Luisa Bertolini

Ultrastructural observations of viral particles within hepatitis C virus-infected human B lymphoblastoid cell line

Hepatitis C virus (HCV)-infected TOFE cells, a human bone marrow-derived B-cell line, were studied by transmission electron microscopy (TEM). We reported the presence of virus-like particles, (VLPs) having a diameter of 45 nm on average, within the cytoplasm of HCV-infected cells and their absence in uninfected cells. The VLPs were mostly seen in dilated cisternae of endoplasmic reticulum (ER) and consisted of an electron-dense core and a membrane envelope with surface projections. In the HCV-infected TOFE cells, examination by TEM revealed tubular structures similar to those observed in liver cells of chimpanzees and humans infected by the virus. In some instances, the outer membrane of the particles was connected to the ER membrane, indicating possible virus budding into the cisternae. Rarely, coated vesicles containing a particle were seen. In most of the infected cells, enlarged cytoplasmic vacuoles filled with degenerative amorphous material were observed. The data suggest a flavivirus-like pattern of HCV morphogenesis in infected non-hepatic cells and identify lymphoblastoid TOFE cells as a valuable system for the study of the sequential steps of the infection process and the morphological effects produced by HCV infection.

Detection of virus-like particles in liver biopsies from HCV-infected patients

In order to directly ascertain the presence of HCV virus infection in livers of patients with HCV chronic hepatitis, we investigated, by transmission electron microscopy (TEM), liver biopsies from 2 adults and 4 children for the presence of virus-like particles (VLPs). The plasmas of these HCV-positive patients were HCV-RNA-positive, with high ALT values. In liver tissue samples examined, we were able to detect plus and minus strands of HCV RNA by strand-specific RT-PCR. Aggregates or single VLPs of about 45 nm in diameter were detectable in variable amounts in endoplasmic cisternae and in hepatocyte cytoplasms of infected patients. These results emphasize the relevance of performing TEM assays to confirm the diagnosis of HCV infection.

Morphological modifications induced by HCV infection in the TOFE human lymphoblastoid cell line

In this study, we analysed by transmission electron microscopy (TEM), sequential details of morphological modifications that accompanied viral morphogenesis in the lymphoblastoid cell line (LCL) TOFE infected in vitro with hepatitis C virus (HCV). As previously reported, we observed virus-like particles (VLPs) in cytoplasmic vesicles mainly located in the perinuclear region of infected cells. In this area, the Golgi apparatus and the endoplasmic reticulum (ER) appeared hyperplastic, remarkably enriched in vesicles and lysosomal structures. Furthermore, only in this perinuclear region, cytopathic-effect(CPE)-like changes seemed to originate, consisting in enlarged cytoplasmic vacuoles filled with degenerative amorphous material containing VLPs. Finally, the complete filling-up of the cytoplasm with these degenerative vacuoles, in addition to cellular lysis displayed by some cells, appeared as the possible terminal pattern of the infectious process. Our data suggest that in vitro HCV-infected TOFE cells undergo typical CPE-like changes that may be connected with virus replication.

Variations in the response of cloned murine friend erythroleukemia cells to different inducers

SummaryCells of the line 3BM-78 derived from murine bone marrow cells infected in vitro with polycythemic Friend leukemia virus (FLV-P) produce virus with spleen focus-forming activity (SFFV) and can be induced to synthesize hemoglobin. Fifteen clones, isolated from this line, have been analyzed in detail for the effect of different inducing agents (dimethylsulfoxide, DMSO; hexamethylene bisacetamide, HMBA; and sodium butyrate, SB) on the synthesis of hemoglobin and virus at the clonal level. All the clones proved to be inducible with one or more of the agents, but the degree of the response depended on the type and concentration of the agent used. In general, the effectiveness of the agent—within the usual range of concentration for induction—both for hemoglobin and for virus synthesis, was in the order HMBA>DMSO>SB. Reverse transcriptase activity was, however, more easily induced than hemoglobin synthesis in that stimulation was seen at lower concentrations of the same inducing agent. This clonal analysis confirmed that virus and hemoglobin production are regulated independently in these erythroleukemic cells chronically infected with FLV-P.

Antibody formation and transient immune complex glomerulopathy in A-Strain mice with C1300 neuroblastoma tumors

SummaryOne to three-month-old A-strain mice, inoculated subcutaneously with 2 × 106 viable syngeneic C1300 neuroblastoma cells (clone NB9R) developed a palpable tumor within 9–12 days and died within 28–30 days. A transient glomerulopathy developed after 16–24 days. Despite a normal histologic appearance, the nephropathy was clearly demonstrated by electron microscopy and was classified as a focal mesangiopathic glomerulonephritis. Deposits of host 7S-G immunoglobulins and C3 complement fragments were detected in these same kidneys by immunofluorescence. Radioimmunoprecipitin determinations on sera obtained from mice at different intervals from tumor cell inoculation, revealed that untreated mice contained circulating antibodies capable of reacting with125I-labeled gp69–71 glycoprotein from Gross murine leukemia virus (MuLV). Antibodies to p30 MuLV antigen and to crude membrane antigen (s) (CMA) solubilized from NB9R cells were found in sera only after tumor cell inoculation. Circulating immune complexes formed by host 7S-G immunoglobulins were clearly detected from day 16 to 22. Antibodies eluted from kidneys with nephropathy were shown to react with NB9R cells in vitro and to react specifically with CMA and the p30 MuLV antigen.

Differences between murine C1300 neuroblastoma clones detected by rosette formation with nerve growth factor-coated sheep red blood cells.

The expression of receptors for nerve growth factor (NGF) on the cell surface was assayed by rosette formation with ligand-coated sheep red blood cells (SRBC). Cell clones derived from the murine C1300 neuroblastoma and from hybrids between a neuroblastoma clone and L cell clones showed a wide variation in the capacity to form rosettes with NGF-coated SRBC. All the neuroblastoma, L cell and hybrid clones formed rosettes with phytohemagglutinin-coated SRBC and none formed rosettes with cytochrome c- or ferritin-coated SRBC or with SRBC not coated with ligand.

TOFE human-B-cell-line-based adsorption-inhibition assay to detect HCV neutralizing antibodies

We previously demonstrated that the human lymphoblastoid B-cell line (LCL) TOFE, derived from normal human bone marrow, is permissive to HCV infection. In this report we developed an in vitro HCV adsorption-inhibition assay based on TOFE cells, to reveal the presence of neutralizing antibodies in sera from acutely infected patients.

Susceptibility of human lymphoblastoid B-cell line CE to HIV1 infection

In this preliminary report, we provide evidence that the human B-lymphoblastoid cell line (LCL) CE, bone-marrow-derived, previously reported to be permissive to hepatitis C virus, is also permissive to HIV1 infection. HIV1 genomes were detectable in cell supernatants, virus RNA transcripts and proviral DNAs in cell extracts at different times post-infection. Therefore, we propose this LCL cell line as a tool for exploring the mutual interactions of the two viruses in double-infected cells.