n the past 10 years, remarkable strides have been made in the understanding of the natural history and pathogenesis of hepatitis B virus (HBV) infection. In this article we will review these advances, with particular reference to the implications for antiviral therapy. Clinical and epidemiologic studies began to differentiate among various types of acute hepatitis in the decades after World War II. The groundbreaking studies of Krugman and colleagues in 1967 firmly established the existence of at least two types of hepatitis, 1 one of which (then called serum hepatitis, and now called hepatitis B) was parenterally transmitted. Links to the virus responsible for this form of hepatitis were derived by serologic studies conducted independently by Prince and colleagues 2-4 and by Blumberg and colleagues. 5 Blumberg and colleagues, searching for serum protein polymorphisms linked to diseases, identified an antigen (termed Au) in serum from patients with leukemia, leprosy, and hepatitis, though the relationship of this antigen to hepatitis was initially unclear. By systematically studying patients with transfusionassociated hepatitis, Prince and coworkers independently identified an antigen, termed SH, that appeared in the blood of these patients during the incubation period of the disease, and further work established that Au and SH were identical. 6,7 The antigen represented the hepatitis B surface antigen (HBsAg). 8,9 These seminal studies made possible the serologic diagnosis of hepatitis B and opened up the field to rigorous epidemiologic and virologic investigation. classification and structure Hepatitis B virus (HBV) is the prototype member of the Hepadnaviridae (hepatotropic DNA virus) family. Hepadnaviruses have a strong preference for infecting liver cells, but small amounts of hepadnaviral DNA can be found in kidney, pancreas, and mononuclear cells. However, infection at these sites is not linked to extrahepatic disease. 10-13 HBV virions are double-shelled particles, 40 to 42 nm in diameter (Fig. 1A), 14 with an outer lipoprotein envelope that contains three related envelope glycoproteins (or surface antigens). 15 Within the envelope is the viral nucleocapsid, or core. 16 The core contains the viral genome, a relaxed-circular, partially duplex DNA of 3.2 kb, and a polymerase that is responsible for the synthesis of viral DNA in infected cells. 17 DNA sequencing of many isolates of HBV has confirmed the existence of multiple viral genotypes, each with a characteristic geographic distribution. 18 In addition to virions, HBV-infected cells produce two distinct subviral lipoprotein particles: 20-nm spheres (Fig. 1B) and filamentous forms of similar diameter i history virologic features
We are researchers who have published analyses of nucleic acid sequence variation of hepatitis C virus (HCV) and associated virological and clinical significance. We are concerned that our investigations are hampered by the lack of a consensus nomenclature for variants of HCV and that this leads to confusion when results from different laboratories are compared. Furthermore, because there are no consistently applied criteria by which new genotypes are defined, investigators assign new type descriptions to novel sequence variants on an ad hoc basis without agreement from
The aim of the following experiments was to provide an objective immunologic criterion for the diagnosis of serum hepatitis, as well as a possible means of screening for carriers of the agent of this disease. An antigen that reacted in the immunodiffusion test with serum from multiply transfused patients was detected in the blood during the incubation period prior to the onset of major chemical or clinical abnormalities. Double blind experiments suggest that this antigen is specific for serum hepatitis virus.Materials and Methods.-Clinical specimens: Sera from cases of transfusion-induced viral hepatitis, which we have collected, were obtained as part of a long-term study involving biweekly follow-up of transfused patients at The New York Hospital. Patients volunteering to participate in this study provided blood samples prior to transfusion and at least biweekly for a period of 6 months or more following transfusion.Test serum: The reference "antiserum" used in the majority of the studies to be described, hereinafter referred to as serum S, was obtained from a 24-year-old male patient with hemophilia who has received more than 10,000 units of blood, fresh-frozen plasma, and cryoprecipitate during the course of treatment for bleeding episodes. He has