient with TTV and HBV co-infection and three of the seven patients with TTV and HCV infection had alanine aminotransferase (ALT) levels higher than 90 Ul-1, but only two of the 10 isolated TTV infections had a mild ALT elevation. These results show that prevalence of TTV was high in blood donors and hospitalized patients, and isolated TTV infection is not related to significant ALT elevation.

An outbreak of enterically transmitted non-A, non-E viral hepatitis.

Patients with isolated serum transaminase elevations of unknown cause are common in China. An outbreak of such disease took place in a technicians' school during 1996. To define the epidemic and determine the etiology, a study was carried out, which included investigation of epidemiological, clinical and histological features. The symptoms of this disease were mild. The major clinical feature was transaminase elevation, and all serum markers of known hepatitis viruses were negative. Although the course of disease in most patients was self-limiting, in a few it was prolonged and relapsed. Histological findings were mild portal hepatitis or non-specific reactive hepatitis. The disease first appeared in 1994, and this outbreak occurred after October 1996. A total of 381 people were affected and the prevalence was as high as 60.7%. Casual contact and small-scale food transmission were considered to be risk factors for infection and the epidemic was under control 2 months later following the introduction of preventive measures for gastroenteric infection. Viral genomic fragments from the so-called transfusion-transmitted virus (TTV) were detected in acute-phase sera and stool samples collected 2 weeks before onset. Therefore, this disease outbreak might be another form of enterically transmitted viral hepatitis, not related to hepatitis A and E.

In situ investigation of Fas/FasL expression in chronic hepatitis B infection and related liver diseases.

To evaluate Fas/FasL expression in hepatitis B virus-related chronic liver disease, liver biopsies from 44 such cases were studied immunohistochemically. FasL was detected in the infiltrating lymphocytes and both FasL and Fas were found in the hepatocytes. The Fas and FasL-positive cells were mostly found at the advancing edges of interphase hepatitis, and Fas/FasL expression was closely correlated with the inflammatory activity. Unexpectedly, FasL was also expressed in liver cirrhotic nodules, particularly in those with hepatocellular carcinoma with or without inflammation. These results suggest that the factors which induce hepatocyte transformation might also trigger FasL expression and promote FasL/Fas-mediated apoptosis.

[Correlation between HBV pre-C/C gene mutations and advanced pathologic activity in chronic hepatitis B].

To investigate the relationship of the HBV preC/C gene mutations with the disease persistence and exacerbation, the sequence of this region was analysed with direct sequencing by using the polymerase chain reaction product of the DNAs extracted from 24 such cases. A mutant with preC stop codon at the site 28 was detected in all the 11 anti-HBe-positive patients with different severity. This stop 28 mutant also coexisted with wild virus in seven of 18 HBeAg-positive patients. C gene mutations clustered in a small segment of codon 84-101, of those especially codon 97 was substituted in 11 of 15 mutants. There were 2, 6 and 20 codon mutations in 8 mild, 6 moderate and 10 severe cases respectively. In addition, the deletion of 144bp was also found in this region in a moderate case and that of 96bp in a severe case. Follow up of six patients revealed a close correlation between increase of genetic variation and advance of pathology.

[Distribution of pre-core A83 point mutation in hepatitis B virus infections].

We designed a mispairing primer, which could introduce a Bsu36I restriction site into the amplified fragment of A83 mutant. Following PCR, the 102bp was diagnosed by the restriction endonuclease, and the variation was detected by demonstration of 82bp and 20bp bands on gel electrophoresis (RFLP). With the method, 77 cases of acute and chronic HBV infections were analysed. Among those, 31 (65%) mutants were detected in 48 anti-HBe-positive cases, and in 29 HBeAg-positive cases, 11 (38%) were co-infected with mutated and wild isolates. No pre C defect was found in acute hepatitis B and chronic asymptomatic carriers, suggesting that mutation occurs only after immune selection. The HBeAg defective variant appears to be involved in the loss of virus tolerance, and therefore in the pathogenesis of acute exacerbation of chronic carriage as demonstrated in this study. The various chronic liver diseases had an approximate mutation frequency, and it seems that A83 mutation nearly makes HBV infection persist.

Etiological investigation of acute post-transfusion non-A, non-B hepatitis in China.

