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R G Webster

Publications and source records attributed to R G Webster.

At least 145 records · Page 8Linked to original sources
PubMed1994

Emergence of a potentially pathogenic H5N2 influenza virus in chickens.

Highly pathogenic influenza A viruses periodically infect both humans and nonhuman animals, including chickens. To gain insight into the origin of influenza outbreaks in poultry, we investigated two H5N2 viruses, A/chicken/Pennsylvania/13609/93 (Ck/PA/93) and A/chicken/Florida/25717/93 (Ck/FLA/93), that had been isolated in live-bird markets in Pennsylvania and Florida during surveillance studies in 1993. Phylogenetic analysis of the HA genes of these isolates, as well as H5N2 viruses isolated from ruddy turnstone surfbirds in 1991 (A/ruddy turnstone/Delaware/244/91 [RT/DE/91]) and other known H5 strains, indicated that Ck/PA/93 and Ck/FLA/93 share a common ancestor with RT/DE/91 and did not originate from A/chicken/Pennsylvania/1370/83 (Ck/PA/1370/83), which devastated chicken populations in 1983-1984. Both isolates were nonpathogenic in chickens by experimental infection and their HA protein (HA0) could not be cleaved into HA1 and HA2 without trypsin. The sequences at the HA cleavage sites of Ck/PA/93 and Ck/FLA/93 (R-K-T-R) appear to be intermediate between those of virulent and avirulent viruses, raising the possibility that a single mutation could promote virulence in chickens. We therefore recommend eradication of such viruses as soon as they appear.

Amino Acid Sequence↗
PubMed1994

Infection of children with avian-human reassortant influenza virus from pigs in Europe.

Pigs have been proposed to act as the intermediate hosts in the generation of pandemic human influenza strains by reassortment of genes from avian and human influenza virus strains. The circulation of avian-like H1N1 influenza viruses in European pigs since 1979 and the detection of human-avian reassortants in pigs raises the question of whether these viruses actually have the potential to transmit and cause disease in humans. We now report the serologic and genetic characterization of two human influenza A viruses (A/Netherlands/5/93 [H3N2] and A/Netherlands/35/93 [H3N2]) that caused influenza in children in The Netherlands in 1993. The results show that these viruses are human-avian ressortants that were generated and currently still are circulating in European swine. This shows the pivotal role that pigs can play in the generation and transmission of avian influenza virus genes to humans and their potential to generate a new human pandemic strain.

Animals↗
PubMed1994

Influenza virus subtypes in aquatic birds of eastern Germany.

We report the findings of a 12-year surveillance study (1977-89) of avian influenza A viruses in eastern Germany. Viruses were isolated directly from feral ducks (n = 236) and other wild birds (n = 89); from domestic ducks (n = 735) living on a single farm; and from white Pekin ducks (n = 193) used as sentinels for populations of wild aquatic birds; mainly sea birds. The efficiency of virus isolation was 9.9% overall, with considerable variability noted among species: 8.7% in wild ducks, 0.9% in other feral birds and 38% in Pekin ducks. Use of sentinel ducks in wild pelagic bird colonies improved virus detection rates fivefold, suggesting that this approach is advantageous in ecological studies. Among the 40 different combinations of hemagglutinin (HA) and neuraminidase (NA) subtypes we identified, H6N1 predominated (23.6% for all avian species), followed by H4N6 (11%). Among individual species, the frequency profiles favored H2N3 (20.8%) and H4N6 (20.3%) in feral ducks; H7N7 (22.3%), H4N6 (24.4%) and H2N3 (10.4%) in Pekin ducks used as sentinels; and H6N1 (34.8%) and H6N6 (15.1%) in domestic ducks maintained on a single farm. By relying on sentinel birds for serological assays, it was possible to trace an "influenza season" in feral swan populations, beginning in August and continuing through the winter months. Comparison of subtype distribution of influenza viruses for Europe and North America showed significant differences. This supports the fact of two geographically distinct gene pools of influenza viruses in birds connected with their distinct flyways of each hemisphere. The high frequency of isolation of H2 influenza viruses is of considerable interest to those interested in the recycling of this subtype in humans. Similarly the frequent isolation of H7N7 influenza viruses raises concern about reservoirs of potentially pathogenic influenza virus for domestic poultry. Our results confirm the existence of a vast reservoir of influenza A viruses in European aquatic birds, which possesses sufficient diversity to account for strains that infect lower animals and humans.

