Biomedical subjects
Publications and source records attributed to R G Webster.
Influenza A viruses of the H13N2 and H13N9 subtypes were isolated from the lung and hilar node of a pilot whale. Serological, molecular, and biological analyses indicate that the whale isolates are closely related to the H13 influenza viruses from gulls.
Single-radial-immunodiffusion (SRD) provides a sensitive and reproducible in vitro assay for haemagglutinin (HA) concentration in inactivated influenza vaccines. The use of SRD for human influenza vaccine standardization and application for equine and avian influenza vaccines is discussed. In clinical trials, vaccine HA concentration measured by SRD has been shown to be directly related to antibody responses and to protection against challenge. The use of SRD may considerably reduce the usage of animals for potency testing of veterinary influenza vaccines.
The epidemiological features of the H5N2 outbreak of influenza in poultry were studied by sequencing the HA genes of several viruses isolated during the epidemic. Comparison of the nucleotide sequences of the HA genes indicated there was a single introduction of virulent virus. The variation rate (silent mutations) in the HA gene of the virulent Ck/Penn virus was 9.0 or 14.4% per 10 years depending on the viruses compared and was similar to that in H3 HA gene of human influenza A virus. The virulent and avirulent viruses isolated after October 1983 were derived from a common ancestoral virus and the virulent virus did not supersede the avirulent virus, instead, the virulent and avirulent viruses coexisted and evolved separately during the course of the epidemic. The evolutionary changes in the HA of H5N2 viruses that occurred during the epidemic permitted us to establish that a virus (A/Chick/Washington/84) that was isolated 8 months after the last H5N2 virus had been isolated from poultry in Pennsylvania belonged to the family of potentially dangerous H5N2 viruses and was a direct descendent of the virus that spread to Maryland and Virginia. All of the virulent Ck/Penn viruses retained the amino acid changes at residues 13 and 69 in the HA.
The genes coding for the neuraminidase (NA) enzyme in antigenic variants of influenza virus X-7(F1) (which contains the NA of A/RI/5+/57) are shorter than other N2 neuraminidase genes. When the parent X-7(F1) virus was cloned at limiting dilution prior to selection of variants with monoclonal antibodies, a minor component with a shorter neuraminidase gene was cloned out, and this deletion was retained in all the variants. Sequence analysis has shown that there is a single deletion, 54 nucleotides long, which occurs in the coding region of the NA. The 18 deleted amino acids are from the stalk region of the protein, which is a thin structure containing the four polypeptide chains of the tetrameric enzyme which separates the enzymatically and antigenically active "head" from the hydrophobic sequence embedded in the viral membrane. The deletion can be seen as a shortened stalk when rosettes of detergent-released NA are examined by electron microscopy, which confirms that the NA stalk is a highly extended structure. Although the more abundant hemagglutinin (HA) would probably extend beyond the shortened NA molecule on the surface of the virus, the NA head is still accessible to all of the available monoclonal antibodies to the different antigenic sites on the molecule. Enzymatic activity toward a small substrate molecule, N-acetyl neuraminyl lactose, is the same for both the parental X-7(F1) virus and the strain having the deleted NA; however, the latter virus is slower to cleave a large substrate, fetuin, and elutes from red cells at a reduced rate.
In 1943 Burnet reported on changes in the hemagglutinating properties of human influenza virus which occurred during adaptation of the virus to growth in chicken eggs. Only recently has direct evidence been presented that these changes affect the antigenic properties of the virus. Schild et al. (G. C. Schild, J. S. Oxford, J. C. deJong, and R. G. Webster (1983), Nature (London) 303, 706-709) demonstrated that egg adaptation of influenza B virus selects variants which are antigenically distinct from virus grown from the same source in mammalian cells. The molecular changes in the hemagglutinin (HA) of influenza B virus associated with adaptation to growth in eggs have now been identified. A specific glycosylation site at the distal tip of the HA of influenza B virus grown exclusively in mammalian cell culture is lost or altered during egg adaptation. Since the HA functions in adsorption of virus to cells, it is concluded that removal or modification of an oligosaccharide structure at this position is required for influenza B virus to attach to and infect the allantois cells of the egg and that this has important implications for the antigenic configuration of the molecule.
