Richard T.C. Huang

Influenza viruses cause hemolysis and fusion of cells.

Abstract Influenza viruses have been found to cause extensive hemolysis and fusion of cells. It can be shown that an optimal pH exists for each virus strain at which hemolysis and fusion activities become most apparent and for these activities proteolytic activation of hemagglutinin is required. Furthermore, it can be demonstrated that the hemolytic activity differs among virus strains and that under optimal conditions influenza viruses hemolyse more efficiently than paramyxoviruses.

Fusion between cell membrane and liposomes containing the glycoproteins of influenza virus

Abstract Glycoproteins of influenza virus strains were incorporated into liposomes by a dialysis procedure, using octylglucoside as detergent. Liposomes containing either the cleaved or uncleaved hemagglutinin of the virus were tested for fusion activity with cellular membranes. Electron microscopic examination as well as microinjection studies revealed that liposomes containing the cleaved hemagglutinin could fuse with cell membranes. In contrast, liposomes containing the uncleaved hemagglutinin were merely adsorbed to the cell surface and fusion occurred only after treatment with trypsin. Native virus particles with the cleaved hemagglutinin could be shown to fuse with liposomes containing cellular receptors of influenza virus. From these results and the known correlation existing between cleavage of hemagglutinin and infectivity of influenza virus, it is suggested that fusion may be an important step in penetration of the nucleocapsid of influenza virus into host cells.

The function of the neuraminidase in membrane fusion induced by myxoviruses

Abstract Interaction of cell membranes with liposomes containing the glycoproteins of myxoviruses (ortho- and paramyxoviruses) was investigated. Liposomes containing the individual or mixed viral glycoproteins were reacted with chicken embryo cells and observed for fusion by electron microscopy. It was found that the presence of neuraminidase is essential for fusion between the liposomes and cells. A hypothesis explaining the cooperative role of neuraminidase for myxovirus-induced membrane fusion is presented.

Association of the envelope glycoproteins of influenza virus with liposomes—a model study on viral envelope assembly☆

Abstract Envelope glycoproteins of virus N (an avian influenza virus) were artificially associated with liposomes, using octylglucoside as detergent which could be readily removed by dialysis. The prepared liposomes, about 0.1-1 μm in diameter, contained specific hemagglutinin and neuraminidase activities. The total amounts of proteins incorporated into liposomes were about the same irrespective of the lipid composition. Similarly, the neuraminidase activity of liposomes remained unchanged by altering lipids. However, the hemagglutinating and cell-adsorbing properties of liposomes varied greatly depending on the composition of lipids. As both lipid and protein components can be altered, the present system may be elaborated for the study of protein-lipid interactions and functions of viral membranes.

Isolation and characterization of the gangliosides butter milk

Abstract Approx. 1–2% of the total lipid content of butter milk is ganglioside, consisting of neuraminosyl lactosyl ceramide (hematoside) and dineuraminosyl lactosyl ceramide (20% and 50% of the total ganglioside, respectively), and at least three other unidentified components. The structural analysis was performed by periodate oxidation and gas chromatography. The positions of attachment of neuraminic acid were further confirmed by the neuraminidase action of the fowl plague and Newcastle disease viruses. The fatty acids of the gangliosides were mainly C 16:0 , C 18:0 , C 18:0 and smaller amounts of C 14 to C 20 acids, resembling those of the milk glycerophosphatides. Preliminary studies of the sphingosines have shown that they are C 14 to C 18 normal and branched bases.

