Virus-like particles have been visualized by electron microscopy in the liver hepatocytes of two out of four chimpanzees inoculated with two different human non-A, non-B hepatitis-implicated plasmas. The first two chimpanzees, which were infected with the same inoculum (inoculum A), developed liver histological features characteristic of mild acute viral hepatitis as well as rises in their serum alanine aminotransferase levels. Clusters of intranuclear particles, 22 k 2 nm in diameter, were visualized in one of every fifth hepatocytes in one of these two chimpanzees. Intracytoplasmic circular membrane fusions, 100 t o 350 nm in diameter, were observed in the hepatocytes of the other chimpanzee receiving inoculum A.The second two animals were infected with a different human plasma containing infectious NANB material (inoculum B). Both animals developed mild increased serum enzyme levels that were variable but generally elevated throughout the course of this study (220 days); minor, equivocal histologic changes were also observed. Intranuclear and intracytoplasmic particles, 20 + 1 nm and 37 nm in diameter, respectively, were visualized in the liver of one of the two chimpanzees infected with inoculum B; intranuclear particles were observed in one of seventy-five hepatocytes and intracytoplasmic particles were observed less frequently. In this same animal, intracytoplasmic circular membrane fusions, similar, if not identical to those described above, were also noted. The intranuclear particles observed in animals receiving inocula A and B were essentially identical, as were the cytoplasmic membrane fusions. Thus, both inocula produced nuclear and cytoplasmic ultrastructural changes. The intracytoplasmic particle clusters (37 nm) were seen in only one animal and demonstrated a high degree of homogeneity, as evidenced by their presence in highly ordered crystalline arrays. All chimpanzees were screened for and found to be negative for evidence of hepatitis A and B, as well as for Epstein-Barr virus or cytomegalo- 2 Burketal virus-induced hepatitis. Previous studies have suggested that nuclear and cytoplasmic ultrastructural changes, similar to those described herein, result from two distinct NANB infectious agents. In contrast, our data suggest that both morphologic changes may be caused by a single agent.
Liver wedge biopsies were obtained from chimpanzees during the acute phase of experimental non-A, non-B hepatitis infections. Primary chimpanzee hepatocytes were maintained for over 4 weeks in vitro with a serum-free medium supplemented with growth factors and hormones. The de novo synthesis and secretion of plasma proteins characteristic for differentiated primate hepatocytes were sustained under these culture conditions. Immunocytochemical staining for a non-A, non-B hepatitis-associated antigen revealed expression of this cytoplasmic marker during the culture period, indicating a persistence of the infection in vitro. Tissue culture medium derived from the hepatocyte cultures was used to inoculate a nonimmune chimpanzee. The animal subsequently displayed an increase in the serum levels of alanine aminotransferase, the development of histopathologic alterations indicative of viral hepatitis, and the appearance of liver cell cytoplasmic tubules diagnostic for non-A, non-B hepatitis. Concentrated tissue culture medium examined by electron microscopy contained virus-like particles with an average diameter of 39-46 nm, which exhibited an envelope and inner 37-nm core structure.
Monolayer cultures of ARH‐77 cells, a human myeloma cell line propagated in vitro, display a variety of morphologic entities ranging from small lymphocytes to classic plasma cells. The cells show intense pyronin and periodic acid‐Schiff affinity but are negative for colloidal iron, sudan black, and naphtol AS‐D chloroacetate esterase. The cells exhibit phenotypic markers pertaining to each stage of the B‐cell lineage. They fail to display sheep erythrocyte and bovine erythrocyte—IgG antibody complex rosettes, common acute lymphocytic leukemia (ALL) antigens and T‐cell antigens, but most cells display surface complement receptors, Ia‐like antigens, and surface and intracytoplasmic Ig. Monoclonal antibodies were negative for T‐antigens, myelomonocytic cell antigens, leukemia‐associated antigens, and BA‐1 and OKT‐10 antigens. However, 100% of the cells were positive with OKT‐9 and B3/25 antibodies that are specific for transferrin receptors. About 50% to 80% of the cells were positive for surface membrane immunoglobulin (kIgG) and about 10% to 50% for cytoplasmic immunoglobulin (kIgG). Virtually all cells were positive when tested for nuclear Epstein‐Barr virus antigens.
The hepatitis B virus core antigen, including the precore sequence (HBcAg-p25), was expressed at very high levels in bacteria. Three expression vectors were constructed in which the synthesis of HBcAg-p25 was controlled by the tac promoter, and the number of nucleotides between the bacterial ribosome binding site and the precore initiation codon was varied in order to maximize HBcAg-p25 synthesis. The relative amount of HBcAg-p25 polypeptide expressed by the different vectors was estimated by SDS-polyacrylamide gel electrophoresis and immunoblot. HBcAg-p25 was associated with an insoluble fraction of bacterial extracts and required ionic detergents for solubilization. Comparison by ELISA of the immunoreactivity of HBcAg with and without the precore sequence suggested that human anti-HBcAg IgG preferentially recognizes HBcAg lacking the precore sequence.
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