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R G Webster

Publications and source records attributed to R G Webster.

At least 199 records · Page 11Linked to original sources
PubMed1991

Isolation of a simian immunodeficiency virus related to human immunodeficiency virus type 2 from a west African pet sooty mangabey.

Two of 25 healthy pet sooty mangabey (SM) monkeys (Cercocebus atys) living in West Africa were seropositive by immunoblot when surveyed for antibody to simian immunodeficiency virus of macaques (SIVmac). SIVsmLIB1 was isolated from one of the pet sooty mangabeys. Nucleotide sequence data showed that this isolate is a member of the SIVsm/human immunodeficiecy virus type 2 (HIV-2)/SIVmac group of primate lentiviruses. Furthermore, sequence comparisons revealed extensive genetic diversity among SIVsm isolates similar to that observed previously in SIV isolates from naturally infected African green monkeys. These observations provide additional evidence for monkey-human cross-species transmission of SIVsm as the source of HIV-2 infection of human.

Animals↗
PubMed1990

Measurement of anti-influenza neuraminidase antibody using a peroxidase-linked lectin and microtitre plates coated with natural substrates.

Neuraminidase-induced removal of sialic acid from natural substrates (desialylation) unmasks saccharides that are specifically recognized by the lectin peanut agglutinin (PNA). We demonstrate that, when a neuraminidase substrate is coated on to the wells of a microplate, it is possible to quantitate the binding of PNA to the desialylated substrate using a peroxidase-conjugated PNA (Po-PNA). The amount of bound PNA correlated directly with the amount of sialic acid removed from the substrate and therefore with the neuraminidase activity. By reacting with specific epitopes that are located near to the enzyme active site, anti-neuraminidase antibodies are capable of inhibiting the virus-induced desialylation of the substrate. Such antibodies therefore reduce the binding of Po-PNA. The advantage of this assay is that since different natural substrates for neuraminidase (erythrocytes, fetuin or gangliosides) can be used to coat the microplates, the capacity of anti-neuraminidase antibody to inhibit the neuraminidase activity towards different types of sialoglycoconjugates can be evaluated. Anti-hemagglutinin or non-specific anti-neuraminidase antibody have no interfering reactivity.

Animals↗
PubMed1990

Conditional expression of foreign genes by temperature-sensitive mutants of vaccinia virus.

To assess the utility of two temperature-sensitive (ts) mutant vaccinia viruses as vectors for the conditional in vitro expression of recombinant foreign genes, we have studied the kinetics of expression of foreign genes incorporated into these viruses. At nonpermissive temperature, 40 degrees C, these viruses were defective either in DNA synthesis or in virus assembly. Foreign gene expression was affected by the nature of the ts lesion and by the nature of the vaccinia promoter positioned upstream from the foreign gene. With both vector viruses, a foreign gene controlled by the p7.5 early-late promoter was expressed at both 33 degrees and 40 degrees C. With the DNA synthesis-defective vector virus, foreign gene expression controlled by the p11 DNA synthesis-dependent late promoter was inhibited at 40 degrees C, but could be turned on by shift to 33 degrees C. This ts expression system provides an alternative to use of drugs that inhibit DNA synthesis as a means for experimental manipulation of gene expression. Both vector viruses can be used with existing vaccinia virus expression technology.

DNA, Recombinant↗
PubMed1990

New crystalline forms of neuraminidase of type B human influenza virus.

New crystalline forms of tetrameric neuraminidase heads from two strains (B/Lee/40 and B/mem/89) of type B human influenza virus were obtained and the crystals diffracted using X-rays to 2.5 A resolution without lattice disorder. The new B/Lee/40 crystalline form is tetragonal, space group P42(1)2, with unit cell dimensions a = 123.8 A, c = 71.8 A. The B/mem/89 crystalline form is also tetragonal, space group I422, with unit cell dimensions a = 122.9 A, c = 164.4 A. There is one neuraminidase monomer per asymmetric unit in both forms.

Crystallization↗
PubMed1990

Lack of persistence of influenza virus genetic information in ducks.

