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R G Webster

Publications and source records attributed to R G Webster.

At least 235 records · Page 13Linked to original sources
PubMed1988

Origin of the hemagglutinin gene of H3N2 influenza viruses from pigs in China.

Influenza viruses of the H3N2 subtype similar to Aichi/2/68 and Victoria/3/75 persist in pigs many years after their antigenic counterparts have disappeared from humans (Shortridge et al. (1977). Science 19, 1454-1455). To provide information on the mechanism of conservation of these influenza viruses in pigs, the hemagglutinin (HA) of four isolates from swine derived from Taiwan and Southern China were analyzed antigenically and genetically. The reactivity pattern of these viruses with a panel of monoclonal antibodies indicates that the HAs of these swine viruses were antigenically closely related to duck H3 and early human H3 viruses. Sequence analysis of the H3 genes from three swine viruses revealed that the swine H3 genes are more closely related to the duck genes than to early human H3 virus (A/Aichi/2/68). The degree of sequence homology of these genes is extremely high (more than 96.5%). Furthermore, the deduced amino acid sequence of the three swine HAs at residues 226 to 228 in the proposed receptor-binding site is Gln-Ser-Gly and is common with the majority of avian influenza viruses. These findings indicate that these H3 viruses may have been introduced into pigs from ducks. The HA gene of the fourth swine influenza virus from Southern China was genetically equally related to avian and early human H3 strains although the sequence through the receptor-binding pocket (226-228) was typical of a human H3 virus, suggesting that either this swine HA gene was derived from ducks or an early human H3 virus was introduced into the pig population where the virus accumulated substantial mutations. The present strains revealed genetic heterogeneity of swine H3 influenza viruses in nature.

Amino Acid Sequence↗
PubMed1988

Localization of RNA polymerases on influenza viral ribonucleoproteins by immunogold labeling.

Monospecific antisera for the influenza polymerase proteins and high resolution immunoelectron microscopy have been used to investigate the topographical distribution of the polymerase molecules on influenza ribonucleoproteins (RNPs). Antibodies to PB1, PB2, and PA identify a single polymerase binding site located at, or very close to, the end of each RNP. Double labeling experiments confirm that all three polymerases are at the same end of each RNP and that they are in close association.

DNA-Directed RNA Polymerases↗
PubMed1988

Antigenic and structural characterization of multiple subpopulations of H3N2 influenza virus from an individual.

Influenza viruses grown in embryonated chicken eggs frequently possess antigenically distinguishable hemagglutinin (HA) compared to virus from the same source grown in mammalian cell culture. To further investigate the extent of variation among viruses from an individual, viruses were isolated from throat washes collected over a 48-hr period during infection with influenza virus designated A/Mem/6/86 (H3N2). Viruses were isolated from limit dilutions in eggs and mammalian Madin-Darby canine kidney (MDCK) cells and the antigenic, structural, and receptor-binding properties of these viruses were determined. Viruses which could be isolated in MDCK cells were present at 10- to 100-fold higher frequency in the original sample than viruses which could be isolated in eggs. The HA of virus clones isolated in MDCK cells were antigenically and structurally identical. In contrast, viruses from the same source, selected at limit dilution in eggs, could be divided into three distinct subpopulations based on the distinguishable antigenic and structural characteristics of their HA molecules. The three groups of egg-grown viruses could be distinguished from each other, and from MDCK cell-grown viruses, not only by a panel of anti-HA monoclonal antibodies, but also by immune ferret sera raised to H3N2 virus strains of recent years and sera raised to the different egg-grown clones themselves. Of these groups, group 1 and group 2 egg-grown viruses each represented a minor subpopulation of viruses which could be isolated in eggs, while viruses of the third antigenic phenotype were the most frequently isolated in eggs. Amino acid substitutions in the HA of egg-grown viruses occurred in antigenic and receptor-binding sites of the molecule. Group 1 viruses each possessed two amino acid substitutions in their HA molecules at residues 193 and 229 in HA1. Group 3 viruses, which displayed altered receptor specificities compared to MDCK cell-grown viruses and other egg-grown viruses, possessed a single amino acid substitution at residue 145 in HA1. The HA of the group 2 egg-grown viruses appeared structurally identical, yet displayed marked differences in antigenic and receptor-binding properties, compared to viruses isolated in MDCK cells. These results demonstrate that multiple, distinct subpopulations of virus can be isolated from a single patient during an infection with influenza and highlights the potential problems in selecting the most appropriate virus for epidemiological and vaccine purposes since selection could result in the use of viruses that are not representative of those which predominate in a human population.