Animals↗
PubMed1994

Protection of ferrets against influenza challenge with a DNA vaccine to the haemagglutinin.

Immunization of ferrets with a plasmid DNA expressing influenza virus haemagglutinin (pCMV/H1 DNA) provided complete protection from challenge with the homologous A/PR/8/34 (H1N1) influenza virus. Delivery of DNA-coated gold beads by gene gun to the epidermis was much more efficient than intramuscular delivery of DNA in aqueous solution. The antibody response induced by DNA delivered by gene gun was more cross-reactive than DNA delivered in aqueous solution or after natural infection. This novel approach to vaccination against influenza may afford broader protection against antigenic drift than that provided by natural infection.

Animals↗
PubMed1994

Sialoglycoproteins that bind influenza A virus and resist viral neuraminidase in different animal sera.

Sialoglycoproteins that are resistant to degradation by viral neuraminidase can effectively neutralize influenza A viruses, because they bind irreversibly to the viruses. To detect such proteins in animal sera, we developed an immunochemical assay based on Western blotting techniques. We assessed the binding activity of sialoglycoproteins in sera from nine different animals toward the A/Aichi/2/68 (H3N2) and A/PR/8/34 (H1N1) strains of influenza virus, with or without viral and bacterial neuraminidase treatment. Using this assay, we found that animal sera contain a spectrum of sialoglycoproteins defined by differing abilities to bind influenza A viruses and to resist the viral neuraminidase. Structural analysis of these inhibitors would provide useful information for the development of anti-influenza virus compounds.

Animals↗
PubMed1994

Potential for transmission of avian influenza viruses to pigs.

Pandemic strains of influenza A virus arise by genetic reassortment between avian and human viruses. Pigs have been suggested to generate such reassortants as intermediate hosts. In order for pigs to serve as 'mixing vessels' in genetic reassortment events, they must be susceptible to both human and avian influenza viruses. The ability of avian influenza viruses to replicate in pigs, however, has not been examined comprehensively. In this study, we assessed the growth potential of 42 strains of influenza virus in pigs. Of these, 38 were avian strains, including 27 with non-human-type haemagglutinins (HA; H4 to H13). At least one strain of each HA subtype replicated in the respiratory tract of pigs for 5 to 7 days to a level equivalent to that of swine and human viruses. These results indicate that avian influenza viruses with or without non-human-type HAs can be transmitted to pigs, thus raising the possibility of introduction of their genes into humans. Sera from pigs infected with avian viruses showed high titres of antibodies in ELISA and neutralization tests, but did not inhibit haemagglutination of homologous viruses, cautioning against the use of haemagglutination-inhibition tests to identify pigs infected with avian influenza viruses. Co-infection of pigs with a swine virus and with an avian virus unable to replicate in this animal generated reassortant viruses, whose polymerase and HA genes were entirely of avian origin, that could be passaged in pigs. This finding indicates that even avian viruses that do not replicate in pigs can contribute genes in the generation of reassortants.

Animals↗
PubMed1994

Antigenicity of the N8 influenza A virus neuraminidase: existence of an epitope at the subunit interface of the neuraminidase.

To locate antigenic epitopes on the N8 neuraminidase (NA), we generated a panel of 97 monoclonal antibodies (MAbs), 66 of which inhibited NA activity (NI antibodies). Three groups of NI MAbs were identified from their different reactivities with escape mutants. Group 1 antibodies recognized the peptide loop containing residues 344 to 346, which appears to be an immunodominant region on the rim of the enzyme center of the N8 NA. Group 2 antibodies recognized a novel epitope containing residues 150, 199, 367, 399, and 400 (N2 numbering). From the location of these residues on the three-dimensional structure of the N8 NA, the epitope appears to be located at the interface of two adjacent monomers in the tetrameric NA, one contributing residues 150 and 199 and the other contributing residues 367 and 399 to 400. The available evidence indicates that the MAbs of this group react with the NA only after it is fully assembled. The third group of antibodies recognized the peptide loops containing residues 367 and 399 to 400. All of the amino acid substitutions in N8 escape mutants which affect the NI activity of antibodies were located in the peptide loops known to form epitopes in the N2 and N9 subtypes, indicating that antigenic regions in the NA head inducing NI antibodies appear to be similar among different subtypes of influenza A viruses. The MAbs used in this study will be valuable in studying the role of each N8 NA epitope in host immune defense systems and in the kinetics analysis of the biosynthesis of the enzyme.

