Biology of hepatitis B virus.

Immunochemical investigations of the viral antigens and molecular characterization of the viral DNA have elucidated the nature of the hepatitis B virus infection underlying acute, chronic, and oncogenic disorders of the liver in man. Cloning and sequencing of viral DNA have made possible studies on the structure of the genome and on certain aspects of the biology of the virus, hitherto constrained for a lack of tissue culture systems and laboratory animal models useful in its propagation.

Hemagglutination assay for antigen and antibody associated with viral hepatitis.

Hemagglutination assays are described for measuring hepatitis-associated Australia antigen and antibody. Red cells coated with isolated antigen, with chromic chloride as a coupling agent, are used for detection of antibodies. Detection of the antigen in serums depends on inhibition of hemagglutination. The test has the sensitivity and rapidity of the best tests available, is simpler to perform, and lends itself to large-scale screening of blood donors.

Australia antigen (hepatitis B antigen): a conformational antigen dependent on disulfide bonds.

Reduction and alkylation of purified hepatitis-associated Australia antigen (hepatitis B antigen) resulted in a total loss of serologic activity. The reduced and alkylated protein formed a single band with a sedimentation coefficient of 31S on analytical ultracentrifugation, and no subunits were detected by Sephadex gel filtration. Although this preparation induced a delayed hypersensitivity response when injected into guinea pigs, it failed to stimulate humoral antibody formation. The data suggest that hepatitis B antigen is a conformational antigen critically dependent upon the disulfide bonds of the protein moiety.

Hepatitis B "e" antigen: an apparent association with lactate dehydrogenase isozyme-5.

Serums containing the "e" antigen of hepatitis B virus were subjected to electrophoresis in polyacrylamide gel. An extra band appeared in the lactate dehydrogenase isozyme pattern, but this band was undetectable in serums containing antibodies to the e antigenic determinant. Prior separation of the lactate dehydrogenase isozyme-5 fraction by chromatography of serum on minicolumns of diethylaminoethyl-Sephadex-A50 improved electrophoretic identification of the extra band. Neutralization with antibodies to the e antigen as well as by antibodies to the homologous d or y component of the hepatitis B surface antigen removed the extra band; antibodies to the lactate dehydrogenase isozyme-5 removed both the normal and the extra enzymatic band of isozyme-5. This feature of the e antigen provides an assay system for laboratory diagnosis of potential clinical usefulness and suggests its possible role in pathogenesis of hepatocellular injury.

The molecular biology of hepatitis B virus.

Infection with the hepatitis B virus (HBV) or related hepadnaviruses is associated with a wide spectrum of liver diseases, including hepatocellular carcinoma (HCC). In this article we review the current state of knowledge about the structure, genetic organization and life cycle of HBV. The mechanisms of viral pathogenesis and HCC development remain poorly understood, but new approaches may soon begin to shed light on these areas.

Detection, semiquantitation, and genetic variation in hepatitis C virus sequences amplified from the plasma of blood donors with elevated alanine aminotransferase.

Hepatitis C virus (HCV) is the predominant etiologic agent of posttransfusion non-A, non-B hepatitis, characterized by undulating elevation of alanine aminotransferase (ALT) and chronic liver disease. A commercial enzyme-linked immunosorbent assay detected antibodies to HCV (anti-HCV) in 11 specimens among 101 nontransfusable plasma units obtained from asymptomatic, volunteer blood donors with elevated levels' of ALT. Using a combined reverse-transcription polymerase chain reaction (RT-PCR) assay developed by us, HCV RNA was detected in 0.6 ml of plasma from 8 of 11 (73%) of the anti-HCV-positive but in none of the 90 anti-HCV-negative specimens. The relatively low concentration of HCV RNA could be detected in the remaining three anti-HCV-positive specimens when 2.4 ml of plasma was analyzed. The plasma concentration of virions was estimated to range from 10(2) to 5 x 10(7)/ml. Direct sequencing performed on the PCR-amplified HCV cDNAs (210 base pairs) from three specimens revealed heterogeneity between 2.5 and 8.6% at the nucleotide level and less than 4% at the amino acid level. Our findings demonstrate that RT-PCR can be performed with 2.4 ml of plasma, providing an assay for the direct detection of HCV RNA and confirming the existence of an asymptomatic carrier state for HCV infection in the apparently healthy anti-HCV-positive donors.

Hepatitis B viral markers in patients with primary hepatocellular carcinoma in Taiwan.

