Search PubMed⌕ Search

Biomedical subjects

R G Webster

Publications and source records attributed to R G Webster.

At least 289 records · Page 16Linked to original sources
PubMed1984

Inhibition of T cell proliferation by antibodies to synthetic peptides.

While T cell proliferation to antigen in the presence of antigen-presenting cells is well known to be readily inhibited by antibodies directed against Class II major histocompatibility complex (MHC) (Ia/HLA-DR) products, it has not been possible to inhibit proliferation by antibodies directed against the antigen. Because of the implications of these observations for targets of T cell recognition, this phenomenon was reinvestigated using human T cell clones, recognizing a small (24 amino acid) synthetic peptide (termed p20) derived from the influenza hemagglutinin-1 molecule. It was found that proliferation of clones to p20 was inhibited efficiently (less than 90%), using p20 as antigen, and rabbit anti-p20. Inhibition was possible either by coculturing p20 antigen and antibody to p20 with cloned T cells and antigen-presenting (E-) cells, or by pulsing antigen-presenting cells with antigen prior to a brief incubation with antibody before washing the E- cells and using them to stimulate cloned T cells. These results do not indicate why previous attempts had failed, but in view of the different techniques available now (cloned T cells, small synthetic polypeptides, and antibody raised against polypeptide) we investigated the influence of these parameters. It was found that, using cloned T cells, the form of the antigen was of importance, as antibody inhibition of the response to hemagglutinin or whole influenza A was much less apparent. These differences were interpreted as being due to greater access of anti-p20 to p20 than to hemagglutinin or influenza. If uncloned T cell lines were used, inhibition was also much harder to detect. This was interpreted as masking of inhibition of the response of some clones in the line by interleukin 2-induced recruitment.

Animals↗
PubMed1984

Persistence of Q strain of H2N2 influenza virus in avian species: antigenic, biological and genetic analysis of avian and human H2N2 viruses.

The characteristics of an avian influenza virus were compared in detail with those of human Asian (H2N2) influenza viruses. Antigenic analysis by different antisera against H2N2 viruses and monoclonal antibodies to both the hemagglutinin and neuraminidase antigens showed that an avian isolate, A/duck/München/9/79 contained hemagglutinin and neuraminidase subunits closely related to those of the early human H2N2 viruses which had been prevalent in 1957. However, this avian virus gave low HI titers with absorbed and non-absorbed antisera to different human H2N2 viruses isolated in 1957. Like human Q phase variant, such as A/RI/5-/57 (H2N2), hemagglutination of the above avian strain was not inhibited by the purified non-specific gamma-inhibitor from guinea pig serum. Growth behavior at restrictive temperature (42 degrees C) clearly differentiate the avian H2N2 virus from human influenza viruses, showing that the former virus grew well in MDCK cells at 42 degrees C but not the latters. Genomic analysis of these viruses revealed that the oligonucleotide map of H2N2 virus isolated from a duck was quite different from those of human H2N2 viruses from 1957 to 1967. The oligonucleotide mapping also indicated that different H2N2 influenza virus variants had co-circulated in humans in 1957.

Animals↗
PubMed1984

Antigenic and biological characterization of influenza virus neuraminidase (N2) with monoclonal antibodies.

