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1.
Semin Respir Infect ; 6(1): 3-10, 1991 Mar.
Article in English | MEDLINE | ID: mdl-1887165

ABSTRACT

Each year, influenza infections cause significant morbidity and mortality in spite of intense research and control efforts. Virus is transmitted primarily by aerosolization of respiratory secretions, and infection can be caused by a small number of viral particles. Following inhalation, virus is deposited on the mucous blanket overlying the target epithelial cell. Mucociliary clearance is the normal mechanism by which deposited particulates are removed; immunoglobulins at the airway surface also play an important role in neutralization of virus. Influenza virus is very efficient at overcoming host defenses, primarily due to the function of two surface glycoproteins, hemagglutinin and neuraminidase. Both glycoproteins have antigenic sites that undergo variation. Hemagglutinin also contains cell binding sites, while neuraminidase prevents interference of mucins with cell binding. Although other factors may contribute to infectivity of the influenza virus, antigenic variation of hemagglutinin is the single most important factor. Influenza vaccination is the most effective means presently available to decrease the attack rate and impact of this infection on society.


Subject(s)
Orthomyxoviridae/pathogenicity , Hemagglutinins, Viral/physiology , Humans , Immunity , Influenza, Human/immunology , Influenza, Human/transmission , Neuraminidase/physiology , Orthomyxoviridae/analysis , Orthomyxoviridae/physiology , Pneumonia/microbiology , Pneumonia/physiopathology
2.
Virology ; 177(2): 807-11, 1990 Aug.
Article in English | MEDLINE | ID: mdl-2371783

ABSTRACT

We present evidence that the hemagglutinin (HA) of influenza B virus and the glycoprotein of influenza C virus (HEF) are acylated. The fatty acid linkage is sensitive to treatment with hydroxylamine and mercaptoethanol, which points to a labile thioester-type linkage. The HA of influenza B virus contains mainly palmitic acid, whereas the HEF glycoprotein of influenza C virus is acylated with stearic acid which has not been observed before as the prevailing fatty acid in viral or cellular acyl proteins.


Subject(s)
Fatty Acids/analysis , Gammainfluenzavirus/analysis , Hemagglutinins, Viral/isolation & purification , Influenza B virus/analysis , Orthomyxoviridae/analysis , Viral Envelope Proteins/isolation & purification , Acylation , Amino Acids/metabolism , Animals , Cell Line , Fatty Acids/metabolism , Glucosamine/metabolism , Hemagglutinin Glycoproteins, Influenza Virus , Hydroxylamine , Hydroxylamines , Influenza B virus/metabolism , Gammainfluenzavirus/metabolism , Mercaptoethanol , Myristic Acid , Myristic Acids/analysis , Palmitic Acid , Palmitic Acids/analysis , Stearic Acids/analysis , Viral Envelope Proteins/biosynthesis
3.
Avian Dis ; 33(4): 649-53, 1989.
Article in English | MEDLINE | ID: mdl-2619660

ABSTRACT

A virus adsorption-elution (viradel) procedure was modified and evaluated for the concentration of influenza virus from water. Influent pH, flow rate, eluent pH and composition, and a second-step concentration method were evaluated. The viradel procedure in combination with a chicken erythrocyte adsorption technique resulted in up to 3200-fold concentration of influenza virus from 100 liters of tap water.


Subject(s)
Orthomyxoviridae/isolation & purification , Virology/methods , Water Microbiology , Adsorption , Animals , Hydrogen-Ion Concentration , Orthomyxoviridae/analysis
4.
J Virol ; 63(9): 3622-33, 1989 Sep.
Article in English | MEDLINE | ID: mdl-2668560