The presence of hepatitis B viral markers in patients with primary hepatocellular carcinoma (PHC) was studied retrospectively at the Taiwan Veterans General Hospital in Taipei, Taiwan. Serum samples from 102 PHC patients and from 100 control individuals were tested for hepatitis B surface antigen (HBsAg), antibody to HBsAg (anti-HBs), antibody to hepatitis B core antigen (anti-HBc), hepatitis Be antigen (HBeAg), and antibody to HBeAg (anti-HBe). Of the 102 PHC patients, 72 (71%) were positive for HBsAg. Nine (9%) additional patients were positive for anti-HBc alone in high titer, 19 (19%) had both anti-HBc and anti-HBs, and 9 (9%) had HBsAg, anti-HBc, and anti-HBs. In the 100 controls, 12 (12%) were HBsAg-positive, whereas 22 (22%) had anti-HBc alone and 50 (50%) had both anti-HBc and anti-HBs. Only 4 (4%) controls and no PHC patients had anti-HBs alone. Of the HBsAg-positive patients with PHC, 17 (29%) had HBeAg and 36 (61%) had anti-HBe. The alpha-fetoprotein (AFP) levels above 400 ng/ml were found in 44% of the PHC patients. Values of AFP above 1 x 10(5) ng/ml were more frequently detected in PHC patients who were HBsAg-positive. Categorization of the geographic origins of the families whose members had PHC revealed that most families had originated from southern China. This study confirms that hepatitis B viral markers are frequently present in Chinese patients with PHC.

Use of polymerase chain reaction for the early detection of HIV infection in the infants of HIV-seropositive women.

Forty-two infants of HIV-seropositive women were evaluated to determine the value of polymerase chain reaction (PCR) in the early detection of HIV infection. All infants less than 6 months old had a simultaneous PCR and culture for HIV. There was an 88% concordance between the two techniques. PCR results showed an excellent correlation with clinical outcome; no PCR-negative patient has subsequently been found to be infected. Occasional false-positive or equivocal PCR results did occur. There was one false-negative culture. PCR is a rapid and sensitive diagnostic test for the early diagnosis of HIV infection in infants at risk, but at present it should be performed in conjunction with other diagnostic tests and good clinical follow-up.

Transcription and replication of human immunodeficiency virus-1 in B lymphocytes in vitro.

We and others have shown that HIV-1 transcription and replication in vitro are increased by the binding of transcriptional enhancer DNA sequences in the HIV-1 long terminal repeat (LTR) to a cellular protein designated Nuclear Factor-kappa B (NF-kappa B), a trans-acting transcription factor which is present in activated but not in resting T cells. Because NF-kappa B is also expressed in resting B cells we suggested that B cells might provide an optimal intracellular environment for HIV-1 transcription. Using a transient transfection assay, we show that the HIV-1 LTR is indeed more efficiently transcribed in the B cell line Raji than in the T cell line Jurkat, and that this effect maps to the transcriptional enhancer (NF-kappa B binding site) of the LTR. Furthermore, we show that Raji cells which have been rendered CD4-positive by gene transfection are susceptible to HIV-1 infection, and that viral gene expression and replication are more efficient in these CD4-positive B cells than in CD4-positive T cells. These findings demonstrate the crucial role of the transcriptional enhancer in regulating HIV-1 expression and, since receptors for HIV-1 are expressed by some Epstein-Barr virus (EBV)-positive B cells, they also suggest that HIV-1 replication could occur in EBV-positive B cells in vivo.

Persistent immune complexes and abnormal CD4/CD8 ratios in HIV infection.

We assessed the immunopathologic role of circulating immune complexes in human immunodeficiency virus infection by evaluating the data base and the serum bank of the San Francisco Men's Health Study, a longitudinal clinical and epidemiological investigation conducted since 1983. A group of 4,276 sera from 1,023 (including 811 homosexual/bisexual) men were tested for circulating immune complexes. We used a modification of the commercially available enzyme immunoassay test, based on monoclonal anti-C1q antibodies coupled to the solid phase, for capturing circulating immune complexes from the test serum, followed by detection of circulating immune complexes with either anti-IgG or with anti-IgM probes. Although persistent IgM and IgG circulating immune complexes were most frequently encountered in human immunodeficiency virus-seropositive homosexual/bisexual men, they were not an indicator of disease progression as assessed by abnormalities in the absolute numbers or ratios of CD4- and CD8-positive T cells, or clinical signs and symptoms of AIDS/ARC.

In situ hybridization and immunocytochemistry for improved assessment of human immunodeficiency virus cultures.