The etiology of acute post-transfusion hepatitis (PTH) non-A, non-B (NANB) in China was investigated with the combination of advanced techniques, including a second generation of enzyme immunoassay for detection of the antibodies to hepatitis C virus (anti-HCV), a reverse transcription and nested polymerase chain reaction (RT-nPCR) for HCV RNA, and a PCR for hepatitis B virus DNA. Of the 57 patients who were diagnosed as acute PTH-NANB, 46 (80.7%) were positive for anti-HCV and 41 (71.9%) had HCV RNA. Combining together, 53 (93.0%) were seropositive for anti-HCV and/or HCV RNA. Surprisingly, 18 of these with HCV markers were also positive for HBV DNA, although they were negative for HBsAg, suggesting that a portion of the patients with acute PTH NANB were coinfected by both HCV and HBV. In addition, 4 (7%) of the patients with acute PTH-NANB had no detectable HCV and HBV markers with the use of the above-mentioned techniques. These results indicate that etiologic agents(s) other than HCV and HBV may also cause acute PTH-NANB or that the current techniques may still not be sensitive enough to detect trace levels of HCV and HBV markers.

Correlation between the prevalence of serum HBV DNA and immunoserologic HBV markers in the subjects with or without hepatitis.

The prevalence of serum HBV DNA, detected by polymerase chain reaction, and that of immunoserologic HBV markers (HBsAg, HBeAg, anti-HBs, and anti-HBc), determined by immunoassays, were compared among three groups of subjects: (A) chronic active hepatitis B patients, (B) chronic asymptomatic HBV carriers, and (C) normal individuals. Except five of the normal individuals, all of the subjects are positive for anti-HBc while some of them were also positive for other immunoserologic HBV markers, such as anti-HBs, HBsAg, and HBeAg. Serum HBV DNA were detected in 81% in group A, 52% in group B, and 20% in group C. In both group A and B, serum HBV DNA were detected in all the subjects with anti-HBc+/HBsAg+/HBeAg+. However, the percentage of seropositive HBV DNA in the subjects with anti-HBc+/HBsAg+ in Group A was much higher than that in Group B. Interestingly, the percent of serum HBV DNA+ in the individuals with anti-HBc+ only was markedly higher than that in the subjects with anti-HBc+/anti-HBs+ in both Group A and C, suggesting that anti-HBs may play a role in the inhibition of HBV replication and clearance of HBV virion from blood. Above serological profiles will provide important information concerning the significance of serum HBV DNA detection in judgement of HBV replication in the individuals with or without HBV infection. Cautions should be taken to clarify those so called normal individuals who have no symptoms of hepatitis B, no HBsAg in the sera and normal transaminase, but have HBV replication in their bodies.

Hepatitis B virus DNA in sera of virus carriers positive exclusively for antibodies to the hepatitis B core antigen.

The prevalence of serum HBV DNA in individuals positive for anti-HBc alone was determined by the polymerase chain reaction in two groups with endemic HBV infection from Canton (group A) and Hainan (group B), provinces of China. Twenty-one out of 294 individuals in group A (7.2%) and 193 out of 1995 in group B (9.7%) were positive for anti-HBc but negative for other markers of ongoing or past HBV infection (HBsAg and anti-HBs). HBV DNA was detected in 6/21 sera in group A (28.6%) and 68/193 in group B (35.2%) in their initial serum specimen. One of the six HBV-DNA-positive individuals in group A became negative after 6 months and four of the 58 positive in group B became negative at 4 years of follow-up. All of the individuals remained positive for anti-HBc and negative for anti-HBs, but one of them became positive for HBsAg on follow-up. None of the anti-HBc- and HBV-DNA-positive subjects had symptoms of liver diseases. They were, therefore, defined as chronic asymptomatic HBV carriers with undetectable HBsAg. This type of carrier should be added to the typical HBsAg-positive carrier, who constitutes about 10-15% of the general Chinese population, to give a more complete estimate of asymptomatic HBV carriers in China.

[Identification of HBV infection in HBsAg-negative chronic active hepatitis with polymerase chain reaction].

To explore the etiological agent(s) of HBsAg negative chronic active hepatitis, serum HBV DNA was tested in thirty-six of such cases with polymerase chain reaction. In total, viral DNA was detected in 24 cases (67%), including 11 of the 12 cases with positive anti-HBc alone, four of the nine cases with positive anti-HBs and nine of the 15 cases without any HBV markers. The results suggest that in cases of HBsAg-negative chronic active hepatitis, the etiology may still be hepatitis B virus, especially in cases with positive anti-HBc. Furthermore, the cause of activated histology might be due to viral replication.