Animals↗
PubMed1988

Protection against lethal influenza with neuraminidase.

The role of the neuraminidase in eliciting protection against a lethal influenza A virus [A/Ck/Penn/1370/83 (H5N2)] infection was investigated in chickens. Isolated N2 neuraminidase administered in adjuvant did not prevent infection but did prevent systemic spread of virus and death of chickens. N2 expressed in a recombinant vaccinia virus protected chickens when administered in adjuvant but was less effective when allowed to replicate and produce pox on the chicken's comb. Chickens vaccinated with isolated N2 in adjuvant or with inactivated H5N2 influenza virus were protected from clinical signs and death after challenge with A/Ck/Penn/1370/83 influenza virus. However, these animals were completely susceptible and died of infection with a heterologous subtype (H7N7) of influenza virus. The role of the different antigenic determinants of the N2 NA was investigated in chickens by passive transfer of monoclonal antibodies. Antibodies to antigenic determinants rimming the enzyme active center reduced disease signs in approximately half of the birds but did not significantly reduce virus levels. Antibodies to one of the two independent antigenic determinants that are distant from the enzyme active center were most effective at reducing virus replication and disease signs. This is surprising because antigenic variants could not be selected in vitro with these antibodies and suggests that they may facilitate clearance of virus. Antibodies to the other determinant that is located distally to the enzyme site were ineffective at providing protection.

Animals↗
PubMed1988

Protective immunity against avian influenza induced by a fowlpox virus recombinant.

Fowlpox virus, the prototypic virus of the genus Avipoxvirus has a natural host range limited to avian species. As such, fowlpox virus provides a suitable candidate for the development of a species-specific recombinant viral vector. This paper reports the development of a fowlpox virus recombinant expressing the haemagglutinin molecule from a highly virulent avian influenza virus. On immunization of chickens and turkeys with the recombinant, protection is afforded against a lethal challenge with either the homologous or a heterologous influenza virus strain.

Animals↗
PubMed1988

Sequence requirements for cleavage activation of influenza virus hemagglutinin expressed in mammalian cells.

Cleavage of the hemagglutinin (HA) in tissue culture systems has been correlated with virulence of avian influenza viruses. To examine the structural requirements for cleavage of the HA, the HA gene from a virulent H5 influenza virus was expressed in mammalian cells (CV-1), and the cleavage site of the HA was explored by using site-specific mutagenesis. The expressed HA protein exhibited normal cleavage, transport to the cell membrane, and ability to adsorb and to fuse erythrocytes at pH 5. Site-specific mutagenesis of the HA directly established that (i) most of the basic amino acids at this site are critical for cleavage activation; (ii) besides the connecting peptide sequence, at least one other structural feature of the HA is required for enzyme recognition; and (iii) the length of the connecting peptide can abrogate the structural feature(s).

Amino Acid Sequence↗
PubMed1988

Posttranslational oligomerization and cooperative acid activation of mixed influenza hemagglutinin trimers.

The influenza virus hemagglutinin (HA) is a well-characterized integral membrane glycoprotein composed of three identical subunits. We have analyzed the formation of mixed trimers in cells expressing two different HA gene products. The results show efficient and essentially random assembly of functional hybrid trimers provided that the HAs are from the same HA subtype. Trimerization is thus a posttranslational event, and subunits are recruited randomly from a common pool of monomers in the endoplasmic reticulum. Mixed trimers were not observed between HAs derived from different subtypes, indicating that the trimerization event is sequence specific. Mixed trimers containing mutant subunits were, moreover, used to establish that the acid-induced conformational change involved in the membrane fusion activity of HA is a highly cooperative event.