Clearance of infectious virus and RNA occurred concurrently after oral infection of ducks with influenza virus. There was no evidence from polymerase chain reaction (PCR) analysis of the hemagglutinin (HA) gene for persistence of viral genetic information. No detectable RNA was found in the spleen indicating processing of antigen near the site of replication.

Animals↗
PubMed1990

Molecular characterization of a new hemagglutinin, subtype H14, of influenza A virus.

Two influenza A viruses whose hemagglutinin (HA) did not react with any of the reference antisera for the 13 recognized HA subtypes were isolated from mallard ducks in the USSR. Antigenic analysis by hemagglutination inhibition and double immunodiffusion tests showed that the HAs of these viruses are similar to each other but distinct from the HAs of other influenza A viruses. Nucleotide sequence analysis showed that these HA genes differ from each other by only 21 nucleotides. However, they differ from all other HA subtypes at the amino acid level by at least 31% in HAI. Thus, we propose that the HAs of these viruses [A/Mallard/Gurjev/263/82 (H14N5) and A/Mallard/Gurjev/244/82 (H14N6) belong to a previously unrecognized subtype, and are designated H14. Unlike any other HAs of influenza viruses, the H14 HAs contained lysine at the cleavage site between HA1 and HA2 instead of arginine. Experimental infection of domestic poultry and ferrets with A/Mallard/Gurjev/263/82 (H14N5) showed that the virus is avirulent for these animals. Based on comparative sequence analysis of different HA genes, it is suggested that differences of 30% or more at the amino acid level in HA1 constitute separate subtypes. Phylogenetic analysis of representatives of each HA subtype showed that H14 is one of the most recently diverged lineages while H8 and H12 branched off early during the evolution of the HA subtypes. These latter two subtypes (H8 and H12) have been isolated very infrequently in recent years, suggesting that these old subtypes may be disappearing from the influenza reservoirs in nature.

Amino Acid Sequence↗
PubMed1990

Derivation of the nucleoproteins (NP) of influenza A viruses isolated from marine mammals.

The nucleoprotein (NP) genes of influenza viruses were sequenced from a variety of virus isolates derived from marine mammals: whales from the Pacific and Atlantic oceans, seal and gull from the Western Atlantic, and a tern from the Caspian Sea. In comparison to published NP sequences, we found pairs of NPs derived from avian and marine mammal isolates to be closely related, e.g., the gull-whale and mallard-seal pairs from the Atlantic Coast of the USA and the tern-Pacific Ocean whale pair of the Eastern Hemisphere. Our analysis suggests that influenza viruses have been independently introduced into marine mammals from avian sources for each of our three examples. Furthermore, the closeness of the relationship in these avian-mammalian NP pairs indicates that the introductions are relatively recent. The sequences of these marine mammal NPs are avian-like and can be clearly distinguished from human NPs. Our results provide further support of interspecies transmission of influenza A viruses from the avian host reservoir directly to mammalian hosts.

Amino Acid Sequence↗
PubMed1990

Antigenic, sequence, and crystal variation in influenza B neuraminidase.

The neuraminidase (NA) genes of influenza B viruses B/Maryland/59, B/Hong Kong/8/73, B/Singapore/222/79, B/Oregon/5/80, B/USSR/100/83, B/Victoria/3/85, B/Leningrad/179/86, B/Memphis/6/86, and B/Memphis/3/89 have been sequenced. The deduced amino acid sequences show high variability in the stalk domain of the NA, but a surprising degree of sequence conservation in the head region which carries all the antigenic and enzyme activity. The variable region coding for the neuraminidase stalk also translates into a variable section in the overlapping NB polypeptide, which is coded from a second reading frame that overlaps the first 100 amino acids of NA. The influenza B NAs are antigenically distinguishable with monoclonal antibodies in neuraminidase-inhibition tests, even when there is only one amino acid sequence difference. However, seven of nine escape mutants selected with monoclonal antibodies were distinguished only by the antibody used for selection. When NA heads of influenza B viruses are crystallized, there are remarkable differences in crystal morphology between neuraminidases which have very few sequence changes.

Amino Acid Sequence↗
PubMed1990

Evolutionary changes in influenza B are not primarily governed by antibody selection.

