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R G Webster

Publications and source records attributed to R G Webster.

At least 253 records · Page 14Linked to original sources
PubMed1987

Molecular analyses of the hemagglutinin genes of H5 influenza viruses: origin of a virulent turkey strain.

Comparative sequence analysis of the hemagglutinin (HA) genes of a highly virulent H5N8 virus isolated from turkeys in Ireland in 1983 and a virus of the same subtype detected simultaneously in healthy ducks showed only four amino acid differences between these strains. Partial sequencing of six of the other genes and antigenic similarity of the neuraminidases established the overall genetic similarity of these two viruses. Comparison of the complete sequence of two H5 gene sequences and partial sequences of other virulent and avirulent H5 viruses provides evidence for at least two different lineages of H5 influenza virus in the world, one in Europe and the other in North America, with virulent and avirulent members in each group. In vivo studies in domestic ducks showed that all of the H5 viruses that are virulent in chickens and turkeys replicate in the internal organs of ducks but did not produce any disease signs. Additionally, both viruses isolated from turkeys and ducks in Ireland were detected in the blood. These studies provide the first conclusive evidence for the possibility that fully virulent influenza viruses in domestic poultry can arise directly from viruses in wild aquatic birds. Studies on the cleavability of the HA of virulent and avirulent H5 viruses showed that the principles established for H7 viruses (F. X. Bosch, M. Orlich, H. D. Klenk, and R. Rott, 1979, Virology 95, 197-207; F. X. Bosch, W. Garten, H. D. Klenk, and R. Rott, 1981, Virology 113, 725-735) also apply to the H5 subtype. These are (1) only the HAs of virulent influenza viruses were cleaved in tissue culture in the absence of trypsin and (2) virulent H5 influenza viruses contain a series of basic amino acids at the cleavage site of the HA, whereas avirulent strains contain only a single arginine with the exception of the avirulent Chicken/Pennsylvania virus. Thus, a series of basic amino acids at the cleavage site probably forms a recognition site for the enzyme(s) responsible for cleavage.

Amino Acid Sequence↗
PubMed1987

Antigenic and genetic conservation of H3 influenza virus in wild ducks.

The hemagglutinins of H3 influenza viruses isolated from migratory ducks on the Pacific flyway in Japan during the period 1977 to 1985 were analyzed antigenically and genetically. Antigenic analysis using monoclonal antibodies to the hemagglutinins of A/Aichi/2/68 (H3N2) and A/duck/Hokkaido/8/80 (H3N8) viruses showed that antigenic drift occurred extensively in human strains, whereas the hemagglutinins of duck viruses were highly conserved. It was also found that the hemagglutinins of duck viruses were antigenically closely related to that of human 1968 H3 prototype strains. Nucleotide sequence analysis of seven duck H3 hemagglutinin genes showed a limited number of changes among the six duck isolates and between these duck isolates and Aichi/68. The deduced amino acid sequence revealed amino acid changes randomly distributed throughout the molecule and not confined to antigenic sites. These findings indicate that the duck virus hemagglutinin genes are conserved in nature and that viruses of different lineages cocirculate.

Amino Acid Sequence↗
PubMed1987

Effects of site-specific mutation on structure and activity of influenza virus B/Lee/40 neuraminidase.

A cDNA copy of the neuraminidase (NA) gene of a high-growing reassortant influenza virus which has the hemagglutinin of B/Hong Kong/8/73 and the NA of B/Lee/40 was cloned into plasmid pUC 9 and subcloned into a late-replacement SV40 vector so that the NA gene could be expressed in eukaryotic cells. The expressed protein was antigenically and enzymatically active. To study structure-function relationships in the B/Lee NA, particularly in comparison to the known structure of influenza A (N2) NA, specific mutations were introduced using synthetic oligonucleotides. Mutation of an apparently unpaired cysteine residue to serine at position 251 had no effect on protein transport or folding as judged by cell-surface reactivity with monoclonal antibodies, and the NA retained enzyme activity, confirming that this Cys is not essential for correct folding of the polypeptide. Mutation of Trp 364 to Leu abolished detectable enzyme activity, while mutation of Thr 368 to Val reduced enzyme activity to less than 25% of wild-type levels. Neither mutation affected antigenic properties. Therefore it is likely that both these side chains extend into the NA active site pocket. The results of these experiments are in accord with similarities in the structure of the B/Lee NA compared with that of influenza A (N2) NA. Although there is about 70% amino acid sequence difference between influenza A and B NAs, residues in the active site are highly conserved. Our in vitro mutagenesis experiments help to confirm the tentative alignment of sequences and have identified conserved amino acid side chains involved in enzyme activity.