Animals↗
PubMed1988

Retrovirus-expressed hemagglutinin protects against lethal influenza virus infections.

An influenza virus hemagglutinin gene, H7, has been expressed in a replication-competent Schmidt-Ruppin Rous sarcoma virus-derived vector. This virus, P1/H7, expressed a glycosylated precursor of the H7 protein which was processed to a mature form and transported to the cell surface. The expressed H7 glycoprotein could not be detected in P1/H7 virus particles. A P1/H7 stock which expressed 5 to 10% of the level of H7 observed in influenza virus-infected chicken embryo fibroblasts was used to immunize 1-month-old chickens. This immunization resulted in low or undetectable levels of hemagglutination-inhibiting and neutralizing antibody. Despite the low serum response, challenge with a highly pathogenic H7N7 virus revealed complete protection against lethal infection.

Animals↗
PubMed1988

Protection of chickens from lethal influenza infection by vaccinia-expressed hemagglutinin.

To study the immune response of the chicken to specific influenza proteins, we have constructed a recombinant vaccinia virus containing the hemagglutinin gene of influenza A/Turkey/Ireland/1378/83 (H5N8). In mammalian cell culture the hemagglutinin expressed by this recombinant virus was full-length, cleaved into HA1 and HA2 in the absence of trypsin, and transported to the cell surface, confirming that other virus products are not required for cleavage activation. Chickens inoculated through the wing web with the live recombinant virus produced extremely low levels of hemagglutination-inhibiting or infectivity-neutralizing antibody. However, they were protected from lethal H5 influenza virus challenge. Protection extended to the antigenically distinct virulent H5 viruses, Chicken/Pennsylvania/1370/83 and Chicken/Scotland/59. Chemically bursectomized vaccinated chickens were not protected, whereas normal chickens with very low antibody levels (less than 10) obtained by passive transfer were protected in a dose-dependent fashion. This indicates that despite the low antibody titers induced by vaccination, protection was mediated by antibody.

Animals↗
PubMed1987

Site-directed mutation of the active site of influenza neuraminidase and implications for the catalytic mechanism.

Different isolates of influenza virus show a high degree of amino acid sequence variation in their surface glycoproteins. Conserved residues located in the substrate-binding pocket of the influenza virus neuraminidase are therefore likely to be involved in substrate binding or enzyme catalysis. In order to study the structure and function of the active site of this protein, a full-length cDNA clone of the neuraminidase gene from influenza A/Tokyo/3/67 was subcloned into aN M13 vector and amino acid substitutions were made in selected residues by using the oligonucleotide mismatch technique. The mutant neuraminidase genes were expressed from a recombinant SV40 vector, and the proteins were analyzed for synthesis, transport to the cell surface, and proper three-dimensional folding by internal and surface immunofluorescence. The mutant neuraminidase proteins were then assayed to determine the effect of the amino acid substitution on enzyme activity. Twelve of the 14 mutant proteins were correctly folded and were transported to the cell surface in a manner identical with that of the wild type. Two of these have full enzyme activity, but seven mutants, despite correct three-dimensional structure, have completely lost neuraminidase activity. Two mutants were active at low pH. The properties of the mutant enzymes suggest a possible mechanism of neuraminidase action.

Binding Sites↗
PubMed1987

Intestinal replication of influenza A viruses in two mammalian species. Brief report.

The sites of replication of influenza A viruses in ferrets and pigs were studied. The majority of the swine, equine, and avian influenza A viruses tested were recovered from the intestinal tract of ferrets as well as from the respiratory tract; most of the human influenza viruses studied were recovered only from the respiratory tract. In contrast with ferrets, only Hong Kong/1/68 (H 3 N 2) influenza virus was recovered from the intestinal tract of pigs. Despite the large biological variability found in ferrets and in pigs, the results do establish that the majority of influenza viruses have the potential to replicate in the intestinal tissues of some mammals. Additionally, the study suggests that there are differences among the influenza A viruses in tissue tropism in different mammals. Both viral and host genetic factors determine the tissue tropism of influenza viruses in mammals.