Sequence analysis of the neuraminidase (NA) genes of influenza virus X-7(F1) and of 12 variants selected with monoclonal antibodies has been used to define in physical terms the antigenic structure of this NA, which was operationally established by R. G. Webster, L. E. Brown, and W. G. Laver (1984, Virology 135, 30-42). X-7(F1) is a reassortant virus containing the NA of the early Asian (H2N2) isolate A/RI/5+/57, and the results of antigenic and sequence analysis of X-7(F1) and of variants selected with monoclonal antibodies have been combined with a similar analysis of the A/Tokyo/3/67 NA (H2N2, M. R. Lentz, G. M. Air, W. G. Laver, and R. G. Webster (1984), Virology 135, 257-265) to obtain a model of antibody binding to N2 NAs. The selection process was biased, however, since only those monoclonal antibodies which inhibited NA activity could be used to select variants. Most of the changes in the variants selected with monoclonal antibodies occur in those parts of the polypeptide chain which encircle the enzyme active site pocket in the three-dimensional structure (P. M. Colman, J. N. Varghese, and W. G. Laver (1983), Nature (London) 303, 41-44). The results suggest that in general the antibody binds to a site on the NA which includes those amino acid side chains which are altered in monoclonal variants. There are, however, several aspects of the antigen-antibody interaction which are not easily explained, and which will probably only be fully elucidated by X-ray crystallographic analysis of NA-antibody complexes.
To define the sequence changes that occurred in an avian influenza virus neuraminidase (NA) during the evolution of virulence, we have studied the NA of the virulent and avirulent A/Chick/Penn/83 (H5N2) influenza viruses. A comparison of the deduced amino acid sequence from these viruses shows that the virulent strain, which evolved from the avirulent by the accumulation of point mutations (Bean et al., 1985), acquired four amino acid changes in the NA: one in the transmembrane segment, one in the stalk, and two in the head. A comparison of the deduced amino acid sequences with those of the human N2 NAs indicates a 20-amino acid deletion in the stalk of the Chick/Penn/83 NA. Antigenic analysis of the NAs from the avirulent and virulent Chick/Penn/83 virus shows they are antigenically very closely related, but can be distinguished with two monoclonal antibodies at a site which probably involves at least one of the amino acid changes in the NA head. Antigenic analysis also shows the Chick/Penn/83 NAs are closely related to the NAs of other N2 avian influenza viruses isolated between 1965 and 1984, supporting previous studies which indicate a relative antigenic stability of the NA among avian N2 influenza viruses. The Chick/Penn/83 NAs are the first N2 NA genes of an avian virus to be sequenced. These NAs are antigenically closely related to the 1957 human N2 NAs, and show a high degree of amino acid sequence homology with the prototype 1957 human N2 NA. These data give further support to the view that the 1957 human H2N2 viruses were at least partially derived from an avian source.
We have probed the antigenic structure of the influenza B virus hemagglutinin (HA) with monoclonal antibodies specific for the HA of influenza B virus, B/Oregon/5/80. Seventeen laboratory-selected antigenic variants of this virus were analyzed by hemagglutination-inhibition (HI) assays or ELISA and an operational antigenic map was constructed. In addition, the monoclonal antibodies were tested in a competitive binding assay to construct a topological map of the antigenic sites. In contrast to the influenza A virus HA, only a single immunodominant antigenic site composed of several overlapping clusters of epitopes was defined by the HI-positive antibodies. Three variants could be distinguished from the parental virus with polyclonal antisera by HI and infectivity reduction assays suggesting that changes in this antigenic site may be sufficient to provide an epidemiological advantage to influenza B viruses in nature. In addition, two nonoverlapping epitopes of unknown biological significance were identified in the competitive binding analysis by two monoclonal antibodies with no HI activity and little or no neutralizing activity. We previously identified single amino acid substitutions in the HAs of the antigenic variants used in this study (M. T. Berton, C. W. Naeve, and R. G. Webster (1984), J. Virol. 52, 919-927). These changes occurred in regions of the molecule which, by amino acid sequence alignment, appeared to correspond to proposed antigenic sites A and B on the H3 HA of influenza A virus. Correlation with the antigenic map established in this report, however, demonstrates that the amino acid residues actually contribute to a single antigenic site on the influenza B virus HA and suggests significant differences in the antigenic structures of the influenza A and B virus HAs.