Amino Acid Sequence↗
PubMed1987

Host cell-mediated variation in H3N2 influenza viruses.

The influence of the host cell on the selection of antigenic variants of influenza A H3N2 viruses and the relevance of host cell selection to the induction of immunity by these viruses have been investigated. Influenza viruses were isolated from human clinical samples during a single epidemic, were passaged in mammalian Madin-Darby Canine Kidney (MDCK) cells or in embryonated hens eggs, and were tested for antigenic variability in the hemagglutinin (HA) molecule with a panel of monoclonal antibodies. In many cases, the HA of virus cultivated in eggs was antigenically distinct from the HA of virus from the same individual grown in mammalian cells. Viruses recovered from different individuals were antigenically similar to each other when grown in mammalian cell lines yet were antigenically heterogeneous when cultivated in eggs. The HA genes of viruses isolated from different individuals during the epidemic were shown, by sequence analysis, to differ from each other by five or six amino acid residues. Sequence analyses of the HA genes of MDCK cell-grown and egg-grown virus obtained from the same individual demonstrated that the molecular changes between antigenically distinct HAs of MDCK cell- and egg-grown A/Mem/12/85 virus involved a single amino acid substitution at residue 156 in HA1, which lies at the tip of the HA molecule and immediately adjacent to the receptor-binding site. However, the amino acid sequences of HAs from MDCK-grown and egg-grown viruses (A/Mem/2/85) isolated from a second individual were identical although these viruses exhibited antigenic differences when examined with anti-HA monoclonal antibodies. Therefore, single amino acid changes in the HA molecule may not be the sole cause of antigenic changes in the HA observed between pairs of MDCK cell-grown and egg-grown viruses and genes other than that encoding the HA may contribute to the host cell-mediated antigenic variation of these viruses. Nevertheless, antigenic differences between viruses grown in eggs and MDCK cells did not influence their ability to protect, since ferrets infected with either live egg-grown or MDCK-grown virus were protected equally well from challenge with virus grown in either host cell type.

Amino Acid Sequence↗
PubMed1987

Crystals of antibodies complexed with influenza virus neuraminidase show isosteric binding of antibody to wild-type and variant antigens.

We describe here, for the first time, crystals of antibodies bound to a viral antigen which diffract X-rays to beyond 3 A. Crystals have been grown of Fab fragments of monoclonal antibody NC41, complexed with influenza virus neuraminidase (NA) of the N9 subtype and with a variant of N9 NA having a sequence change of Asn to Asp at position 331. This reduces, but does not abolish, the binding of NC41 antibody (in the case of another variant, Ser 371 to Leu, binding of NC41 antibody appears to be abolished). We are presenting data on the three-dimensional structure of these two complexes which indicates that NC41 antibody binds isosterically to the wild type and variant neuraminidase molecules.

Antibodies, Monoclonal↗
PubMed1987

Glycosylation affects cleavage of an H5N2 influenza virus hemagglutinin and regulates virulence.

Based on nucleotide sequence analysis of the hemagglutinin (HA) gene from the virulent and avirulent A/chicken/Pennsylvania/83 influenza viruses, it was previously postulated that acquisition of virulence was associated with a point mutation that resulted in loss of a glycosylation site. Since there are two potential glycosylation sites in this region of the HA molecule and since all Asn-Xaa-Thr/Ser sequences in the HAs of different strains are not necessarily glycosylated, the question remained open as to whether either one of these sites was glycosylated. We now provide direct evidence that a site-specific glycosylation affects cleavage of the influenza virus HA and thus virulence. We have identified the glycosylation sites on the HA1 subunit from the virulent and avirulent strains by direct structural analysis of the isolated proteins. Our results show that the only difference in glycosylation between the HA1s of the virulent and avirulent strains is the lack of an asparagine-linked carbohydrate on the virulent HA1 polypeptide at residue 11. Further, we show that the HA1s of both the avirulent and virulent viruses are not glycosylated at one potential site, while all other sites contain carbohydrate. Amino acid sequence analysis of the HA1 of an avirulent revertant of the virulent strain confirmed these findings.