Amidohydrolases↗
PubMed1994

Subtype cross-reactive, infection-enhancing antibody responses to influenza A viruses.

Antibody-dependent enhancement of the uptake of influenza A virus by Fc receptor-bearing cells was analyzed by using virus strains of the three human influenza A virus subtypes, A/PR/8/34 (H1N1), A/Japan/305/57 (H2N2), and A/Port Chalmers/1/73 (H3N2). Immune sera obtained from mice following primary infection with an H1N1, H2N2, or H3N2 subtype virus neutralized only virus of the same subtype; however, immune sera augmented the uptake of virus across subtypes. Immune sera from H1N1-infected mice augmented uptake of the homologous (H1N1) and H2N2 viruses. Antisera to the H2N2 virus augmented the uptake of virus of all subtypes (H1N1, H2N2, or H3N2). Antisera to the H3N2 virus augmented the uptake of the homologous (H3N2) and H2N2 viruses. These results show that subtype cross-reactive, nonneutralizing antibodies augment the uptake of influenza A virus strains of different subtypes. Antibodies to neuraminidase may contribute to the enhanced uptake of viruses of a different subtype, because N2-specific monoclonal antibodies augmented the uptake of both A/Japan/305/57 (H2N2) and A/Port Chalmers/1/73 (H3N2) viruses.

Animals↗
PubMed1993

DNA vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations.

Plasmid DNAs expressing influenza virus hemagglutinin glycoproteins have been tested for their ability to raise protective immunity against lethal influenza challenges of the same subtype. In trials using two inoculations of from 50 to 300 micrograms of purified DNA in saline, 67-95% of test mice and 25-63% of test chickens have been protected against a lethal influenza challenge. Parenteral routes of inoculation that achieved good protection included intramuscular and intravenous injections. Successful mucosal routes of vaccination included DNA drops administered to the nares or trachea. By far the most efficient DNA immunizations were achieved by using a gene gun to deliver DNA-coated gold beads to the epidermis. In mice, 95% protection was achieved by two immunizations with beads loaded with as little as 0.4 micrograms of DNA. The breadth of routes supporting successful DNA immunizations, coupled with the very small amounts of DNA required for gene-gun immunizations, highlight the potential of this remarkably simple technique for the development of subunit vaccines.

Animals↗
PubMed1993

Neutralization and infection-enhancement epitopes of influenza A virus hemagglutinin.

We studied 18 mAb specific for the H3 hemagglutinin (HA) to analyze the relationships between neutralizing and infection-enhancing epitopes on the influenza HA. The mAb could be separated into three groups based on their neutralization (N) and enhancement (E) activity in assays with the prototype virus; group I (N+E+), group II (N+E-) and group III (N +/- E+). A representative mAb from each group was analyzed for its effect on the infectivity of a group of escape mutants, selected with mAb to three sites on the H3 HA, and wild-type H3 viruses to define the relationship between neutralizing epitopes and infection-enhancing epitopes. A group I mAb (N+E+), which recognized site A on the HA, neutralized virus infection at high concentrations of antibody and enhanced virus infection at low concentrations. A group II mAb (N+E-), which recognized site B, had high neutralizing but no enhancing activity. The failure of this mAb to enhance virus uptake was a result of the inability of the Fc portion of virus-mAb complexes to bind to Fc receptor. The addition of anti-murine IgG as a second antibody to these virus-mAb complexes augmented virus uptake. A group III mAb (N +/- E+), which recognized site C, had enhancing but little neutralizing activity. This is the first definition of distinct epitopes that induce neutralizing and/or enhancing mAb.

Amino Acid Sequence↗
PubMed1993

Molecular and biological changes in the cold-adapted "master strain" A/AA/6/60 (H2N2) influenza virus.

The live cold-adapted (ca) A/AA/6/60 influenza vaccine is being commercially developed for worldwide use in children and is being used as a model for other live vaccines. Although it has been proven safe and immunogenic, the molecular basis of cold adaptation has never been determined. To identify sequence changes responsible for cold adaptation, we have compared the sequence of the master ca vaccine strain to its progenitor wild-type virus, wt A/AA/6/60 E2 (wt2). Only 4 nt differences encoding 2 aa differences were found in three gene segments. Computer-predicted RNA folds project different secondary structures between the ca and wt2 molecules based on the two silent differences between them. Genes coding for the acidic polymerase, matrix, and nonstructural proteins are identical between the two viruses. The few differences found in the ca A/AA/6/60 virus after its long stepwise passage at 25 degrees C in primary chicken kidney cells suggest that cold adaptation resulted in greater genetic stability for the highly variable RNA genome.