Antigenic analysis of human and avian H2 influenza virus was carried out with monoclonal antibodies to the HA molecules of H2 influenza viruses isolated in the early stage of an H2 pandemic. The study revealed antigenic differences between inhibitor sensitive (Japan+/57, RI+57) and inhibitor resistant strains (Japan-/57, Ri-/57). This indicates that the receptor-binding specificity of the haemagglutinin can markedly influence the antigenic analysis obtained with monoclonal antibodies in HI test. Minor antigenic differences (microheterogeneity) could be detected between different H2 influenza viruses isolated in 1957. Minor antigenic variation continued in the H2 viruses until 1961, but significant antigenic drift occurred in 1962 so that viruses isolated after that date reacted with few monoclonal antibodies. Analysis of avian H2 influenza viruses suggested antigenic differences between the different avian H2 haemagglutinin, but no correlation between the year of isolation and the progressive antigenic drift similar to that seen in the human strains was found.
A long-term prospective study was conducted to evaluate the incidence of specific complications following extracapsular cataract extraction. Eight hundred forty-two consecutive extracapsular cataract extractions were performed over a ten-year period with a minimum follow-up time of one year. The mean follow up period for this series was 32.2 months. The incidence of retinal detachment in the entire population was 1.4%. In those eyes with a cataract as the only ocular abnormality and with no surgical complications, the frequency was 1.0%. The incidence in eyes following uncomplicated procedures, with no other ocular pathology and with an intact posterior capsule was 0.8%. The incidence of opacification of the posterior capsule requiring capsulotomy was 16.7%. The mean time interval for a secondary capsulotomy was 24.3 months. A positive correlation between this time interval and patient age was established.
We report the safety and antigenicity of influenza A vaccines in seronegative children one to seven years of age. A natural H1N1 challenge that occurred shortly after completion of the vaccination program permitted an evaluation of efficacy. Twenty-eight subjects were inoculated with live cold-adapted (ca) influenza A/Washington/897/80 (H3N2), 29 with ca influenza A/California/10/78 (H1N1), 24 with inactivated whole-virus influenza A/Bangkok/79 (H3N2), and 30 with a placebo. The ca vaccines were well tolerated, whereas the inactivated vaccine caused adverse reactions in about one-third of the children. Fifty-seven percent of the ca H1N1 recipients showed serological responses, contrasted with 84% and 100% of subjects receiving the ca or inactivated H3N2 vaccines, respectively. None of the 16 children with induced H1N1 antibody developed clinically apparent influenza-like illness, compared with eleven of the 51 initially seronegative children who did not receive the ca H1N1 vaccine and with four of the 12 who failed to respond. Results of the efficacy field trial suggest protection against infection and symptomatic illness in children inoculated with ca H1N1, despite its failure to stimulate high levels of hemagglutinin-inhibiting antibody.