Competitive radioimmunoassays using monoclonal antibodies established that the neuraminidase of A/RI/5+/57 (H2N2) influenza can be divided into four overlapping antigenic regions. Antigenic regions 1 and 4 are sufficiently far apart so that there was no competition between antibodies for these sites. Region 1 is conserved in neuraminidases from N2 viruses over a 10-year period, while the other regions changed antigenically during this time. The antibodies belonging to groups 2 and 3 completely inhibited catalytic activity on fetuin substrate, whereas antibodies in groups 1 and 4 inhibited weakly or not at all. Antigenic region 2 can be further divided into four overlapping areas (2a, 2b, 2c, and 2d) based on the reactivity patterns of monoclonal antibodies with antigenic variants, chemically modified neuraminidase, and the ability of the antibodies to inhibit enzyme activity of different molecular weight substrates. Previous studies [R. G. Webster, V. S. Hinshaw , and W. G. Laver (1982) Virology 117, 93-104; D. C. Jackson and R. G. Webster (1982) Virology 123, 69-77] characterized only region 2 of the neuraminidase molecule. Each of the monoclonal antibodies inhibited virus release from MDCK cells when incorporated in an agar overlay, and some antibodies in each group inhibited hemagglutination by intact virus, but only antibodies in group 2 neutralized virus in embryonated eggs and permitted selection of antigenic variants. The results indicate that antibodies to some antigenic sites on the neuraminidase may inhibit virus release more efficiently than others, depending on their relation to the enzyme active center. None of the monoclonal antibodies inhibited the hemolytic activity of viruses possessing N2. Based on antigenic mapping and biological properties of the monoclonal antibodies, a topographical map of the neuraminidase can be constructed. It is proposed that antigenic regions 1 and 4 are spacially separated and, based on their failure to inhibit biological activity, may be located on the bottom surface of the molecule; region 3 may be on the top surface of the molecule but at some distance from the catalytic center. Antigenic region 2 probably encompasses most of the top surface of the molecule; region 2d being closest to the enzyme center, with subregions 2a and 2b adjacent to it on the top surface. Chemical treatment of the neuraminidase with trinitrobenzenesulfonic acid (TNBS) causes modification of the 2b region, confirming the antigenic mapping results.

Animals↗
PubMed1984

Sequence of the neuraminidase gene of influenza virus A/Tokyo/3/67 and previously uncharacterized monoclonal variants.

A full-length cDNA copy of the neuraminidase (NA) gene of influenza strain A/Tokyo/3/67 was cloned into the plasmid pBR322, and the nucleotide sequence of the gene was determined. In addition, the sequence changes in six variants of A/Tokyo/3/67 selected with various monoclonal antibodies (Ab) to the NA were determined by dideoxy sequencing of the vRNA. In five of the monoclonal variants, a single change occurred, resulting in an amino acid substitution at residue 344. Arginine in the parent virus changed to every amino acid possible with a single nucleotide change. In another variant, arginine at position 253 changed to serine, a change that also occurred in field strains. All variants so far sequenced that were selected by monoclonal Ab to A/Tokyo/3/67 virus changed at position 344, except one which changed at residue 368. Both of these positions are in clusters of residues that vary considerably in field strains, the clusters being 344-347 and 368-370. Analysis of the three-dimensional crystal structure of the NA of A/Tokyo/3/67 shows that these clusters are directly adjacent on the protein, and likely comprise a single antigenic site. A total of three or four antigenic sites have been proposed for the NA protein, based on antigenic mapping with monoclonal Ab [R. G. Webster, V. S. Hinshaw , and W. G. Laver (1982) Virology 117, 93-104]. Variants selected by Ab to Tokyo/67 NA all change in this single antigenic site, whereas variants selected by Ab to other strains change in other regions. It is possible that, although there may be three or four antigenic sites on the NA molecule, there may be a single, dominant antigenic site for each strain.

Amino Acid Sequence↗
PubMed1984

Influenza virus neuraminidase with hemagglutinin activity.