Animals↗
PubMed1987

Distribution of sequence differences in influenza N9 neuraminidase of tern and whale viruses and crystallization of the whale neuraminidase complexed with antibodies.

Neuraminidase genes from A/tern/Australia/G70C/75 (H11N9) and A/whale/Maine/1/84 (H13N9) influenza viruses have been sequenced. Seventy-two nucleotide changes were found, 17 of which result in changes in the amino acid sequence of the neuraminidase; 3 in the stalk region and 14 in the heads. To our surprise, all of the sequence changes in the head region are located on the base of the neuraminidase tetramer, resulting in conservation of antigenic sites on top of the neuraminidase which vary extensively in human influenza virus neuraminidase. Whale N9 neuraminidase, like tern N9 neuraminidase, possesses high levels of hemagglutinating activity but, unlike the tern neuraminidase, failed to form large well-ordered crystals. However, when the neuraminidase was complexed with Fab fragments of monoclonal antibodies, which were made against the tern N9 neuraminidase, large crystals of the complexes were obtained which diffract X-rays to beyond 3 A.

Amino Acid Sequence↗
PubMed1987

Molecular analysis of the hemagglutinin genes of Australian H7N7 influenza viruses: role of passerine birds in maintenance or transmission?

In 1985 a fowl plague-like disease occurred in chickens in Lockwood, Victoria, Australia and caused high mortality. An H7N7 influenza virus was isolated from the chickens (A/Chicken/Victoria/1/85); additionally, an antigenically similar virus was isolated from starlings (A/Starling/Victoria/5156/85) and serological evidence of H7N7 virus infection was found in sparrows. Antigenic analysis with monoclonal antibodies to H7, oligonucleotide mapping of total vRNA, and sequence analysis of the HA genes established that the chicken and starling influenza viruses were closely related and probably came from the same source. There was high nucleotide sequence homology (95.3%) between the HA genes of A/Chick/Vic/85 and a fowl plague-like virus isolated from chickens in Victoria 9 years earlier [A/Fowl/Vic/76 (H7N7)]. The sequence homologies indicated that the A/Chick/Vic/85 and A/Fowl/Vic/76 were derived from a common recent ancestor, while another recent H7N7 virus, Seal/Mass/1/80 originated from a different evolutionary lineage. Experimental infection of chickens and starlings with A/Chick/Vic/1/85 (H7N7) was associated with high mortality (100%), transmission to contact birds of the same species, and virus in all organs. In sparrows one-third of the birds died after infection and virus was isolated from most organs; transmission to contact sparrows did not occur. In contrast, the H7N7 virus replicated in ducks and spread to contact ducks but caused no mortality. These studies establish that the host species plays a role in determining the virulence of avian influenza viruses, and provide the first evidence for transmission of virulent influenza viruses between domestic poultry and passerine birds. They support the hypothesis that potentially virulent H7N7 influenza viruses could be maintained in ducks where they cause no apparent disease and may sometimes spread to other wild birds and domestic poultry.

Amino Acid Sequence↗
PubMed1987

Structural changes in the haemagglutinin which accompany egg adaptation of an influenza A(H1N1) virus.

Antigenic changes are often induced in the haemagglutinin (HA) of human isolates of influenza A(H1N1) and B viruses during their isolation and propagation in embryonated hens' eggs. In this report we describe molecular changes in the HA of an A(H1N1) virus which accompany egg adaptation. The HA1 amino acid substitutions of egg-adapted variants are located in the vicinity of the receptor binding site. This suggests that egg-adapted variants are selected on the basis of altered receptor site specificity with concomitant effects on the antigenic configuration of the HA molecule. In this study, at least three antigenically distinct groups of egg-adapted variants were observed. These observations have implications for the indiscriminate use of egg-adapted viruses in sero-epidemiological studies and vaccine production.

Animals↗