The authors characterized the early intracellular events involved in human immunodeficiency virus (HIV) replication after in vitro inoculation into cultures of susceptible human T-cell lines and phytohemagglutinin-stimulated peripheral-blood mononuclear cells (PMCs). Within 24 hours of infection, in situ hybridization with HIV DNA probe detected cytoplasmic viral RNA. Viral core antigen was detected in infected cells over the subsequent two to ten days by means of an immunocytochemical assay employing monoclonal antibodies. Several days later, cell-free virus was detected by both reverse transcriptase assay and a p25gag antigen-capture assay. When these methods were applied to monitor cultures of ten sero-positive persons' PMCs, a similar progression of virus replication was apparent: cytoplasmic viral RNA was detected in infected PMCs by day 3, with the subsequent appearance of intracellular viral proteins (days 6-9) and cell-free virus (days 12-21). In situ hybridization and immunocytochemistry offer complementary, sensitive, and specific approaches for monitoring the early stages of acquired immune deficiency syndrome virus replication in vitro.

Transfusion-related transmissible diseases: detection by polymerase chain reaction-amplified genes of the microbial agents.

The detection of blood-borne microbes by PCR has broadly and rapidly progressed in the past 5 years as briefly described in this article. This progress has been largely because of the scientific developments made at Roche Molecular Systems by Sninsky et al through collaborations with academic and Government institutions. This unprecedented cooperation propels the continuing work at Roche Molecular Systems to bring the PCR technology into routine laboratory diagnosis. Whether the success of EIA in virtual elimination of hepatitis and retroviral infections can be matched by the cost-effectiveness of putative application of PCR in screening blood supply remains to be determined.

Interaction of aflatoxin and hepatitis B virus in the pathogenesis of hepatocellular carcinoma.

Aflatoxin-B1 (AFB) and chronic hepatitis B virus (HBV) infection epidemiologically correlate with the geographic distribution of hepatocellular carcinoma (HCC). Integration of HBV DNA into the cellular genome of HCCs and the in vivo formation of adducts between AFB and nucleic acids lead us to suggest that hepatocytes with integrated HBV DNA preferentially accumulate AFB; the AFB-adducts formed may then initiate cell transformation by modifying the expression of critical host genes. The altered molecular biology of liver cells in HCC is evidenced by the fact that HBV does not replicate in HCC tissues or cell lines. The effect of AFB on the expression of cellular genes such as endogenous retrovirus(es) and possibly cellular oncogene(s) can be analyzed in HCC cell lines with and without integrated HBV DNA. In addition, human HCC tissues can be probed for HBV sequences and AFB-DNA adducts at the single-cell level. The presence of HBV and AFB can be correlated with the expression of putative transforming genes, providing a new insight into the interaction between liver cells, HBV and AFB in the pathogenesis of HCC.

Diagnosis of viral hepatitis.

Acute viral hepatitis is a serious infectious disease of the liver caused by several viruses and characterized by acute necrosis of hepatocytes and inflammation. In this article, recent advances in hepatitis serology and virology are briefly reviewed, and practical, up-to-date laboratory tests are outlined.

Immunologic mechanisms in hepatitis B assayed by antigen-binding lymphocytes.

Immune mechanisms in hepatitis B virus (HBV) infection were investigated in 16 persons with and without hepatitis using tests for HBS Ag, anti-HBS, anti-HBC and 125I-labeled HBS Ag binding lymphocytes (ABL) in peripheral blood. Anti-HBC, which is an evidence of a recent or current HBV infection, was detected in all HBS Ag positive sera. High counts of ABL correlated with the presence of anti-HBS in serum but not with anti-HBC or with HBS Ag. In patients with chronic hepatitis, and in asymptomatic carriers of HBS Ag, there was a trend towards low counts of ABL, which may represent partial tolerance ot HBS Ag in carriers of this particle. Further work on ABL for HBS Ag and HBC Ag should enhance our understnading of immunologic responses to the antigens of the hepatitis B virus.

Immunobiology of persistent blood-borne viral infections.

Host-virus interactions have co-evolved to play an interactive role in the pathogenesis of viral infections and their disease outcome. Host responses to viral infections, including the cell-mediated and humoral immune responses, have been a subject of intensive research in virology and immunology. Definition of specific cellular receptors for cellular entry of the agents, the rates of their intracellular viral replication, the rates of turnover of circulating virions, persistence of viral infection possibly due to inadequate immune responses, and continued formation of circulating immune complexes provide the framework for our current understanding of the immunopathology of virally induced disease. Among the multiple blood-borne viruses (BBV) transmissible through transfusion, hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency viruses (HIV-1/-2) are relatively more important than several other viruses. Not only do they establish asymptomatic persistent infections with occasional oncogenic sequelae, but they also cause significant morbidity and mortality when transmitted through transfusion of blood and blood products. Molecular characterization of these agents and their in vitro inactivation and removal from blood have become key issues in contemporary transfusion safety since the advent of AIDS. Because many of the BBV are associated with white blood cells that have no therapeutic benefit in haemotherapy, simple filtration-removal of leukocytes from donated blood confers a dual benefit of immunological and virological safety in transfusion medicine.