Isolated intact influenza virus neuraminidase (NA) molecules of the N9 subtype have been found to possess hemagglutinin (HA) activity which, at equivalent protein concentration, was fourfold higher than that of isolated hemagglutinin molecules of the H3 subtype. The amino-terminal sequence of the N9 NA is the same as in neuraminidases of the eight other influenza A virus NA subtypes previously reported. Viruses possessing N9 NA therefore have two different HA activities and antibody to either HA or NA alone was incapable of inhibiting hemagglutination by the virus. However, antibody to the HA of an H1N9 virus neutralized its infectivity as effectively as it neutralized H1N1 or H1N2 viruses whose neuraminidases have no HA activity. (Antibodies to N9 NA did not neutralize the infectivity of viruses with N9 neuraminidase). 2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid inhibited N9 NA activity but had no effect on the HA activity of the isolated N9 NA. One interpretation of this result would be that the HA and NA activities are located in separate sites. Pronase-released N9 NA heads form crystals suitable for X-ray diffraction studies and preliminary data to 2.9 A establish the space group as cubic, I432 with cell dimension a = 184 A. Data extend to beyond 1.9 A resolution, and these will be collected in the future.

Animals↗
PubMed1984

Is virulence of H5N2 influenza viruses in chickens associated with loss of carbohydrate from the hemagglutinin?

The A/Chick/Penn/83 (H5N2) influenza virus that appeared in chickens in Pennsylvania in April 1983 and subsequently became virulent in October 1983, was examined for plaque-forming ability and cleavability of the hemagglutinin (HA) molecule. The avirulent virus produced plaques and cleaved the HA only in the presence of trypsin. In contrast, the virulent virus produced plaques and cleaved the HA precursor into HA1 and HA2 in the presence or absence of trypsin. The apparent molecular weight of the HA1 from the avirulent virus was higher than that from the virulent virus, but when the viruses were grown in the presence of tunicamycin, the molecular weights of HA were indistinguishable. Two of nine monoclonal antibodies to the HA of the avirulent virus indicate that there is at least one epitope on the HA that is different between the virulent and avirulent viruses. The amino acid sequences of the HAs from the two viruses were compared by sequencing their respective HA gene. The nucleotide sequence coding for the processed HA polypeptide contained 1641 nucleotides specifying a protein of 547 amino acids. The amino acid sequences of the virulent and avirulent viruses were indistinguishable through the connecting peptide region, indicating that the difference in cleavability of the H5 HA is not directly attributed to the amino acid sequence of the connecting peptide. Four of seven nucleotide changes resulted in amino acid changes at residues 13, 69, and 123 of HA1 and at residue 501 of the HA2 polypeptide. Since there were no deletions or insertions in the amino acid sequence of the virulent or avirulent viruses, the possibility exists that the difference in molecular weight is due to loss of a carbohydrate side chain in the virulent strain. The amino acid change in the virulent strain at residue 13 is the only mutation that could affect a glycosylation site and this is in the vicinity of the connecting peptide. It is postulated that the loss of this carbohydrate may permit access of an enzyme that recognizes the basic amino acid sequences and results in cleavage activation of the HA in the virulent virus.

Amino Acid Sequence↗
PubMed1984

From the National Institutes of Health. Summary of a meeting on the origin of pandemic influenza viruses.

Influenza type A virus periodically undergoes major antigenic shifts in which the hemagglutinin (HAG) and sometimes the neuraminidase (NA) antigens are replaced by HAG and NA antigens of another subtype. Three such shifts have taken place since the virus was first isolated, and all appear to have occurred in China. The way in which these "new" influenza type A viruses suddenly appear (or reappear) in the human population is not known. At a meeting held in Beijing, China, on November 10-12, 1982, participants discussed the latest findings on the molecular biology of influenza viruses and on aspects of their ecology that may offer insight into the factors responsible for the origin of pandemic influenza viruses. Information obtained in earlier studies has provided some clues about how the antigenic shifts may occur. For example, the H3N2 virus has been found to be a recombinant deriving seven of its eight genes from an H2N2 strain and gene 4 (which encodes for the HAG) from some other virus, possibly an avian influenza virus of the H3 subtype [1-3]. In addition, studies of the genome of the H1N1 virus that appeared in Anshan, China, in 1977 have shown that this virus almost certainly underwent no replication for 27 years. This finding suggests that the virus existed in an animal reservoir during this period [4, 5].