had no episodes of icteric hepatitis, but it was presumed that he had been multiply exposed to the virus or viruses of serum hepatitis. Serum S was chosen for these studies because the patient's multiple exposure was thought to ensure a hyperimmune status. Subsequently, four other sera from multiply transfused patients have been found to react in a manner similar to serum S. For some experiments, the serum was concentrated by ethanol fractionation. To each milliliter of serum to be concentrated, 8 ml of 30% ethanol in 0.1 Ml NaCl, 0.01 M tris(hydroxymethyl)aminomethane (Tris), 0.001 Ml ethylenediaminetetraacetate (EDTA), (pH 7.0 at -7oC) were added. This mixture was held at -70C and lyophilized. The dried globulin fraction was then rehydrated with distilled water to 0.1 the original volume of serum employed.Immunodiffusion technique: Double diffusion in agar gel was done by a micro-Ouchterlony technique.1 Nonspecific precipitation reactions between adjacent wells were eliminated by the use of 0.9% agarose dissolved in a buffer composed of 0.1 M NaCl, 0.01 M Tris (pH 7.6 at 250C), and 0.001 M EDTA containing 1 mg/ml protamine sulfate.Protamine sulfate has been recently suggested as a means of decreasing virus-agar interaction.2 Plates were incubated in a humid atmosphere at room temperature and read daily for 7 days. Strong reactions were evident after overnight incubation, while weaker reactions required 2 or 3 days' incubation and intensified for several days.Clinical chemical methods: Serum glutamic pyruvic transaminase (SGPT) was assayed by a kinetic spectrophotometric method with the Gilford multiple method sample recording spectrophotometer.3 Serum lactic dehydrogenase (LDH) enzymes were assayed by the method of Amador et al.4 with the same instrument. Serum LDH...
Genomic RNA from the human prototype strain H of the hepatitis C virus (HCV-H) has been molecularly cloned and sequenced. The HCV-H sequence reported consists of 9416 nucleotides including the 5' and 3' untranslated regions. HCV-H shows 96% amino acid identity with the American isolate HCV-1 but only 84.9% with the Japanese isolates HCV-J and HCV-BK. In addition to the hypervariable region (region V) previously identified in the putative E2 domain, three other variable domains were identified: region V1 (putative El), region V2 (putative E2), and region V3 (putative NS5). These regions appear rather conserved (86-100%) among the American isolates (HCV-1 and HC-J1) or among various Japanese isolates (HCV-J, HCV-BK, HCV-JH, and HC-J4) but show striking heterogeneity when the two subgroups are compared (42-87.5% amino acid difference). A structural similarity between the 5'-terminal hairpin structure of HCV and of poliovirus was observed. This study further suggests the existence of at least two genomic subtypes of HCV and confirms a distant relationship between HCV and pestiviruses.
Hepatitis C virus (HCV) in highly infectious sera has been shown to be predominantly associated with low-density lipoproteins. To determine whether the association is specific to low-density lipoproteins (LDL) or very low-density lipoproteins (VLDL), we fractionated HCV-containing plasma by a column chromatographic procedure known to separate these classes. Hepatitis C virus RNA detected by polymerase chain reaction (PCR) was associated primarily with the very low-density (VLDL) fraction. However, it could not be ruled out that virus-associated LDL may have eluted with this fraction. Hepatitis C virus virions isolated from sera having sufficient titre for visualization by electron microscopy are generally coated with antiviral antibodies, therefore we utilized the lipid association to isolate antibody-free virions. Very low-density lipoproteins were isolated by ultracentrifugal flotation and then treated with deoxycholate to release the virions. These were then isolated in a highly purified form by centrifugation in a sucrose gradient. The 1.10-1.11 g ml-1 region of the gradients contained 60-70 nm particles. Particles with similar surface structure but having a diameter of only 30-40 nm constituted about 30% of the total. The latter may represent defective interfering particles. The identity of both small and large particles with HCV virions and associated particles was confirmed by their trapping on grids by an anti-HCV E2 monoclonal antibody, and by their aggregation by rabbit antiserum to an amino-terminal peptide of E1. Thus, both E1 and E2 epitopes are displayed on the surface of intact HCV virions.
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