ABSTRACT

A panel of 16 monoclonal antibodies recognizing M protein (M1) of influenza virus was generated. Competition analyses resulted in localization of 14 monoclonal antibodies to three antigenic sites. Three monoclonal antibodies localized to site 1B recognized a peptide synthesized to M1 (residues 220 to 236) with enzyme-linked immunosorbent assay titers equivalent to or greater than that seen with purified M1; therefore, site 1B is located near the C terminus of M1. Sites 2 and 3 localize to the N-terminal half of M1. Antigenic variation of M proteins was seen when the monoclonal antibodies were tested against 14 strains of type A influenza viruses. Several monoclonal antibodies showed specific recognition of A/PR/8/34 and A/USSR/90/77 M proteins and little or no reactivity for all other strains tested. Immunofluorescence analysis with the monoclonal antibodies showed migration of M protein to the nucleus during the replicative cycle and demonstrated association of M protein with actin filaments in the cytoplasm. Use of a vaccinia virus recombinant containing the M-protein gene demonstrated migration of M protein to the nucleus in the absence of synthesis of gene products from other influenza virus RNA segments.


Subject(s)
Antibodies, Monoclonal/immunology , Orthomyxoviridae/analysis , Viral Matrix Proteins/immunology , Amino Acid Sequence , Animals , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique , Mice , Mice, Inbred BALB C , Viral Matrix Proteins/analysis
5.
FEBS Lett ; 250(2): 487-92, 1989 Jul 03.
Article in English | MEDLINE | ID: mdl-2753145

ABSTRACT

Fusion of influenza virus with human erythrocytes at pH 5.2 was followed by fluorescence microscopy using a cooled slow-scan CCD camera. The high sensitivity of the CCD permits repetitive digital imaging of the same cells with minimal photobleaching. The experimental conditions were such that only a small number of virus particles were adsorbed per cell. Quantitative analysis of the data indicated that for most cells only a single fusion event took place. This was, however, sufficient to cause haemolysis within 30 min at 20-22 degrees C for about 60% of cells. There was a highly variable time lag between fusion and haemolysis. The lateral diffusion coefficient of virus particles on the cell surface when bound at pH 7.4 was less than 2 x 10(-13) cm2.s-1. The technique should be of value for more detailed studies of the dynamics of viral and other membrane fusion events.


Subject(s)
Erythrocytes/microbiology , Orthomyxoviridae/analysis , Erythrocytes/analysis , Hemolysis , Humans , Microscopy, Fluorescence/instrumentation
6.
Nature ; 333(6172): 426-31, 1988 Jun 02.
Article in English | MEDLINE | ID: mdl-3374584

ABSTRACT

The three-dimensional structures of influenza virus haemagglutinins complexed with cell receptor analogues show sialic acids bound to a pocket of conserved amino acids surrounded by antibody-binding sites. Sialic acid fills the conserved pocket, demonstrating that it is the influenza virus receptor. The proximity of the antibody-binding sites suggests that antibodies neutralize virus infectivity by preventing virus-to-cell binding. The structures suggest approaches to the design of anti-viral drugs that could block attachment of viruses to cells.


Subject(s)
Hemagglutinins, Viral/metabolism , Orthomyxoviridae/analysis , Receptors, Virus/metabolism , Sialic Acids/metabolism , Antibodies/metabolism , Antiviral Agents/metabolism , Binding Sites , Binding Sites, Antibody , Hemagglutinin Glycoproteins, Influenza Virus , Hemagglutinins, Viral/genetics , Models, Molecular , N-Acetylneuraminic Acid , Protein Binding , Protein Conformation , Receptors, Virus/immunology
7.
Virus Res ; 10(2-3): 177-91, 1988 May.
Article in English | MEDLINE | ID: mdl-3414182