Animals↗
PubMed1984

Are seals frequently infected with avian influenza viruses?

Influenza A virus isolates of the H4N5 subtype (which has previously been detected only in birds) were recovered from harbor seals dying of viral pneumonia on the New England coast from June 1982 through March 1983. When these isolates were compared with other mammalian and avian viruses in serological assays and RNA-RNA competitive hybridization, it was found that the seal viruses were most closely related antigenically and genetically to recent avian virus strains and were readily distinguishable from mammalian viruses, including H7N7 isolates recovered from seals in 1980. Unlike any previous isolates from mammals, these recent seal viruses replicate in the intestinal tracts of ducks, a characteristic of avian viruses. The association of avian viruses with influenza outbreaks in seals suggests that transmission of avian viruses to seals is occurring in nature. Potentially, this may be an example of the adaptation of avian viruses to mammals, which would represent an intermediate step in the evolution of new mammalian strains.

Animal Diseases↗
PubMed1984

Antigenic structure of the influenza B virus hemagglutinin: nucleotide sequence analysis of antigenic variants selected with monoclonal antibodies.

We report here the complete nucleotide sequence of the hemagglutinin (HA) gene of influenza B virus B/Oregon/5/80 and, through comparative sequence analysis, identify amino acid substitutions in the HA1 polypeptide responsible for the antigenic alterations in laboratory-selected antigenic variants of this virus. The complete nucleotide sequence of the B/Oregon/5/80 HA gene was established by a combination of chemical sequencing of a full-length cDNA clone and dideoxy sequencing of the virion RNA. The nucleotide sequence is very similar to previously reported influenza B virus HA gene sequences and differs at only nine nucleotide positions from the B/Singapore/222/79 HA gene (Verhoeyen et al., Nucleic Acids Res. 11:4703-4712, 1983). The nucleotide sequences of the HA1 portions of the HA genes of 18 laboratory-selected antigenic variants were determined by the dideoxy method. Comparison of the deduced amino acid sequences of the parental and variant HA1 polypeptides revealed 16 different amino acid substitutions at nine positions. All amino acid substitutions resulted from single-point mutations, and no double mutants were detected, demonstrating that as in the influenza A viruses, single amino acid substitutions are sufficient to alter the antigenicity of the HA molecule. Many of the amino acid substitutions in the variants occurred at positions also observed to change in natural drift strains. The substitutions appear to identify at least two immunodominant regions which correspond to proposed antigenic sites A and B on the influenza A virus H3 HA.

Amino Acid Sequence↗
PubMed1984

Cytolytic T lymphocyte and antibody responses to synthetic peptides of influenza virus hemagglutinin.

Two peptides corresponding to HA1(181-204) and HA2(103-123) of the A/Japan/305/57 influenza virus hemagglutinin (HA) were chemically synthesized by solid-phase methods and were tested for their ability to generate murine secondary anti-influenza cytolytic T lymphocytes (CTL) in vitro and to bind monoclonal anti-HA antibodies. Peptide HA1(181-204) could only generate CTL in the presence of helper factors contained in supernatant fluids from either Concanavalin A-stimulated mouse spleen cultures or WEHI-3 cells grown in vitro. Peptide HA2(103-123) stimulated the induction of anti-influenza CTL independent of helper factors, but the stimulation was also greatly increased if helper factors were added. A 10-fold molar excess of peptide HA2(103-123) was required to obtain optimal CTL activation over the quantities required in the HA1(181-204) system. This molar ratio remained unchanged, even in the presence of helper factors. Induction of influenza-specific CTL was antigen-dependent in both systems, even though some killing of noninfected target cells was also occasionally observed. Our results suggest that synthetic peptides can be recognized as antigenic determinants in the generation of H-2-restricted anti-viral CTL capable of killing appropriately infected target cells. The inability of peptide HA1(181-204) to generate sufficient help for CTL development suggests that certain regions of the HA can be recognized by CTL precursors, but not by all of the required helper cells. Peptide HA1(181-204) also reacted with three monoclonal anti-HA antibodies as well as mouse anti-influenza (A/Japan/305/57) immune sera. This antibody reactivity suggests the possibility of a shared antigenic epitope or region between T and B cells, and therefore provides new insight in our understanding of viral antigenicity.

