Biomedical subjects
Publications and source records attributed to F Superti.
The effect of a mixed poliovirus-rotavirus infection in HT-29 cells, a gut tumour derived cell line highly susceptible to both viruses, has been analyzed. The obtained results showed an increase of poliovirus multiplication in cells super-infected or co-infected with rotavirus, whereas the pre-infection with poliovirus had an interfering effect on rotavirus replication.
We investigated the effect of different polyions on the early phases of SA-11 rotavirus infection in susceptible LLC-MK2 cells in order to clarify the influence of electrostatic interactions in rotavirus binding to cell membranes and to select antiviral compounds able to prevent viral attachment. When added during the viral attachment step, polymers having positive charge (protamine, protamine sulphate, DEAE-dextran, histone and poly-L-lysine hydrobromide) enhanced virus infection whereas those having negative charge (mucin, heparin, heparan sulphate, alpha-1-acid glycoprotein and dextran sulphate) inhibited the viral replication. The effect of polyanions on SA-11 rotavirus and on cell membrane receptors has also been examined. Results obtained indicated that while mucin and alpha-1-acid glycoprotein act directly on virus particles, the target of heparin, heparan sulphate and dextran sulphate is the host cell membrane.
During the period May 1987-January 1989, faecal samples from 417 paediatric inpatients admitted to the main paediatric hospital in Rome were screened by direct electron microscopy and rotavirus enzyme-linked immunosorbent assay. Rotaviruses were detected in 18.2% of cases and adenoviruses in 7%, whereas astroviruses were found in 1% of cases. Different percentages of rotavirus excretors were revealed by enzyme-linked immunosorbent assay and electron microscopy. This discrepancy seems to be due to false positive results introduced by enzyme-linked immunosorbent assay. Analysis of electron microscopy-positive samples by rotaviral RNA polyacrylamide gel electrophoresis showed different electropherotypes of rotavirus among which a single, largely predominant long electropherotype (55.4%) was revealed. Short electropherotype subgroup I rotaviruses were demonstrated in about 10.7% of samples.
Astroviruses are intestinal pathogens associated with gastroenteritis in man and animals. The mechanism of internalization into host cells has not been reported previously. The cell entry pathway of serotype 1 human astrovirus into 293 cell line was studied biochemically and morphologically. Viral infection was monitored by indirect immunofluorescence. Infected cells were treated with the lysosomotropic agents ammonium chloride, methylamine, and dansylcadaverine or the ionophore monensin to raise the intraendosomal and intralysosomal pH. All drugs tested inhibited the early stages of infection whereas they did not interfere with the viral binding to the plasma membrane. The presence of astrovirus particles was detected by electron microscopy in coated pits and later in coated vesicles. The data indicate adsorptive endocytosis as the most probable mechanism by which astroviruses enter susceptible cells.
An investigation of SA-11 rotavirus binding to human serum lipoproteins was carried out. Various subclasses of lipoproteins, purified by ultracentrifugal flotation, and apoproteins were tested for their activity in inhibiting viral infectivity and hemagglutination. All tested lipoprotein subclasses (very low, low and high density, lipoproteins; VLDL, LDL, HDL, HDL1) were shown to interact with SA-11 rotavirus: VLDL and LDL were the most active in preventing rotavirus replication, whereas HDL and HDL1 inhibited viral hemagglutination to a greater extent. Moreover, A1 and A2 apoproteins were effective towards both viral infectivity and hemagglutination. Results obtained are in agreement with a preferential interaction of VP7 or VP4 proteolytic products with low density lipoproteins and of VP8* with high density lipoproteins. Binding of SA-11 to lipoproteins or apoproteins was also quantified by an enzyme-linked immunosorbent assay procedure and lipoproteins-virus interaction was visualized by electron microscopy.