Further Studies on the Role of Neuraminidase and the Mechanism of Low pH Dependence in Influenza Virus-induced Membrane Fusion

The role of neuraminidase and the mechanism of low pH dependence in influenza virus-induced membrane fusion have been studied further using fowl plague virus (FPV, H7N1). Two specific anti-FPV neuraminidase antisera obtained from chickens immunized with recombinant virus strains inhibited viral neuraminidase activity without influencing its haemagglutinating activity. These sera totally inhibited the FPV-induced fusion of erythrocytes and partially reduced haemolysis. But both fusion and haemolysis activities could be restored by external addition of Vibrio cholerae neuraminidase, indicating participation of neuraminidase in FPV-induced membrane fusion. With regard to low pH-dependent fusion by influenza virus, it was found that erythrocytes of various species showed different pH optima for haemolysis by FPV and that erythrocytes could be sensitized for fusion and haemolysis by FPV at neutral pH if they had been pretreated with a low pH buffer. These results demonstrated that surface properties of erythrocytes rather than that of the virus are critical in the low pH-dependent fusion and haemolysis by influenza viruses.

Antiviral activity of some natural and synthetic sugar analogues

A number of natural and synthetic sugar analogues have been tested for their antiviral activity, using an influenza virus strain as a model. Hemagglutinating titres (HA) and cytopathic effect (CPE) were surveyed to estimate the virus production. It was found that introduction of the benzyl group into these sugars generally causes them to become antivirally active. Substitution with methyl, acetyl, uridyl and thiocyanyl groups or derivatization with azido, isopropylidene and benzylidene groups were without effect. All sugars containing the 2‐deoxy‐2‐acetamido group were inactive.

Sphingolipids of influenza viruses

Total lipid of four egg grown influenza viruses (A2-Asia, A2-England, A2-Taiwan and fowl plague virus) were extracted with chloroform-methanol. After mild alkali treatment of the extracts, glycosphingolipids and sphingomyelin were separated by a silicic acid column, and finally purified by thin layer chromatography. Fatty acid, sphingosine and carbohydrate components of individual lipid classes were then analysed by gas-liquid chromatography. Nearly identical results were obtained with all viruses investigated. Approximately 20% of the total lipid was monohexosylceramide, distributed equally between glucosyl- and galactosyl- analogues. Lactosylceramide and oligohexosylceramides were found in much smaller concentrations (approx. 2%). About 15% of the total lipid was attributed to sphingomyelin. A large proportion of fatty acids (around 25% in sphingomyelin and 60% in glycolipids) belonged to the long chain (C19-C26) normal- and 2-hydroxy series. C18-sphingosine was found to be the only base present in all lipid classes investigated.

Cytotoxic T cell lysis of target cells fused with liposomes containing influenza virus haemagglutinin and neuraminidase.

The lytic activity of secondary cytotoxic lymphocytes against influenza A virus was tested on cells which had been fused with liposomes containing the haemagglutinin and the neuraminidase of an avian influenza A virus (fowl plague virus, FPV). Fusion was obtained solely by the activity of the haemagglutinin and neuraminidase incorporated into the liposomes, without the need for any additional fusion factor. Highly reproducible lysis of these FPV-liposome target cells by influenza A-specific cytotoxic cells was found. In contrast, target cells containing the glycoproteins HN and F of Newcastle disease virus (NDV) were not lysed. In almost all experiments effector cell populations capable of lysing target cells also lysed the natural killer cell (NK)-sensitive cell line YAC-1. However, high NK activity alone was not sufficient to lyse target cells fused with liposomes containing the viral surface glycoproteins. To our knowledge this is the first report where after artificial introduction of viral surface components into cell membranes (either by fusion or by transfection) lysis of target cells was monitored also for non-specific lysis mediated by NK-like cells. Both the H-2 restriction and the virus specificity of lysis of FPV-liposome target cells indicate that influenza virus haemagglutinin and possibly neuraminidase do function as target antigens for influenza-specific T cells.

Productive infection of chick embryo cells by influenza viruses tightly bound on substratum.

To test whether penetration of influenza viruses could occur at the plasma membrane of host cells, virus particles were tightly bound on Concanavalin A-coated substratum of plastic culture plates and then overlayed with embryo cells.Under these conditions, endocytosis of the viruses was prevented but the cells were found to be effectively infected. The results indicate, that infection by influenza viruses can occur through fusion between the viral membrane and the host cell plasma membrane.