Animals↗
PubMed1983

Antigenic variation in the influenza A virus nonstructural protein, NS1.

The antigenic structure of the nonstructural (NS1) protein encoded by influenza type A virus was examined using monoclonal antibodies prepared against purified NS1 inclusions isolated from the cytoplasm of infected cells. Topographical analysis by competitive radioimmunoassay indicated that three different overlapping antigenic regions were present on the NS1 of A/WSN/33 (H1N1). Immunoprecipitation studies using infected cell lysates showed that antigenic determinants on A/WSN/33 NS1 are common to NS1 proteins encoded by a wide range of viruses of human, swine, equine, and avian origin. Several avian strains, however, were found to encode antigenically variant NS1 proteins which had either extensive changes in one or more antigenic regions or small changes in epitopes within a region suggestive of antigenic drift. There was no correlation between surface antigen subtype and the antigenic profile of the NS1 protein. The antigenic relationships of NS1 proteins shown in this study are in agreement with the available sequence data.

Animals↗
PubMed1983

Phenotypic variation in influenza virus reassortants with identical gene constellations.

To determine which gene segments of influenza A viruses are responsible for the property of tissue tropism, reassortants were produced between the avian influenza strain, A/Mal/NY/6750/78 [H2N2] (Mal/NY), and a human strain, A/Udorn/307/72 [H3N2] (Udorn). The avian strain replicates in the intestinal tract of ducks and the human strain does not. Eight reassortants were shown by hybridization analysis to have the same gene constellation, having received hemagglutinin gene segment 4 from Udorn and the remaining seven gene segments from Mal/NY. With one exception, all reassortants containing the Udorn HA were restricted in their ability to traverse the digestive tract of ducks and replicate therein. The exception, reassortant R2, replicated to high titers in the intestinal tract. The R2 virus was shown to possess a hemagglutinin molecule that was antigenically distinguishable from the Udorn parent with polyclonal and monoclonal antibodies. The virus was "nonavid" in reaction with antihemagglutinin antibodies and was phenotypically similar to avian influenza viruses. The results suggest that the R2 hemagglutinin has undergone mutation(s) altering tissue tropism and antigenic properties of the virus. These studies illustrate the importance of the hemagglutinin gene in determining tissue tropism and present an example of phenotypic variation in a virus population with the same gene constellation but do not exclude a requirement for other gene products.

Animals↗
PubMed1983

Sequence of the hemagglutinin gene of influenza virus A/Memphis/1/71 and previously uncharacterized monoclonal antibody-derived variants.

A cDNA copy of the hemagglutinin (HA) gene of Mem/1/71HA Bel/42NA was cloned into the plasmid pBR322. The cloned gene contained the coding information for all of the HA except for the hydrophobic membrane insertion sequence of the C-terminus of HA2. The cloned HA DNA was used to make HA-specific primers from restriction fragments for internal priming on the viral RNA. These primers were used to sequence the complete HA gene of Mem/1/71, and to determine nucleotide sequence changes in four previously uncharacterized monoclonal antibody generated variants of Mem/1/71 (Webster, Laver, Air, Ward, Van Wyke, and Gerhard, Ann. N.Y. Acad. Sci. 354, 142-161, 1980).

Antibodies, Monoclonal↗
PubMed1983

Altered tissue tropism of human-avian reassortant influenza viruses.