Human peripheral blood mononuclear cell substrate for propagating wild type HIV-1.

Molecularly derived HIV proteins fail to induce broadly neutralizing antibodies (NAB) and cytotoxic T cell (CTL) responses, which are considered necessary for any effective vaccination. Natural virion proteins of the primary isolates of HIV-1 (pi-HIV), grown in human peripheral blood mononuclear cells (PBMC) followed by viral inactivation and pooling to reflect the antigenic diversity of the prevalent strains in a given population, would provide a novel polyvalent HIV vaccine (HIVAX) capable of inducing both HIV-NAB and CTL. Proven technologies are harnessed to provide a framework for advancing human blood-derived HIVAX. Fresh leukocytes, recovered by gravity flow elution from leukocyte depletion blood filters, or "buffy coats", routinely removed in preparing blood components for transfusion, provide an abundant and safe cell substrate following ficoll-separation of viable PBMC. The low content of wild-type HIV (wt-HIV) in infected plasma can be chaperoned by dendritic cells through their DC-SIGN receptor for gp120 and efficiently expanded in co-culture with CD4-enriched cell substrate in a medium containing human AB serum as a substitute for foetal calf serum. Dimethyl methylene blue (DMMB), ethylene diiimine (EDI), or psoralens can be used to inactivate the virion RNA selectively and the unbound chemicals and their products are removable by molecular sieving. Additional inactivation by physical methods would include proven hydrostatic pressure cycling technology (PCT) and flash pasteurization at 60 degrees C for less than 10 minutes to totally inactivate infectivity of the virions. Our empirical strategy is to pool five different HIV isolates of the prevalent subtype B (U.S.A.) or C (Southern Africa), augmented by other subtypes A, C/B, D, E, and 'X' (new emerging subtypes), to provide a polyvalent HIV vaccine (HIVAX). Immunochemical quantification of the gp120, gp41, p24 and p31 antigen content of HIVAX ensures consistency of the product, and safety is ensured by failure to amplify HIV nucleic acid sequences by RT-PCR and to demonstrate infectivity in animal models. Ultimate efficacy HIVAX must be shown by human clinical trials in the high-risk populations.

Properties of cyanovirin-N (CV-N): inactivation of HIV-1 by sessile cyanovirin-N (sCV-N).

Cyanovirin-N (CV-N) is a novel anti-HIV protein isolated and characterized from a cyanobacterium Nostoc ellipsosporum. CV-N protein is a single 101 amino acid chain containing two intrachain disulphide bonds and considerable internal sequence duplication, but no significant homology to previously described proteins or to the transcription products of known nucleotide sequences. In solution, CV-N exists largely as a beta-sheet protein with internal two-fold pseudosymmetry. CV-N irreversibly inactivates diverse laboratory strains, primary isolates and clades of HIV-1, as well as strains of HIV-2 and simian immunodeficiency virus (SIV). CV-N binds with extremely high affinity to highly conserved binding site(s) on the viral envelope glycoprotein gp120, preventing virus-to-cell fusion, viral entry and infection of cells. The CV-N binding site appears to overlap, but is not identical with, the unique carbohydrate-dependent epitope 2G12, and may lie predominantly within an immunologically "silent" region of gp120. CV-N is undergoing preclinical development for topical anti-HIV prophylactic (e.g., microbicidal) applications to prevent sexual transmission of HIV. Since CV-N may be immunogenic in humans, methods for using CV-N for ex vivo inactivation of HIV in blood, plasma, or putative vaccines preferably would allow for its exclusion from biologicals for parenteral use. To explore this concept we biotinylated CV-N (bCV-N) and coupled it to streptavidin coated magnetic beads to provide a product which we termed sessile CV-N (sCV-N). When reacted with a laboratory strain and a primary isolate of HIV- 1, the sCV-N completely inactivated 100 TCID50 of the virus. However RT-PCR of the viral extracts indicated that only a fraction of the virus was removed by the sCV-N, leaving behind a relatively larger fraction of non-infectious virus in the supernatant which we designated as replication incompetent virions (RIV). It would be worthwhile investigating the role of RIV as a putative HIV vaccine.

Molecular approaches to the detection of agents in persistent viral infections.

Detection of infection with BBVs transmitted by the blood donated by apparently healthy volunteers is facilitated by several laboratory tests employed in screening out infected blood. Because several of the screening assays are immunological markers of host response to the infectious agents, the detection of viral nucleic acids remains a major goal that may possibly be fulfilled by the advent of PCR.