Susceptibilities of a continuous rhesus monkey kidney cell line (MA-104) and that of a human colon carcinoma cell line (HT-29) to infection by different human and animal rotavirus strains were compared. HT-29 cells appeared to be more sensitive to human rotavirus infection than MA-104 cells, whereas the latter cell line was more susceptible to animal rotavirus replication. The greater sensitivity to human rotavirus infection of HT-29 cells was confirmed by the successful, direct isolation of these viruses from faecal specimens. Human rotavirus infection of HT-29 cells was also followed by transmission electron microscopy. In ultra-thin sections, unenveloped particles of rotaviruses, representing infectious mature virions, were observed in large number. Moreover, many "double-shelled" particles were detected in negative-stained supernatants from infected cultures. Scanning electron microscopy of uninfected HT-29 cells showed that in the presence of Ca++, required for rotavirus growth, they are able to express some of the features of mature intestinal cells. In view of these results, HT-29 cells appear to be a useful in vitro model for the study of rotavirus infection.
Rabies virus is able to infect in vitro a wide range of homeothermic and poikilothermic cells but little is known about its multiplication in arthropod cells. In this research the infection of rabies virus in Aedes pseudoscutellaris cells, a mosquito cell line susceptible to mosquito-borne viruses, was studied. After 60 days of incubation at 26 degrees C up to 70% of infected cells showed the synthesis of both viral nucleocapsid and envelope antigens, although viral yield and cell damage could not be detected. Research performed in order to investigate the role of membrane carbohydrate moieties in rabies virus-mosquito cell interaction suggested the participation of galactose and N-acetylglucosamine whereas sialic acid, known to be a rabies binding site in many homeothermic cell lines, was not involved.
The chemical nature of receptors involved in the attachment of simian rotavirus (SA-11) to a monkey kidney cell line (LLC-MK2) was investigated. Enzymic treatment of cells before virus infection indicated that membrane proteins and phospholipids are not involved in virus attachment, whereas sialic acid and galactose participate in the receptor structure to differing extents. Incubation of SA-11 with bovine brain gangliosides before infection strongly reduced its ability to bind to cell membranes. Similar experiments with individual purified gangliosides from bovine brain showed that virus infection was prevented by preincubation with GM1. Moreover, desialylated cells regained susceptibility to virus infection when coated with whole gangliosides or GM1 immediately after Clostridium perfringens neuraminidase treatment. The binding of SA-11 to whole gangliosides or GM1 was quantified by an ELISA procedure. The results suggest that gangliosides, mainly GM1, are part of the receptor structure for SA-11 of susceptible LLC-MK2 cells.
In the present work the non-antibody inhibitory activity of human serum towards SA-11 rotavirus hemagglutination (HA) has been studied. Among human serum protein components, inhibitory activity was recovered in fractions III (beta-globulin), IV (alpha-globulin) and IV (glycoprotein). Additional experiments performed with purified lipoprotein sub-classes and their lipid and apolipoprotein moieties demonstrated the ability of HDL and, HDL1 to prevent SA-11 HA.
The effect in vitro of some cytoplasmic structure and function inhibitors on the different stages of rabies virus infection was investigated. Treatment of fibroblasts (CER) and human neuroblastoma cells (IMR-32) with substances acting on low pH intracellular compartments (methylamine and monensin) prevented rabies virus genome delivery in the cytosol. An early inhibition of viral infection was also obtained in the presence of B and D cytochalasins and trifluoperazine which interact with microfilament structures. Treatment with colchicine and vinblastine did not affect rabies multiplication, suggesting that microtubules are not involved in this process. However, the multiplication of prebound virions did not take place in the presence of inhibitors of oxidative phosphorylation (sodium azide and CCCP) and of glycolysis (2-deoxy-D-glucose) indicating that rabies virus replication is largely energy-dependent in both host cells examined.
In this study we investigated the activity of halogeno-, cyano- and amidino-isoflavenes, isoflavans and flavans on the multiplication of human astroviruses. These are naked small round viruses which have been recognized as causative agents of human gastroenteritis, and whose capsid proteins are similar to those of picornaviruses. Although all drugs tested caused a dose-dependent reduction of viral antigen synthesis as monitored by immunofluorescence, the chloro derivatives were the most effective.
The relationship between plasma membrane receptor organization and cell susceptibility in vitro was investigated in mammalian, avian, fish, and arthropod cell lines infected with fixed rabies virus. IMR32, HeLa, CER, and EPC cells were widely susceptible to infection with CVS virus, whereas a lower level of specific viral antigens was detectable in A. albopictus cells. In spite of these differences, the amount of infectious virus particles bound to the various cell surfaces was similar. Competition experiments carried out with plasma membranes extracted from ability of these components to bind the virus and to prevent infection. The different cellular permissiveness to rabies infection described here did not correlate with significant differences in number or in chemical structure of the receptor binding sites, but more likely with events following virus adsorption.