Avian influenza viruses replicate to high titers in the cells lining the intestinal tract of birds; however, human strains do not. A series of reassortant viruses with all six internal genes from an avian strain and one or both genes for the surface antigens from a human strain failed to transit and infect the intestinal tracts of ducks. However, these reassortants did replicate in the bursa of ducks after rectal inoculation. These studies provide the first evidence that the hemagglutinin and neuraminidase are critical for the enterotropism of avian viruses but are not essential for replication in other avian tissues.

Animals↗
PubMed1983

Changes in the antigenicity of the hemagglutinin molecule of H3 influenza virus at acidic pH.

In order to determine the location and biological significance of the acid-induced conformational change in influenza virus hemagglutinin (HA) reported by Skehel et al., monoclonal antibodies were prepared to the molecule before and after treatment at pH 5.0. These antibodies together with monoclonal antibodies to the different antigenic regions of the H3 HA were used in immunoprecipitation and ELISA binding studies to show that antigenic changes accompanied the conformational change in the HA. Treatment at pH 5.2 or less exposed new determinants on the HA while two antigenic regions, located at the tip and interface of the molecule at neutral pH, were lost or modified. Antigenic sites in the loop and hinge regions defined by the available monoclonal antibodies were not affected by the conformational change. Monoclonal antibodies specific for the acid-induced conformation efficiently inhibited hemagglutination of the virus at low pH but were extremely poor inhibitors of virus-induced red blood cell hemolysis at its pH optimum of 5.1. These antibodies were unable to neutralize viral infectivity under neutral or acidic conditions. Antibodies specific for the non-acid-treated HA conformation failed to inhibit hemagglutination at low pH values but were able to both inhibit hemolysis of red blood cells and neutralize virus infectivity. Residual unmodified HA after pH 5.0 treatment could explain the inhibition of hemolysis and infectivity by monoclonal antibodies in each of the different antigenic areas.

Animals↗
PubMed1983

Swine influenza-like viruses in turkeys: potential source of virus for humans?

Influenza A viruses (subtype H1N1), recently isolated from turkeys in different areas of the United States, were determined to be closely related to strains typically associated with pigs. This conclusion was based on comparisons of H1N1 isolates from pigs, humans, ducks, and turkeys with polyclonal and monoclonal antibodies, RNA-RNA competitive hybridization, and replication studies. One of the H1N1 isolates from turkeys caused influenza in a laboratory technician, who displayed fever, respiratory illness, virus shedding, and seroconversion. These results suggest that turkeys as well as pigs are involved in the maintenance of these viruses and their transmission to humans.

Animals↗
PubMed1983

Inhibition of virus-induced hemolysis with monoclonal antibodies to different antigenic areas on the hemagglutinin molecule of A/seal/Massachusetts/1/80 (H7N7) influenza virus.

A/seal/Massachusetts/1/80 (H7N7) influenza virus caused maximal hemolysis at pH 5.9 Monoclonal antibodies to each of the four nonoverlapping antigenic areas on the hemagglutinin molecule of the virus inhibited the hemolysis whereas those belonging to two of the groups did not inhibit hemagglutination of the virus. Hemolysis also occurred when the virus was incubated at pH 5.9 prior to addition of erythrocytes. Such hemolysis caused by acid-treated virus was inhibited with the antibodies as well. At pH 5.9, hemagglutination of neither intact virus nor hemagglutinin rosettes was inhibited with any of the monoclonal antibodies, indicating conformational change in the hemagglutinin molecule, at this pH. On the other hand, hemagglutination-inhibition was observed when the antigens were incubated with the monoclonal antibodies at pH 7.0 and then the pH was later shifted to 5.9, suggesting that antibody-binding interferes with the conformational change in the hemagglutinin molecule at pH 5.9. The present findings indicate that antibodies to the hemagglutinin of influenza virus can inhibit virus-induced hemolysis by blocking conformational change in the hemagglutinin molecule and blocking later step of fusion than the conformational change, in addition to blocking attachment of virus to the receptor of erythrocytes.

Animals↗