ABSTRACT

The spike glycoprotein of influenza C/Johannesburg/1/66 was isolated in a soluble form by digestion of MDCK cell-grown virions with bromelain. The whole ectodomain of the glycoprotein could be recovered with an apparent molecular weight of 75,000 daltons determined in SDS-PAGE. Comparison to Triton X-100-isolated glycoprotein revealed that a C-terminal peptide of 3000-4500 daltons must have remained in the viral membrane. When purified by sucrose density gradient centrifugation the glycoprotein sedimented with a sedimentation coefficient of 10 S, indicating a molecular weight of 206,000 daltons, which is consistent with a trimeric structure of the spike molecule. The trimeric form was stabilized in sucrose gradients by Ca2+ ions. Bromelain digestion of virions with uncleaved glycoprotein, grown in MDCK cells without trypsin, produced two disulphide-linked subunits with similar electrophoretic mobilities in SDS-PAGE to the biologically active glycoprotein. The smaller subunit differed from the product cleaved in vivo (gp 30) by the presence of an additional arginine residue at the N-terminus. The soluble glycoprotein appears to possess both receptor-binding and receptor-destroying enzyme activities, as isolated glycoprotein inhibited hemagglutination of intact influenza C virions and showed RDE activity in an in vitro test. Glycoprotein exposed to low pH, which was sensitive to trypsin digestion, also demonstrated both these biological activities. Glycoprotein-mediated hemolysis could not be observed.


Subject(s)
Bromelains/metabolism , Gammainfluenzavirus/analysis , Glycoproteins/isolation & purification , Orthomyxoviridae/analysis , Viral Proteins/isolation & purification , Amino Acid Sequence , Cell Line , Centrifugation, Density Gradient , Electrophoresis, Polyacrylamide Gel , Glycoproteins/analysis , Gammainfluenzavirus/metabolism , Molecular Sequence Data , Molecular Weight , Trypsin/metabolism , Viral Proteins/analysis , Virion/analysis , Virion/metabolism
8.
J Gen Virol ; 69 ( Pt 4): 839-46, 1988 Apr.
Article in English | MEDLINE | ID: mdl-3356980

ABSTRACT

Of the biological activities of influenza C virus, haemagglutination, receptor inactivation and fusion, only the latter has been conclusively correlated with its surface glycoprotein (gp). We have purified the gp by octylglucoside treatment of influenza C virions followed by centrifugation into a sucrose gradient. Evidence was obtained that gp also represents the receptor-destroying enzyme of influenza C virus, which has been characterized as a neuraminate 9-O-acetylesterase: (i) it inactivated the receptors for influenza C virus on chicken erythrocytes; (ii) it had acetylesterase activity as indicated by the release of acetate from bovine submandibulary mucin; (iii) monoclonal antibodies directed against gp inhibited the acetylesterase activity of influenza C virus. Although purified gp was unable to agglutinate chicken red blood cells, it blocked haemagglutination by viruses. This finding as well as the haemagglutination inhibition activity of monoclonal anti-gp antibodies indicate that gp is also responsible for the haemagglutinating activity of influenza C virus. Thus, as the influenza C glycoprotein is the only myxovirus glycoprotein with three different activities, we propose the designation HEF in order to describe its function as a haemagglutinin (H), an esterase (E) and a fusion factor (F).


Subject(s)
Carboxylic Ester Hydrolases/isolation & purification , Gammainfluenzavirus/analysis , Glycoproteins/physiology , Hemagglutinins, Viral/isolation & purification , Orthomyxoviridae/analysis , Viral Fusion Proteins/isolation & purification , Viral Proteins/physiology , Acetylesterase , Animals , Antibodies, Viral/immunology , Carboxylic Ester Hydrolases/pharmacology , Chickens , Erythrocytes , Glycoproteins/immunology , Glycoproteins/isolation & purification , Gammainfluenzavirus/immunology , Receptors, Virus/drug effects , Viral Proteins/immunology , Viral Proteins/isolation & purification
9.
Biochim Biophys Acta ; 939(3): 476-84, 1988 Apr 22.
Article in English | MEDLINE | ID: mdl-2833310