Amino Acid Sequence↗
PubMed1987

Defective interfering virus associated with A/Chicken/Pennsylvania/83 influenza virus.

The A/Chicken/Pennsylvania/1/83 influenza virus, isolated from a respiratory infection of chickens, is an avirulent H5N2 virus containing subgenomic RNAs (W.J. Bean, Y. Kawaoka, J.M. Wood, J.E. Pearson, and R.G. Webster, J. Virol. 54:151-160, 1985). We show here that defective interfering particles are present in this virus population. The virus had a low ratio of plaque-forming to hemagglutinating units and produced interference with standard virus multiplication in infectious center reduction assays. Subgenomic RNAs were identified as internally deleted polymerase RNAs. We have confirmed that this virus protects chickens from lethal H5N2 influenza virus infection. This protective effect appeared to be due to the inhibition of virulent virus multiplication. Additionally, subgenomic RNAs derived from polymerase RNAs were detected in 5 of 18 RNA preparations from animal influenza virus isolates. Therefore, defective interfering particles are sometimes produced in natural influenza virus infections, not just under laboratory conditions. These particles may be capable of suppressing the pathogenic effect of virulent virus infections in nature.

DNA-Directed RNA Polymerases↗
PubMed1987

Antigenic structure and variation in an influenza virus N9 neuraminidase.

We previously determined, by X-ray crystallography, the three-dimensional structure of a complex between influenza virus N9 neuraminidase (NA) and the Fab fragments of monoclonal antibody NC-41 [P. M. Colman, W. G. Laver, J. N. Varghese, A. T. Baker, P. A. Tulloch, G. M. Air, and R. G. Webster, Nature (London) 326:358-363, 1987]. This antibody binds to an epitope on the upper surface of the NA which is made up of four polypeptide loops over an area of approximately 600 A2 (60 nm2). We now describe properties of NC-41 and other monoclonal antibodies to N9 NA and the properties of variants selected with these antibodies (escape mutants). All except one of the escape mutants had single amino acid sequence changes which affected the binding of NC-41 and which therefore are located within the NC-41 epitope. The other one had a change outside the epitope which did not affect the binding of any of the other antibodies. All the antibodies which selected variants inhibited enzyme activity with fetuin (molecular weight, 50,000) as the substrate, but only five, including NC-41, also inhibited enzyme activity with the small substrate N-acetylneuramin-lactose (molecular weight, 600). These five probably inhibited enzyme activity by distorting the catalytic site of the NA. Isolated, intact N9 NA molecules form rosettes in the absence of detergent, and these possess high levels of hemagglutinin activity (W.G. Laver, P.M. Colman, R.G. Webster, V.S. Hinshaw, and G.M. Air, Virology 137:314-323, 1984). The enzyme activity of N9 NA was inhibited efficiently by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, whereas hemagglutinin activity was unaffected. The NAs of several variants with sequence changes in the NC-41 epitope lost hemagglutinin activity without any loss of enzyme activity, suggesting that the two activities are associated with separate sites on the N9 NA head.

Antibodies, Monoclonal↗
PubMed1987

Serological studies with influenza A(H1N1) viruses cultivated in eggs or in a canine kidney cell line (MDCK).

Pairs of influenza A(H1N1) viruses cultivated from the same clinical specimen in canine kidney (MDCK) cells or in embryonated hens' eggs can frequently be distinguished by their reactions with monoclonal antibodies to haemagglutinin and with antibodies in ferret or human sera. Egg-adapted virus, further passaged in MDCK cultures remained "egg-like" in serological characteristics indicating that the differences in their serological reactions were not a direct result of host cell-dependent glycosylation of the haemagglutinin. Haemagglutination-inhibiting (HI) or virus neutralizing antibodies in human sera can be detected more frequently, and to higher titre, in tests employing virus grown exclusively in MDCK cells than in tests with virus adapted to growth in embryonated eggs. Striking differences were detected in the serological reactions in HI tests when sera from ferrets infected with egg-grown virus were tested against a series of strains of influenza A(H1N1) virus isolated in 1983 and adapted to growth in eggs. In contrast, sera from ferrets infected with MDCK-derived virus failed to distinguish serologically between the same viruses that had been passaged exclusively in MDCK cells and also revealed relatively small differences between their egg-adapted counterparts.It was concluded that the cell substrate used for virus isolation and cultivation is a factor that should be considered when interpreting the results of strain characterization of influenza A(H1N1) isolates and in sero-surveys using these viruses.