Cross-linking of T-cell surface molecules CD4 and CD8 stimulates phosphorylation of the lck tyrosine protein kinase at the autophosphorylation site.

p56lck, a lymphocyte-specific tyrosine protein kinase, binds to the cytoplasmic tails of the T-cell surface molecules CD4 and CD8. Cross-linking of CD4 expressed on the surface of murine thymocytes, splenocytes, and CD4+ T-cell lines induced tyrosine phosphorylation of p56lck dramatically. Cross-linking of CD8 stimulated tyrosine phosphorylation of p56lck strongly in murine L3 and GA4 cells, slightly in splenocytes, but not detectably in thymocytes. Differing effects of cross-linking on in vitro tyrosine kinase activity of p56lck were observed. An increase in the in vitro kinase activity of p56lck, when assayed with [Val5]-angiotensin II as an exogenous substrate, was found to accompany cross-linking of CD4 in three cell lines. No stimulation of the in vitro kinase activity, however, was observed after cross-linking of CD8 in L3 cells. The phosphorylation of p56lck at Tyr-394, the autophosphorylation site, was stimulated by cross-linking in all cell lines examined. Tyr-394 was the predominant site of increased tyrosine phosphorylation in two leukemic cell lines. In the other two cell lines, the phosphorylation of both Tyr-394 and an inhibitory site, Tyr-505, was found to increase. In contrast to cross-linking with antibodies, no striking increase in the tyrosine phosphorylation of p56lck was stimulated by antigenic stimulation. Therefore, the effect of antibody-induced aggregation of CD4 and CD8 on the tyrosine phosphorylation of p56lck differs, at least quantitatively, from what occurs during antigen-induced T-cell activation.

Analysis of the sites in p56lck whose phosphorylation is induced by tetradecanoyl phorbol acetate.

p56lck is a member of the src family of tyrosine kinases that is expressed almost exclusively in lymphocytes. Previous studies have shown that treatment of T cells with activators of protein kinase C induces serine phosphorylation of p56lck. We show here that treatment of Jurkat cells with 12-O-tetradecanoyl phorbol-13-acetate also induces threonine phosphorylation of p56lck. Chymotryptic mapping shows that at least three sites in p56lck undergo phosphorylation in TPA-treated Jurkat cells. Cyanogen bromide cleavage analysis demonstrates that these new phosphorylation sites are located in an amino-terminal 32 kDa fragment containing amino acid residues 14-261.

[HBsAg/PRE-S2 expression of viral replication and hepatic inflammation in chronic hepatitis B virus infection].

A close relationship (r = 0.186, P less than 0.025) was found between the serologic level of HBsAg/pre-S2, an envelope protein of hepatitis B virus, and the level of viral replication expressed by HBeAg. The rate of co-appearance and co-absence of both pre-S2 and HBeAg was 75.0%. Among HBeAg-positive cases, the prevalence of pre-S2 and its serologic level were significantly higher during exacerbation than resolution of chronic active hepatitis (73.3 vs 33.3%, 111.4 vs 1.4 ng/ml), and also higher than those among HBeAg-negative cases. Those the prevalence and the level were also higher in chronic active hepatitis than in chronic asymptomatic carriers (26.9 vs 11.5%, 2.95 vs 0.49 ng/ml). Pre-S2 prevalence in the group with highest scoring of histological activity was significantly higher than that in the groups with lower scoring (88.9 vs 40.0 and 36.4%) and the serologic level of pre-S2 also increased along with the scoring. Those data suggest that pre-S2 was associated with hepatic inflammatory activity as well.

Significance of various classes of anti-HBc in individuals with chronic asymptomatic HBV infection.

Among 108 individuals with chronic asymptomatic hepatitis B virus infection, liver biopsies and serological investigation were performed simultaneously to determine the correlations of anti-HBc with the infection level of the liver pathology. The results showed that the prevalence of IgA and IgM anti-HBc, and the titers of IgA, IgM, and total anti-HBc, correlated with the liver damage. IgA anti-HBc was most prominent, being present in the whole group of patients with chronic active hepatitis with cirrhosis, and was absent in the whole group of asymptomatic carriers with normal histology. There were no linkages of IgA and IgM anti-HBc with the infectivity; in addition, the titer of total anti-HBc correlated inversely with the viral replication.

[Immunologic study of a synthetic peptide with HBsAg activity].

A cyclic 24-peptide covering 122-145 residues of the HBsAg sequence was synthesized. This study was intended to probe into the prospect of the peptide as a candidate of hepatitis B vaccine. The results of radioimmunoassay showed positive reactions between anti-HBs and synthetic peptide and between anti-peptide and HBsAg. Using GP28/P23, a major protein dissociated from HBsAg, as antigen, the affinities of anti-peptide and anti-HBs were determined and no significant difference was found; indicating an excellent homology between them. The rabbits were immunized with the peptide 300 micrograms X 3, and with HB vaccine 3 micrograms X 3, both added Freund's adjuvant. The serologic anti-HBs level induced by the peptide was nearly 3.3 orders of twofold dilution lower than that by HB vaccine. When mice were immunized with alum adjuvant 250 micrograms of peptide induced an anti-HBs response roughly equal to 0.25 microgram of HB vaccine. So, the immunogenicity of this peptide was approximately (6-12) X 10(-4) times that of HB vaccine.