ABSTRACT

A prerequisite for the purification of any protein to homogeneity is that the protein is not non-specifically associated with other proteins especially during the final stage(s) of the fractionation procedure. This requirement is not so often fulfilled when nonionic detergents (for instance Triton X-100) are used for solubilization of membrane proteins. The reason is that these detergents are not efficient enough to prevent the protein of interest from forming aggregates with other proteins upon contact with chromatographic or electrophoretic supporting media, which, due to their polymeric nature, have a tendency to induce aggregation of other polymers, for instance, hydrophobic proteins. The aggregation can be avoided if sodium dodecyl sulfate (SDS) is employed as detergent. We therefore suggest that membrane proteins should be purified by conventional methods in the presence of SDS and that the purified proteins, which are in a denatured state, are allowed to renature. There is good change to renature internal membrane proteins since they should not be so susceptible to denaturation by detergents as are water-soluble proteins because the natural milieu of the former proteins is lipids which in fact are detergents. In this paper we present a renaturation method based on the removal of SDS by addition of a large excess of G 3707, a nonionic detergent. By this technique we have renatured a 5'-nucleotidase from Acholeplasma laidlawii and a neuraminidase from influenza virus. The enzyme activities were higher (up to 6-fold) after the removal of SDS than prior to the addition of SDS.


Subject(s)
Acholeplasma laidlawii/analysis , Bacterial Proteins/isolation & purification , Membrane Proteins/isolation & purification , Orthomyxoviridae/analysis , Viral Proteins/isolation & purification , 5'-Nucleotidase , Acholeplasma laidlawii/ultrastructure , Cell Fractionation , Cell Membrane/analysis , Cell Membrane/ultrastructure , Neuraminidase/analysis , Nucleotidases/analysis , Protein Denaturation , Sodium Dodecyl Sulfate
11.
J Gen Virol ; 67 ( Pt 12): 2685-94, 1986 Dec.
Article in English | MEDLINE | ID: mdl-3025345

ABSTRACT

The nucleotide sequence of the gene encoding the fusion (F) glycoprotein of the Beaudette C strain of Newcastle disease virus (NDV) has been determined from cDNA clones obtained from virion RNA. The gene is 1792 nucleotides long, including mRNA start and polyadenylation signals typical of paramyxoviruses. The single open reading frame encodes a polypeptide of 553 amino acids, with a predicted molecular weight of 59042. The F polypeptide has three regions of high hydrophobicity: an N-terminal signal peptide, the N terminus of F1 (known from protein sequencing) and a C-terminal membrane-spanning region by which the F glycoprotein is anchored to the membrane. The cleavage site of F0 is located in a highly basic region of the F polypeptide. Five potential asparagine-linked glycosylation sites are present in the amino acid sequence, of which one is in F2 and the others in F1. Comparison of the NDV F amino acid sequence to those from other paramyxoviruses reveals homology to Sendai virus, simian virus 5 and human respiratory syncytial virus. There is also limited homology between the N terminus of F1 of NDV and the N termini of HA2 of influenza viruses. Post-translational modifications of the NDV F polypeptide are discussed in the light of information provided by the amino acid sequence.


Subject(s)
Genes, Viral , Newcastle disease virus/genetics , Viral Fusion Proteins/genetics , Amino Acid Sequence , Base Sequence , DNA , Hemagglutinins, Viral/analysis , Newcastle disease virus/analysis , Orthomyxoviridae/analysis , Orthomyxoviridae/genetics , Parainfluenza Virus 1, Human/analysis , Parainfluenza Virus 1, Human/genetics , Paramyxoviridae/analysis , Paramyxoviridae/genetics , RNA, Viral/genetics , Respiratory Syncytial Viruses/analysis , Respiratory Syncytial Viruses/genetics , Respirovirus/analysis , Respirovirus/genetics , Viral Fusion Proteins/analysis
12.
FEBS Lett ; 207(2): 266-70, 1986 Oct 27.
Article in English | MEDLINE | ID: mdl-2429866

ABSTRACT

Computer analysis of the intrinsic membrane protein, myelin proteolipid, shows strong sequence similarities between the putative extramembrane segments of the proteolipid protein and a number of viral proteins, several of which infect humans. These similarities are even more striking than those reported previously between viral proteins and the encephalitogenic myelin basic protein (MBP). These findings, along with other reports of molecular mimicry by viruses, suggest that immunological cross-reactions between virus-induced antibodies or T-cells and analogous antigenic determinants (epitopes) in myelin proteolipid could be involved in the pathophysiology of multiple sclerosis or post-infectious demyelinating syndromes.