Influenza A Virus, H1N1 Subtype↗
PubMed1986

Electron and X-ray diffraction studies of influenza neuraminidase complexed with monoclonal antibodies.

Complexes of influenza virus neuraminidase both with antigen-binding (Fab) fragments and with whole monoclonal antibody molecules have been crystallized. Uniformly thin platelet microcrystals suitable for structure analysis by electron diffraction, yielding reflections to approximately 4.3 A resolution, have been grown from one neuraminidase-Fab complex, that of N9 neuraminidase with 32/3 Fab, and thicker crystals of a second neuraminidase-Fab complex (N9 neuraminidase-NC35 Fab) diffract X-rays to approximately 4.0 A resolution. Electron microscope lattice images of microcrystals both of Fab and of immunoglobulin G complexed with neuraminidase have been interpreted in terms of negatively stained images of the respective individual complex protomers. The sites of binding of the antibodies to the antigen are consistent with the notion that single amino acid changes observed in monoclonal variants of neuraminidase occur in binding epitopes for the antibody used for their selection.

Antibodies, Monoclonal↗
PubMed1986

Distribution of hemagglutinin and neuraminidase on influenza virions as revealed by immunoelectron microscopy.

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.

Antibodies, Monoclonal↗
PubMed1986

Molecular changes in A/Chicken/Pennsylvania/83 (H5N2) influenza virus associated with acquisition of virulence.

One of the unresolved questions concerning the acquisition of virulence by the A/Chicken/Pennsylvania/83 (H5N2) influenza virus is which gene segments other than the hemagglutinin (HA) showed changes that were relevant. To answer this question, reassortants were made possessing the hemagglutinin gene of the virulent virus and the seven other genes from the avirulent parent. Since both the virulent and avirulent H5N2 strains are antigenically almost indistinguishable, it was necessary to transfer the genes of interest to a "carrier" virus before the appropriate reassortment could be selected. The gene compositions of the reassortants was established by a combination of sequence analysis and migration on polyacrylamide gels. These analyses established that the avirulent influenza virus present in April 1983 possessed seven of the eight gene segments necessary for virulence; mutation(s) in the HA gene were required for acquisition of virulence. Other viruses such as A/Seal/Mass/1/80 (H7N7) could provide the other genes necessary for virulence. Two changes in the HA have been associated with the acquisition of virulence; these are at amino acid residues 23 and 78 (H3 numbering) (Y. Kawaoka and R.G. Webster, Virology, 146, 130-137 (1985]. Isolation of an amantadine-resistant avirulent revertant virus provided the opportunity to determine which of the two amino acid changes in HA is critical. Sequence analysis of the revertant virus revealed amino acid changes at residues 23 in HA1 and 40 in HA2 (H3 numbering). The change at residue 23 of HA1 is probably associated with reversion to avirulence, of cleavability of HA, and inability to plaque in tissue culture without trypsin; while the change at residue 40 of HA2 may be associated with the amantadine-resistant phenotype. These studies establish that a single critical point mutation in the hemagglutinin gene of the avirulent A/Chicken/Pennsylvania/1/83 (H5N2) was probably all that was required to produce the highly virulent Chicken/Pennsylvania virus; the avirulent virus already possessed the other genes necessary for virulence.

Amino Acid Sequence↗
PubMed1986

Spin-labeling of influenza virus hemagglutinin permits analysis of the conformational change at low pH and its inhibition by antibody.