Influenza B viruses evolve more slowly than human influenza A, but no reasons for the difference have been established. We have analyzed sequence changes in the hemagglutinin and neuraminidase of influenza B viruses (and have determined four hemagglutinin sequences, of B/Bonn/43, B/USSR/100/83, B/Victoria/3/85, and B/Memphis/6/86) in relation to antigenic properties and compared these with similar analyses of variation in influenza A antigens. Independent of the slower rate of change in influenza B antigens, only approximately 30% of nucleotide changes in either the hemagglutinin or neuraminidase gene sequence result in amino acid changes in the protein, whereas in influenza A 50% of nucleotide changes result in altered amino acids. Thus, there is less selection for change, or less tolerance to change, in the influenza B antigens. This is similar to findings with influenza C and findings with influenza A viruses that replicate in lower animals and birds and is closer to the type of variation found in other RNA viruses. We propose that human influenza A is unique in that it is the only virus group in which antibody selection dominates evolutionary change.

Antibodies, Viral↗
PubMed1990

Molecular analysis of the haemagglutinin gene of an avian H1N1 influenza virus.

This study presents the first nucleotide sequence and deduced primary amino acid sequence of a subtype H1 haemagglutinin from the avian influenza virus A/duck/Alberta/35/76 (H1N1). The molecule is structurally, antigenically and molecularly similar to H1 haemagglutinins of human viruses but sequence homology differences indicate that there has not been a recent transfer of haemagglutinin genetic information between them.

Amino Acid Sequence↗
PubMed1990

Evolution of influenza A virus PB2 genes: implications for evolution of the ribonucleoprotein complex and origin of human influenza A virus.

Phylogenetic analysis of 20 influenza A virus PB2 genes showed that PB2 genes have evolved into the following four major lineages: (i) equine/Prague/56 (EQPR56); (ii and iii) two distinct avian PB2 lineages, one containing FPV/34 and H13 gull virus strains and the other containing North American avian and recent equine strains; and (iv) human virus strains joined with classic swine virus strains (i.e., H1N1 swine virus strains related to swine/Iowa/15/30). The human virus lineage showed the greatest divergence from its root relative to other lineages. The estimated nucleotide evolutionary rate for the human PB2 lineage was 1.82 x 10(-3) changes per nucleotide per year, which is within the range of published estimates for NP and NS genes of human influenza A viruses. At the amino acid level, PB2s of human viruses have accumulated 34 amino acid changes over the past 55 years. In contrast, the avian PB2 lineages showed much less evolution, e.g., recent avian PB2s showed as few as three amino acid changes relative to the avian root. The completion of evolutionary analyses of the PB1, PB2, PA and NP genes of the ribonucleoprotein (RNP) complex permits comparison of evolutionary pathways. Different patterns of evolution among the RNP genes indicate that the genes of the complex are not coevolving as a unit. Evolution of the PB1 and PB2 genes is less correlated with host-specific factors, and their proteins appear to be evolving more slowly than NP and PA. This suggests that protein functional constraints are limiting the evolutionary divergence of PB1 and PB2 genes. The parallel host-specific evolutionary pathways of the NP and PA genes suggest that these proteins are coevolving in response to host-specific factors. PB2s of human influenza A viruses share a common ancestor with classic swine virus PB2s, and the pattern of evolution suggests that the ancestor was an avian virus PB2. This same pattern of evolution appears in the other genes of the RNP complex. Antigenic studies of HA and NA proteins and sequence comparisons of NS and M genes also suggest a close ancestry for these genes in human and classic swine viruses. From our review of the evolutionary patterns of influenza A virus genes, we propose the following hypothesis: the common ancestor to current strains of human and classic swine influenza viruses predated the 1918 human pandemic virus and was recently derived from the avian host reservoir.

Amino Acid Sequence↗
PubMed1990

Mechanism of antigenic variation in an individual epitope on influenza virus N9 neuraminidase.

