title: IgG neutralization of type A influenza viruses and the inhibition of the endosomal fusion stage of the infectious pathway in BHK cells.
abstract: We have used three strains of type A influenza virus (A/fowl plaque virus/Rostock/34: H7N1; A/PR/8/34: H1N1; A/X31: H3N2) labeled with the fluorescent probe octadecyl rhodamine B chloride (R18) to study how neutralizing monoclonal IgG, specific for the haemagglutinin, affects the interaction of virus with BHK-21 cells. R18 labels viral lipid and self-quenches in the virion; dilution of R18 by fusion of viral and cellular membranes or by dissolution in detergent activates fluorescence. IgG neutralization reduced attachment to BHK-21 cells to an extent which depended on the virus-antibody concentration, but in no case was the inhibition of virus attachment to cells sufficient to fully account for the amount of neutralization obtained. The majority (> 78%) of attached IgG-neutralized virus became resistant to release by neuraminidase which indicated it was internalized by the cell. The endosomal fusion ability of IgG-neutralized virus which became cell-associated was also inhibited, to an extent which depended on the virus-antibody permutation. Inhibition of endosomal fusion, which is responsible for primary uncoating, was corroborated by immunofluorescence studies which showed that the NP antigen of IgG-neutralized virus, unlike that of infectious virus, did not accumulate in the nucleus. The loss of infectivity attributable to the combined inhibition of attachment and inhibition of fusion was sufficient to account for the extent of neutralization caused by relatively low concentrations of IgG (1/e or 63% neutralization). However it could not, with two possible exceptions, account for the neutralization resulting from higher concentrations of IgG (sufficient to cause > 99.9% neutralization), and we conclude that at least one other mechanism is operating simultaneously, such as that described by Rigg et al., (1989, J. Gen. Virol. 70, 2097-2109).
authors.affiliation: Department of Biological Sciences, University of Warwick, Coventry, United Kingdom.
authors.forename: M C
authors.initials: MC
authors.lastname: Outlaw
authors.forename: N J
authors.initials: NJ
authors.lastname: Dimmock
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology*; D000956:Antigens, Viral / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D004705:Endocytosis; D005455:Fluorescent Antibody Technique; D007074:Immunoglobulin G / Q000276:immunology*; D009980:Influenza A virus / Q000276:immunology* / Q000502:physiology; D008561:Membrane Fusion; D051379:Mice; D009500:Neutralization Tests; D014779:Virus Replication
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; D007074:Immunoglobulin G