Subject(s)
Myelin Proteins , Viral Proteins , Adenoviridae/analysis , Amino Acid Sequence , Cell Membrane/immunology , Cytoplasm/immunology , Epitopes/immunology , Herpesvirus 4, Human/analysis , Humans , Measles virus/analysis , Myelin Proteins/immunology , Myelin Proteolipid Protein , Myelin Sheath/immunology , Orthomyxoviridae/analysis
14.
Virology ; 149(1): 36-43, 1986 Feb.
Article in English | MEDLINE | ID: mdl-3511607

ABSTRACT

Monoclonal antibodies specific for the hemagglutinin (HA) or the neuraminidase (NA) of influenza viruses were used in immunoelectron microscopic studies to determine the distribution of the two surface spikes on the virion. Indirect immunogold staining revealed that the HA is uniformly distributed on the virion while the NA occurs in discrete areas. Crosslinking and low temperature studies argue against redistribution of the HA and NA after antibody attachment and indicate that the NA on influenza virus occurs in patches.


Subject(s)
Hemagglutinins, Viral/analysis , Influenza A virus/analysis , Neuraminidase/analysis , Orthomyxoviridae/analysis , Virion/analysis , Antibodies, Monoclonal , Gold , Hemagglutinin Glycoproteins, Influenza Virus , Hemagglutinins, Viral/immunology , Immunologic Techniques , Influenza A virus/enzymology , Influenza A virus/immunology , Influenza A virus/ultrastructure , Microscopy, Electron , Models, Biological , Neuraminidase/immunology , Orthomyxoviridae/enzymology , Orthomyxoviridae/immunology , Orthomyxoviridae/ultrastructure , Temperature , Virion/enzymology , Virion/immunology , Virion/ultrastructure
15.
Mol Gen Mikrobiol Virusol ; (11): 36-7, 1985 Nov.
Article in Russian | MEDLINE | ID: mdl-3916213

ABSTRACT

Protein A-containing formaldehyde-fixed S. aureus (strain Cowan) was incubated with an antiviral serum or with a monospecific serum against NP protein, washed, and used as immunosorbent in order to isolate viral ribonucleoproteins (nucleocapsids) containing intact viral RNA from the extracts of influenza virus infected [3H]-uridine-labelled cells.


Subject(s)
Orthomyxoviridae/analysis , RNA, Viral/analysis , Ribonucleoproteins/analysis , Animals , Cell Line , Dogs , Immunosorbent Techniques
16.
Vopr Virusol ; 30(6): 668-71, 1985.
Article in Russian | MEDLINE | ID: mdl-4095974

ABSTRACT

Four strains of influenza C virus were studied by cellulose acetate electrophoresis. All of them were found to contain super-capsid proteins destroyed by electrophoresis apparently due to contact with sodium dodecylsulphate. According to Laver's classification, influenza C viruses may be placed with Group 3 viruses. Among major virion proteins of influenza C viruses matrix protein was found to be the most stable forming a separate protein band on cellulose acetate, and it may be readily identified and eluted from paper in preparative amounts.


Subject(s)
Gammainfluenzavirus/analysis , Orthomyxoviridae/analysis , Viral Proteins/analysis , Electrophoresis, Cellulose Acetate/methods , Electrophoresis, Polyacrylamide Gel , Influenza A virus/analysis , Influenza A virus/drug effects , Influenza A virus/isolation & purification , Influenza B virus/analysis , Influenza B virus/drug effects , Influenza B virus/isolation & purification , Gammainfluenzavirus/drug effects , Gammainfluenzavirus/isolation & purification , Peptides/analysis , Peptides/isolation & purification , Sodium Dodecyl Sulfate/pharmacology , Viral Proteins/isolation & purification
17.
Vaccine ; 3(3 Suppl): 182-8, 1985 Sep.
Article in English | MEDLINE | ID: mdl-4060845