Adaptation, Physiological↗
PubMed1993

Recombinant antineuraminidase single chain antibody: expression, characterization, and crystallization in complex with antigen.

The variable heavy (VH) and variable light (VL) genes of NC10, a monoclonal antibody with specificity toward N9 neuraminidase (NA), were cloned and sequenced. A single chain Fv (scFv) fragment of NC10, consisting of VH and VL domains joined by a peptide linker, was designed, constructed and expressed in the E. coli expression vector pPOW. The N-terminal secretion signal PelB directed the synthesized protein into the periplasm where it was associated with the insoluble membrane fraction. An octapeptide (FLAG) tail was fused to the C-terminus of the single chain Fv to aid in its detection and remained intact throughout the protein purification process. NC10 scFv was purified by solubilization of the E. coli membrane fraction with guanidinium hydrochloride followed by column chromatography. The purified NC10 scFv showed binding affinity for its antigen, NA, 2-fold lower than that of the parent Fab. The complex between NA and the scFv has been crystallized by the vapor diffusion method. The crystals are tetragonal, space group P42(1)2, with unit cell dimensions a = b = 141 A, c = 218 A.

Amino Acid Sequence↗
PubMed1993

Genetic reassortment between avian and human influenza A viruses in Italian pigs.

Pandemic strains of influenza A virus arise by genetic reassortment between avian and human viruses. To examine the possibility that pigs serve as "mixing vessels" for such reassortment events (Scholtissek et al., Virology 147, 287-294, 1985), we phylogenetically analyzed the internal protein genes of classic H1N1, avian-like H1N1, and human-like H3N2 viruses circulating among Italian pigs. The results show that human-like H3N2 strains isolated from 1985 to 1989 contained the internal protein genes of avian-like H1N1 viruses, whereas those isolated in 1977 and 1983 did not. Thus, at some time between 1983 and 1985, genetic reassortment took place between avian- and human-like viruses in Italian pigs. This study provides the first evidence supporting genetic reassortment between human and avian viruses in a natural swine environment.

Animals↗
PubMed1993

Phylogenetic analysis of the N8 neuraminidase gene of influenza A viruses.

Phylogenetic analysis of the N8 neuraminidase (NA) genes from 18 influenza A viruses, representing equine and avian hosts in different geographic locations, revealed three major lineages: (i) currently circulating equine 2 viruses; (ii) avian viruses isolated in the Eurasian region, including A/Equine/Jilin/1/89, a recent avian-like N8 isolate found in horses in China; and (iii) avian viruses isolated in North America. Comparison of mutation rates indicated that avian N8 genes have evolved more slowly than their equine counterparts. That is, in both avian lineages, 72% of the nucleotide changes were silent in the terminal branches of the phylogenetic tree, whereas in equine 2 viruses, 59% of the nucleotide changes were silent. This suggests greater selective pressure on the NA gene from the mammalian immune system, leading to progressive evolution. Alternatively, the slower mutation rate for avian N8 genes could reflect a selective advantage gained from a longer, continuous span of evolution. The shape of the phylogenetic tree, the evolutionary rate, and the calculated date of origin for the N8 equine 2 virus lineage were comparable to findings for the equine 2 virus hemagglutinin (HA) gene (Bean et al., J. Virol. 66, 1129-1138, 1992). This suggests that both viral membrane glycoproteins of equine 2 viruses have evolved together and have been subjected to similar levels of selective pressure. Several amino acid residues were found to differ among the three host-specific lineages, but they may not be involved in host restriction of the NA, as they are shared by EQ/Jilin/1/89 and viruses of avian origin. The present findings complement detailed structural information on the N2 and N9 subtypes and should prove valuable in understanding future X-ray diffraction studies of N8 crystals.

Amino Acid Sequence↗
PubMed1993

Origin of the pandemic 1957 H2 influenza A virus and the persistence of its possible progenitors in the avian reservoir.