In April 1983, an influenza virus of low virulence appeared in chickens in Pennsylvania. Subsequently, in October 1983, the virus became virulent and caused high mortality in poultry. The causative agent has been identified as an influenza virus of the H5N2 serotype. The hemagglutinin is antigenically closely related to tern/South Africa/61 (H5N3) and the neuraminidase is similar to that from human H2N2 strains (e.g., A/Japan/305/57) and from some avian influenza virus strains (e.g., A/turkey/Mass/66 [H6N2]). Comparison of the genome RNAs of chicken/Penn with other influenza virus isolates by RNA-RNA hybridization indicated that all of the genes of this virus were closely related to those of various other influenza virus isolates from wild birds. Chickens infected with the virulent strain shed high concentrations of virus in their feces (10(7) 50% egg infective dose per g), and the virus was isolated from the albumin and yolk of eggs layed just before death. Virus was also isolated from house flies in chicken houses. Serological and virological studies showed that humans are not susceptible to infection with the virus, but can serve as short-term mechanical carriers. Analysis of the RNA of the viruses isolated in April and October by gel migration and RNA-RNA hybridization suggested that these strains were very closely related. Oligonucleotide mapping of the individual genes of virulent and avirulent strains showed a limited number of changes in the genome RNAs, but no consistent differences between the virulent and avirulent strains that could be correlated with pathogenicity were found. Polyacrylamide gel analysis of the early (avirulent) isolates demonstrated the presence of low-molecular-weight RNA bands which is indicative of defective-interfering particles. These RNAs were not present in the virulent isolates. Experimental infection of chickens with mixtures of the avirulent and virulent strains demonstrated that the avirulent virus interferes with the pathogenicity of the virulent virus. The results suggest that the original avirulent virus was probably derived from influenza viruses from wild birds and that the virulent strain was derived from the avirulent strain by selective adaptation rather than by recombination or the introduction of a new virus into the population. This adaptation may have involved the loss of defective RNAs, as well as mutations, and thus provides a possible model for a role of defective-interfering particles in nature.
The influenza A virus [A/Chicken/Pennsylvania/1370/83 (H5N2)] that caused up to 80% mortality among chickens provided a model system for testing the efficacy of chemotherapeutic agents against highly virulent influenza virus. Amantadine and rimantadine administered in drinking water were efficacious both prophylactically and therapeutically. However, under conditions simulating natural transmission of virus, amantadine- and rimantadine-resistant viruses arose and were transmitted to other birds in contact with the infected chickens, causing mortality. Simultaneous administration of inactivated H5N2 vaccine and amantadine provided protection. Thus, chemotherapy may be useful in the treatment of a highly pathogenic influenza virus outbreak in humans or other animals when used in combination with vaccine.
Monoclonal antibodies have been found to provide useful additional information for the antigenic analysis of influenza A viruses of the H3N2 and H1N1 subtypes. They have been particularly useful in the interpandemic period when multiple variants circulate concurrently.Apparently heterogeneous isolates can be placed in fairly clear-cut groups on the basis of their reactivity with certain monoclonal antibody preparations. It is thought likely that variants reacting with the least number of monoclones are the most different antigenically from the fully reactive strains.
A 39-year-old woman with progressive left unilateral glaucoma associated with corneal edema, iris changes, and peripheral anterior synechia formation underwent a left trabeculectomy and iridectomy in an attempt to control the intraocular pressure. Shortly after surgery the edema worsened despite low intraocular pressure measurements. A repeat trabeculectomy combined with a full thickness keratoplasty was performed 11/2 years later. The posterior corneal surface was found to be covered by a partially doubled layer of endothelial cells with ultrastructural features resembling epithelium. Similar cells were not noted on the surface of the initial trabecular meshwork and iris specimens, but were seen on the surface of the specimens obtained at the second trabeculectomy. The anterior and middle portions of Descemet's membrane, formed during perinatal and early adult life, appeared normal, but its posterior portion appeared abnormal. The ultrastructural changes in the endothelial cells and the abnormalities observed in Descemet's membrane suggest that the endothelial cells were initially normal but subsequently acquired epithelial-like features as they degenerated and proliferated. The continued presence of corneal edema despite the hypercellularity of the endothelium suggests that the proliferating cells were permeable to aqueous.