Monoclonal antibodies which inhibit influenza virus neuraminidase (NA) and which therefore indirectly neutralize virus infectivity bind to epitopes located on the rim of the active-site crater. The three-dimensional structure of one of these epitopes, recognized by monoclonal antibody NC41, has previously been determined (W. R. Tulip, J. N. Varghese, R. G. Webster, G. M. Air, W. G. Laver, and P. M. Colman, Cold Spring Harbor Symp. Quant. Biol. 54:257-263, 1989). Nineteen escape mutants of influenza virus A/tern/Australia/G70c/75 (N9) NA selected with NC41 were sequenced. A surprising restriction was seen in the sequence changes involved. Ten mutants had a Ser-to-Phe change at amino acid 372, and six others had mutations at position 367. No escape mutants with changes at 369 or 370 were found, although these mutations were selected with other antibodies and rendered the epitope unrecognizable by antibody NC41. Another N9 NA, from A/ruddy turnstone/NJ/85, which differs by 14 amino acids from the tern virus NA, still bound antibody NC41. Epitope mapping by selecting multiple escape mutants with antibody NC41 thus identified only three of the five polypeptide loops on NA that contact the antibody. Escape mutants selected sequentially with three different monoclonal antibodies showed three sequence changes in two loops of the NC41 epitope. The multiple mutants were indistinguishable from wild-type virus by using polyclonal rabbit antiserum in double immunodiffusion tests, but NA inhibition titers were fourfold lower. The results suggest that although the NC41 epitope contains 22 amino acids, only a few of these are so critical to the interaction with antibody that a single sequence change allows selection of an escape mutant. In that case, the variety of amino acid sequence changes which can lead to polyclonal selection of new epidemic viruses during antigenic drift might be very limited.

Amino Acid Sequence↗
PubMed1990

Evolution of the nucleoprotein gene of influenza A virus.

Nucleotide sequences of 24 nucleoprotein (NP) genes isolated from a wide range of hosts, geographic regions, and influenza A virus serotypes and 18 published NP gene sequences were analyzed to determine evolutionary relationships. The phylogeny of NP genes was determined by a maximum-parsimony analysis of nucleotide sequences. Phylogenetic analysis showed that NP genes have evolved into five host-specific lineages, including (i) Equine/Prague/56 (EQPR56), (ii) recent equine strains, (iii) classic swine (H1N1 swine, e.g., A/Swine/Iowa/15/30) and human strains, (iv) gull H13 viruses, and (v) avian strains (including North American, Australian, and Old World subgroups). These NP lineages match the five RNA hybridization groups identified by W. J. Bean (Virology 133:438-442, 1984). Maximum nucleotide differences among the NPs was 18.5%, but maximum amino acid differences reached only 10.8%, reflecting the conservative nature of the NP protein. Evolutionary rates varied among lineages; the human lineage showed the highest rate (2.54 nucleotide changes per year), followed by the Old World avian lineage (2.17 changes per year) and the recent equine lineage (1.22 changes per year). The per-nucleotide rates of human and avian NP gene evolution (1.62 x 10(-3) to 1.39 x 10(-3) changes per year) are lower than that reported for human NS genes (2.0 x 10(-3) changes per year; D. A. Buonagurio, S. Nakada, J. D. Parvin, M. Krystal, P. Palese, and W. M. Fitch, Science 232:980-982, 1986). Of the five NP lineages, the human lineage showed the greatest evolution at the amino acid level; over a period of 50 years, human NPs have accumulated 39 amino acid changes. In contrast, the avian lineage showed remarkable conservatism; over the same period, avian NP proteins changed by 0 to 10 amino acids. The specificity of the H13 NP in gulls and its distinct evolutionary separation from the classic avian lineage suggests that H13 NPs may have a large degree of adaptation to gulls. The presence of avian and human NPs in some swine isolates demonstrates the susceptibility of swine to different virus strains and supports the hypothesis that swine may serve as intermediates for the introduction of avian influenza virus genes into the human virus gene pool. EQPR56 is relatively distantly related to all other NP lineages, which suggests that this NP is rooted closest to the ancestor of all contemporary NPs. On the basis of estimation of evolutionary rates from nucleotide branch distances, current NP lineages are at least 100 years old, and the EQPR56 NP is much older.(ABSTRACT TRUNCATED AT 400 WORDS)

Adaptation, Biological↗
PubMed1990

Direct sequencing of the HA gene of influenza (H3N2) virus in original clinical samples reveals sequence identity with mammalian cell-grown virus.