ABSTRACT

The virus-coded proteins and the genomes of influenza C virus isolates obtained from Chinese pigs in 1981-1982 and of human influenza C virus strains isolated between 1947 and 1981 were compared. Using SDS polyacrylamide gel electrophoresis and one-dimensional peptide mapping we found the virus-coded proteins of the pig influenza C viruses to be similar to those of human influenza C virus strains. The sizes of the genomes of human and pig influenza C viruses were indistinguishable. Genome analysis by oligonucleotide (ON) mapping revealed that the genomes of the pig influenza C viruses were very similar to but not identical with those of human influenza C virus strains. ON changes were found scattered over the whole genome. ON mapping of isolated segments of several influenza C virus strains suggested that two pig strains (C/P/B/10/81 and C/P/B/32/81) are related by a reassortment event which is likely to have occurred in nature. The rate of genome variation in influenza C viruses seemed to be similar to that seen in influenza B, and slower than that recorded for influenza A viruses.


Subject(s)
Gammainfluenzavirus/analysis , Orthomyxoviridae/analysis , RNA, Viral/analysis , Viral Proteins/analysis , Animals , Genes, Viral , Genetic Variation , Humans , Gammainfluenzavirus/genetics , Gammainfluenzavirus/isolation & purification , Oligoribonucleotides/isolation & purification , RNA, Viral/genetics , Swine/microbiology , Viral Proteins/genetics
18.
J Virol ; 54(3): 833-43, 1985 Jun.
Article in English | MEDLINE | ID: mdl-2987535

ABSTRACT

The nonstructural NS2 protein of influenza A/PR/8/34 virus was efficiently expressed in bacteria, and monospecific antisera were prepared against the bacterially synthesized polypeptide. These antisera were cross-reactive among the NS2 proteins of various influenza A viruses. However, they did not react with the NS2 of influenza B/Lee/40 virus nor with other proteins of influenza A viruses such as NS1. Antisera against NS2 were used to determine that the NS2 protein is localized in the cell nucleus during influenza virus infection, as shown by immunofluorescence microscopy. Cells infected with simian virus 40 recombinants containing the influenza virus NS gene revealed that both the NS1 and NS2 proteins appeared in the nucleus, even in the absence of expression of other influenza virus-specific components.


Subject(s)
Bacteria/analysis , Orthomyxoviridae/analysis , Viral Proteins/analysis , Animals , Escherichia coli/metabolism , Fluorescent Antibody Technique , Immune Sera/immunology , Plasmids , Rabbits , Simian virus 40/genetics , Viral Nonstructural Proteins , Viral Proteins/genetics , Viral Proteins/immunology
19.
Cell ; 41(1): 269-78, 1985 May.
Article in English | MEDLINE | ID: mdl-2986845

ABSTRACT

The nucleotide sequence of the Sendai virus (SV) HN (hemagglutinin-neuraminidase) gene was determined. The deduced primary structure of the protein showed only one hydrophobic domain likely to represent the transmembrane region, but at its N terminus. Since the SV F protein is anchored in the membrane at its C terminus, the two SV glycoproteins are thus membrane-anchored in opposite orientations, similar to the two influenza virus (FLU) glycoproteins. Amino acid sequence comparisons of the SV HN and the FLU HA and NA proteins revealed homologies between 100 amino acids of the hemagglutinin region of the FLU HA protein and the C terminus of the SV HN, and between 200 amino acids of the neuraminidase region of the FLU NA and the central region of SV HN. Alignment of the neuraminidase, hemagglutinin, and fusion regions shared by these glycoproteins suggest the structure of a possible ancestral gene.


Subject(s)
Hemagglutinins, Viral/genetics , Neuraminidase/genetics , Orthomyxoviridae/genetics , Parainfluenza Virus 1, Human/genetics , Viral Proteins/genetics , Amino Acid Sequence , Base Sequence , Biological Evolution , Biological Transport , Cell Membrane/metabolism , Cloning, Molecular , Genes, Viral , HN Protein , Hemagglutinin Glycoproteins, Influenza Virus , Hemagglutinins, Viral/analysis , Hemagglutinins, Viral/metabolism , Kinetics , Neuraminidase/analysis , Neuraminidase/metabolism , Orthomyxoviridae/analysis , Viral Envelope Proteins/metabolism , Viral Fusion Proteins , Viral Proteins/analysis , Viral Proteins/metabolism
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