The effect of some cellular function inhibitors on hepatitis A virus (HAV) adsorption and on the successive events of infection in a monkey cell line (Frp/3 cells) was investigated. Treatments of Frp/3 cells with colcemide, vinblastine and cytochalasin D, which affect cytoskeleton organization, indicated that neither microtubules nor microfilaments play an important role in the early events of HAV infection. Monensin, which acts as an ionophore on intracellular vesicle compartments inhibited HAV infection probably at the uncoating step. Inhibition of viral replication to a different degree was observed with both inhibitors of oxidative phosphorylation, such as dinitrophenol and sodium azide, and with an inhibitor of glycolysis, 2-deoxy-D-glucose. However, none of these compounds significantly affected the early steps of infection, thus demonstrating that HAV replication is largely dependent upon cell energy.
The involvement of lipid components in hepatitis A virus (HAV) attachment to host cells has been investigated. Isolated Frp/3 cell membranes and whole lipids, phospholipid and glycolipid fractions extracted from them were able to bind the virus and to prevent infection. Treatment of virus with various phospholipids or glycolipids demonstrated the participation of phosphatidylserine, phosphatidylethanolamine and galactose in HAV binding. Results obtained added further information on the receptor specificity of host cells for HAV.
Lactose-negative Escherichia coli strains were isolated at high frequency from children with diarrhea in Somalia during a 2-year study on diarrheal diseases. Sixty-four of these strains, considered to be a representative sample, were characterized for virulence factors, plasmid profiles, and antibiotic resistance. Of these strains, 5 were recognized as enteroinvasive E. coli (they were serotyped as O135:K-:H-), 6 belonged to classical enteropathogenic E. coli serotypes, 9 were able to adhere to tissue culture cells (of these, 4 showed a pattern of localized adherence and 1 was an enteropathogenic strain), 18 were both adherent and hemolytic, and 8 were simply hemolytic. None hybridized with 32P-labeled heat-labile or heat-stable (a and b) enterotoxin gene probes or produced moderate or high-level cytotoxic effects on HeLa cells. Of the 64 strains examined, 24 produced mannose-resistant hemagglutination with human, chicken, and monkey erythrocytes. One of these was serotyped as O4:K-:H8, and a rabbit O antiserum raised against this strain allowed us to establish that 23 strains had the same O antigen. The 23 O4 strains were hemolytic and were not enterotoxic for rabbit ileal loops, and intact bacteria were able to destroy tissue culture cell monolayers very rapidly. The uniformity of the antibiotic resistance pattern and of the plasmid DNA content, together with the fact that they were isolated in different years and in different children, suggests that the O4 strains must be epidemiologically relevant in Somalia. A possible diarrheagenic role for the adherent-hemolytic E. coli strains is also discussed.
The effect of some cellular function inhibitors on adsorption and successive events of penetration of vesicular stomatitis virus to phylogenetically unrelated permissive cells was investigated. Treatment of HeLa, CER, EPC and Aedes albopictus cells with colcemide and cytochalasin D which affect cytoskeleton organization indicated that microfilaments but not microtubules were involved in the early events of VSV infection. Inhibitors of oxidative phosphorylation such as dinitrophenol and sodium azide, and the glycolysis inhibitor 2-deoxy-D-glucose, did not allow multiplication of prebound virions, demonstrating that VSV replication is largely energy dependent in either host cells examined.
Research has been carried out in order to clarify the chemical nature of cell receptors interacting with a fast growing strain of hepatitis A virus (HAV) producing a cytopathic effect on Frp/3 cells. Cell surface susceptibility to HAV attachment has been studied after treatment with enzymes acting on different chemical groupings. Results obtained showed a lowering of cell susceptibility to HAV infection following the action of phospholipase A2, phospholipase C, trypsin and beta-galactosidase. These data suggested that phospholipids, proteins and galactose participate to the cellular receptorial area for HAV.
Gangliosides from different cells were tested for their inhibiting activity on VSV attachment to CER cells and goose erythrocytes. This inhibiting activity was enhanced when gangliosides were inserted into liposomes, in particular if containing phosphatidylserine.