When influenza (H3N2) viruses from infected individuals are grown in embryonated chicken eggs, viruses are isolated which differ antigenically and structurally from viruses grown in mammalian Madin-Darby canine kidney (MDCK) cell culture [G.C. Schild, J.S. Oxford, J.C. de Jong, and R.G. Webster, Nature (London) 303:706-709, 1983]. To determine which of these viruses is most representative of virus replicating in the infected individual, a region of the HA gene of virus present in original clinical samples was amplified by using the polymerase chain reaction and sequenced directly. Comparison of 170 amino acid residues of HA1 flanking and containing the receptor-binding site and antigenic sites indicated that over this region, the HA of virus replicating in the infected individual was identical to that of virus after growth in MDCK cells and was distinct from the HA of viruses grown in eggs. Therefore, cultivation of human influenza H3N2 virus in mammalian MDCK cells results in a virus similar to the predominant population of virus found in the infected individual.

Amino Acid Sequence↗
PubMed1989

Three-dimensional structures of influenza virus neuraminidase-antibody complexes.

X-ray diffraction analysis of crystals of a monoclonal Fab fragment NC41 bound to a viral antigen, influenza virus neuraminidase, shows an epitope involving five surface loops of the antigen. In addition it reveals an unusual pairing pattern between the domains of light and heavy chains in the variable module of the antibody. We interpret this result to imply that association with antigen can induce changes in the quaternary structure of the Fab, through a sliding of domains at the variable light/variable heavy chains (VL-VH) interface. In addition, Fab binding has altered the conformation of some of the surface loops of the antigen. The structure of the NC10 Fab-neuraminidase complex has now also been solved. It binds an epitope that overlaps the NC41 epitope. In this structure, there is no electron density for the C-module of the Fab fragment, implying it is disordered in the crystal lattice. The implications of these, and other antibody-antigen structures, for immune recognition are discussed.

Antigen-Antibody Complex↗
PubMed1989

Origin of the hemagglutinin on A/Equine/Johannesburg/86 (H3N8): the first known equine influenza outbreak in South Africa.

A severe influenza outbreak occurred in horses in South Africa in 1986. The causative agent was identified as an influenza virus [A/Equine/Johannesburg/86 (H3N8)]. Antigenic analyses of the hemagglutinin (HA) with ferret antisera and monoclonal antibodies showed that the Eq/Johannesburg/86 virus is similar to recent equine H3 viruses. The nucleotide sequence analysis on the HA genes of Eq/Johannesburg/86 and other equine H3 influenza viruses, together with the epidemiological data, clearly demonstrated that the Eq/Johannesburg/86 virus was derived from a virus that had been circulating in horses in the United States in 1986-87. The epidemiological information suggests that the unusually severe influenza outbreak in South Africa may be due to the lack of immunity to these viruses in the horse population.

Amino Acids↗
PubMed1989

Antigenic and molecular characterization of subtype H13 hemagglutinin of influenza virus.

Influenza A viruses with subtype H13 hemagglutinin display an unusual host range. Although common in shorebirds, they are very rare or absent in wild ducks; additionally, H13 viruses have been isolated from a whale. To study the molecular basis for this host range, we have determined the complete nucleotide sequences of the hemagglutinin genes of three H13 influenza viruses from different species or geographical areas: A/gull/Maryland/77, A/gull/Astrachan (USSR)/84, and A/pilot whale/Maine/84. Based on the deduced amino acid sequences, H13 hemagglutinin shares the basic structure of other type A hemagglutinin subtypes such as H3, but has clearly diverged from other completely sequenced subtypes. Unique features of H13 hemagglutinin include the occurrence, near the receptor binding pocket, of residues Arg/Lys-227 and Trp-229 (H3 numbering); the significance of these are unknown. The sequence of the HA1-HA2 cleavage site resembles those of avirulent avian influenza viruses. The whale H13 hemagglutinin is similar to those from gulls, supporting the hypothesis that influenza viruses from avian sources can enter marine mammal populations but are probably not permanently maintained there. Antigenic analysis using a panel of monoclonal antibodies suggests that, like other subtypes, H13 viruses are heterogeneous, with different antigenic variants predominating in the eastern versus the western hemispheres.

Amino Acid Sequence↗