To study the conformational changes in the hemagglutinin (HA) molecule of A/seal/Mass/1/80 (H7N7) (Seal) influenza virus at low pH, a spin-labeling method was used. This method also permits study of antibody interaction with the HA. A synthetic nitroxide compound was used for spin-labeling of tyrosine residues of the isolated HA molecule. Electron spin resonance (ESR) spectra of the spin-labeled HA at various pH values indicated that a conformational transition occurred under acidic conditions, and around pH 5.8 the HA molecule has maximal flexibility. Since virus-induced hemolysis occurs optimally at pH 5.8-5.9, the HA molecule in the maximally flexible conformation is considered to mediate membrane fusion. The ESR spectra of the antibody-bound HA at various pH values revealed that monoclonal antibodies to different regions on the molecule may inhibit the conformational change by different modes. One antibody inhibited the changes in the HA that resulted in flexibility, while the other did not. These results support the assumption that monoclonal antibodies, which failed to inhibit hemagglutination of the virus yet neutralized viral infectivity, inhibited the fusion step in the viral replication process by interfering with a low pH-induced conformational change in the HA molecule (Kida, H., Webster, R.G. and Yanagawa, R. (1983) Arch. Virol. 76, 91-99).

Animals↗
PubMed1986

Assembly of influenza hemagglutinin trimers and its role in intracellular transport.

The hemagglutinin (HA) of influenza virus is a homotrimeric integral membrane glycoprotein. It is cotranslationally inserted into the endoplasmic reticulum as a precursor called HA0 and transported to the cell surface via the Golgi complex. We have, in this study, investigated the kinetics and cellular location of the assembly reaction that results in HA0 trimerization. Three independent criteria were used for determining the formation of quaternary structure: the appearance of an epitope recognized by trimer-specific monoclonal antibodies; the acquisition of trypsin resistance, a characteristic of trimers; and the formation of stable complexes which cosedimented with the mature HA0 trimer (9S20,w) in sucrose gradients containing Triton X-100. The results showed that oligomer formation is a posttranslational event, occurring with a half time of approximately 7.5 min after completion of synthesis. Assembly occurs in the endoplasmic reticulum, followed almost immediately by transport to the Golgi complex. A stabilization event in trimer structure occurs when HA0 leaves the Golgi complex or reaches the plasma membrane. Approximately 10% of the newly synthesized HA0 formed aberrant trimers which were not transported from the endoplasmic reticulum to the Golgi complex or the plasma membrane. Taken together the results suggested that formation of correctly folded quaternary structure constitutes a key event regulating the transport of the protein out of the endoplasmic reticulum. Further changes in subunit interactions occur as the trimers move along the secretory pathway.

Animals↗
PubMed1986

Vaccination as a strategy to reduce the emergence of amantadine- and rimantadine-resistant strains of A/Chick/Pennsylvania/83 (H5N2) influenza virus.

The influenza A virus (A/Chick/Pennsylvania/83 (H5N2) that caused up to 80% mortality in chickens provided a model system for testing the efficacy of chemotherapeutic agents against highly virulent influenza virus. Amantadine and rimantadine administered to chickens in drinking water were efficacious both prophylactically and therapeutically. However, under conditions simulating natural transmission of virus amantadine and rimantadine-resistant viruses arose and were transmitted to contact birds, causing mortality. Simultaneous administration of inactivated H5N2 vaccine and amantadine provided protection. Thus, chemotherapy may be useful in the treatment of a highly pathogenic influenza virus outbreak in humans or other animals when used in combination with vaccine.

Adamantane↗
PubMed1986

Antigenic mapping of an avian H1 influenza virus haemagglutinin and interrelationships of H1 viruses from humans, pigs and birds.

Monoclonal antibodies to the haemagglutinin (HA) of the avian H1 influenza virus A/duck/Alberta/35/76 were used to construct an operational antigenic map of the HA molecule and to study the interrelationships of H1 viruses from different hosts. Haemagglutination inhibition tests between the monoclonal antibodies and variants selected by them provided evidence of four antigenic regions which overlap to varying degrees. Avian H1 influenza viruses displayed a spectrum of reactivities to the monoclonal antibody panel. Representatives of the epidemic strains of human H1 influenza viruses and early swine influenza viruses showed little or no reactivity with the monoclonal antibodies but swine influenza-like viruses isolated from pigs and humans in the last decade reacted with 11 of 17 antibodies. The antigenic similarity of these viruses to many avian isolates suggests that there has been a transfer of HA genetic information between mammalian and avian H1 influenza viruses.

Animals↗