H2N2 influenza A viruses caused the Asian pandemic of 1957 and then disappeared from the human population 10 years later. To assess the potential for similar outbreaks in the future, we determined the antigenicity of H2 hemagglutinins (HAs) from representative human and avian H2 viruses and then analyzed the nucleotide and amino acid sequences to determine their evolutionary characteristics in different hosts. The results of longitudinal virus surveillance studies were also examined to estimate the prevalence of avian H2 isolates among samples collected from wild ducks and domestic poultry. Reactivity patterns obtained with a large panel of monoclonal antibodies indicated antigenic drift in the HA of human H2 influenza viruses, beginning in 1962. Amino acid changes were clustered in two regions of HA1 that correspond to antigenic sites A and D of the H3 HA. By contrast, the antigenic profiles of the majority of avian H2 HAs were remarkably conserved through 1991, resembling the prototype Japan 57 (H2N2) strain. Amino acid changes were distributed throughout HA1, indicating that antibodies do not play a major role in the selection of avian H2 viruses. Phylogenetic analysis revealed two geographic site-specific lineages of avian H2 HAs: North American and Eurasian. Evidence is presented to support interregion transmission of gull H2 viruses. The human H2 HAs that circulated in 1957-1968 form a separate phylogenetic lineage, most closely related to the Eurasian avian H2 HAs. There was an increased prevalence of H2 influenza viruses among wild ducks in 1988 in North America, preceding the appearance of H2N2 viruses in domestic fowl. As the prevalence of avian H2N2 influenza viruses increased on turkey farms and in live bird markets in New York City and elsewhere, greater numbers of these viruses have come into direct contact with susceptible humans. We conclude that antigenically conserved counterparts of the human Asian pandemic strain of 1957 continue to circulate in the avian reservoir and are coming into closer proximity to susceptible human populations.

Americas↗
PubMed1993

Influence of host cell-mediated variation on the international surveillance of influenza A (H3N2) viruses.

Growth of clinical specimens of influenza viruses in eggs can result in the selection of antigenic variants distinct from corresponding viruses grown in mammalian tissue culture. To evaluate the contribution of host cell selection on the antigenic diversity of human influenza isolates, as seen in annual surveillance studies, viruses grown in embryonated eggs were compared by antigenic and genetic analyses with their mammalian tissue culture-grown counterparts. Clinical specimens were gathered from around the world from late 1987 to 1990 and the antigenicity of isolated viruses was assessed by hemagglutination-inhibition assays using immune ferret sera as is currently performed for routine surveillance and the selection of vaccine strains. In addition, viruses were assessed using a panel of anti-H3 HA monoclonal antibodies. The extent of antigenic variation exhibited by the egg-grown strains was far greater than the relative antigenic homogeneity of the tissue culture-grown viruses. Nucleotide sequence analysis of HA1 gene PCR products of 28 MDCK cell and egg derived pairs allowed identification of amino acid substitutions responsible for the antigenic differences observed and the adaptation to growth in eggs. Among these substitutions was a change at amino acid position 186 of HA1 (Ser in tissue culture viruses and lle in egg-grown viruses) which was observed at relatively high frequency. Egg- and MDCK-grown pairs with this single amino acid difference were classified into distinct antigenic groups by ferret sera raised to WHO reference viruses. Given the additional antigenic diversity observed among egg-grown strains, considerable care should be taken in the selection of reference and vaccine strains grown in eggs. Rapid sequence comparisons of MDCK- and egg-grown viruses allow identification of variants arising through egg selection and will prove to be a useful adjunct to antigenic surveillance for the selection of reference and vaccine strains.

Africa↗
PubMed1993

Binding affinity of influenza virus N9 neuraminidase with Fab fragments of monoclonal antibodies NC10 and NC41.

Sedimentation equilibrium centrifugation has been applied to determine the affinity and stoichiometry of the interaction between Fab fragments, derived from monoclonal antibodies NC10 and NC41, with influenza virus neuraminidase N9 isolated from either tern or whale. Although the two neuraminidase epitopes recognized by NC10 and NC41 Fab overlap, crystallographic studies have shown that the modes of binding of each Fab are different. The sedimentation equilibrium experiments described here reveal that the binding affinities are also different, with NC10 Fab binding more strongly to each neuraminidase. Furthermore, comparison of the affinity of binding of each antibody fragment reveals a stronger interaction with tern neuraminidase than with whale neuraminidase. Although the respective epitopes recognized by each antibody on the two antigens are similar, this technique shows that they do nevertheless possess sufficient differences to affect significantly the binding of antibody.

Animals↗