Migratory waterfowl and shore birds harbour a wide range of influenza viruses, some of which have been implicated in influenza outbreaks in mammals and domestic birds. In the present study, a comparison was made of two marshalling areas for different migratory flyways of waterfowl in North America over a 6-8-year period. Virtually all known influenza subtypes were isolated and the predominant subtype changed from year to year. A marked difference between the two locations was that the predominant subtypes circulating were never the same, even though in both areas, most virus isolations were made from the same duck species (mallard duck). Isolations of paramyxovirus were characterized mainly as avian PMV-1.Viruses isolated from ducks included those antigenically related to viruses causing disease in birds and mammals, although the viruses did not necessarily appear in ducks immediately before they appeared in other species. For example, H5N2 isolates antigenically related to the virus causing severe disease outbreaks in chickens in the USA in 1983, were detected in ducks from both areas at different times (1976, 1980 and 1982). These studies indicate that ducks in different areas represent a continual source of orthomyxoviruses and paramyxoviruses of potential disease significance to other species.
Antigenic analysis of human and avian H2 influenza viruses were done with monoclonal antibodies to the HA molecules in hemagglutination inhibition (HI) assays. These studies revealed that the receptor-binding specificity of the hemagglutinin can markedly influence the antigenic analysis obtained with monoclonal antibodies in HI tests. Influenza viruses that are sensitive or resistant to inhibition by horse serum inhibitors showed marked differences in their reactivity with monoclonal antibodies to the hemagglutinin. This was apparent with the A/RI/5+/57 and A/RI/5-/57 strains of H2N2 viruses isolated by Choppin and Tamm (1960a), half of the panel of different monoclonal antibodies failed to inhibit hemagglutination of the RI/5- variant, whereas all of the 18 monoclonal antibodies inhibited RI/5+. These findings have important implications in the antigenic analysis of influenza viruses where HI assays are conventionally used to determine the extent of antigenic drift in nature. Antigenic differences were detectable between different human H2 influenza virus isolates from 1957 that were sensitive to inhibition by horse serum, indicating that minor antigenic variation occurs within the first year of appearance of the new subtype. Minor antigenic variation continued in the H2 viruses until 1961, but by 1962 antigenically distinguishable variants that could be discriminated with both monoclonal antibodies and postinfection ferret antisera predominated. Analysis of avian H2 influenza viruses with a panel of monoclonal antibodies indicated that antigenic variation occurs and that multiple different variants cocirculate in the population. There was no progressive antigenic change in the avian H2 influenza viruses with time, as was found with the human H2N2 strains. Topographical mapping of the H2 hemagglutinin by selection of antigenic variants with monoclonal antibodies and analysis of their reactivity patterns by HI showed overlap between the epitopes examined. These results may reflect restriction in the antibody repertoire of the mice used in preparation of the monoclonal antibodies or that the H2 hemagglutinin does not have such discrete nonoverlapping antigenic regions found in the early H3 influenza virus.
Monoclonal antibodies were used to compare the antigenicities of the haemagglutinins of two receptor binding mutants of X-31 (H3N2) influenza virus. The mutants which differed from each other in recognizing sialic acid in either alpha 2-6 linkage or alpha 2-3 linkage to galactose also differed exclusively at residue 226 of the HA1 polypeptides of their haemagglutinins (G. N. Rogers, J. C. Paulson, R. S. Daniels, J. J. Skehel, I. A. Wilson, and D. C. Wiley, Nature (London) 304, 76-78, 1983). The results obtained indicate that whereas the majority of antihaemagglutinin monoclonal antibodies react with both mutants equally well, a number which specifically recognize residues 193, 199, 219, and 229 of HA1 of the alpha 2-6 linkage-binding mutant fail to recognize the alpha 2-3 linkage-binding haemagglutinins. The results are discussed with reference to the three-dimensional structure of the haemagglutinin and in relation to differences in antigenicity observed in the haemagglutinins of viruses grown in cells of different types.