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Biomedical subjects

R G Webster

Publications and source records attributed to R G Webster.

At least 91 records · Page 5Linked to original sources
PubMed1998

Evidence for zanamivir resistance in an immunocompromised child infected with influenza B virus.

Zanamivir, a neuraminidase inhibitor, has shown promise as a drug to control influenza. During prolonged treatment with zanamivir, a mutant virus was isolated from an immunocompromised child infected with influenza B virus. A hemagglutinin mutation (198 Thr-->Ile) reduced the virus affinity for receptors found on susceptible human cells. A mutation in the neuraminidase active site (152 Arg-->Lys) led to a 1000-fold reduction in the enzyme sensitivity to zanamivir. When tested in ferrets, the mutant virus had less virulence than the parent; however, it had a growth preference over the parent in zanamivir-treated animals. Despite these changes, the sensitivity of the mutant virus to zanamivir assessed by a standard test in MDCK cells was unaffected. These data indicate that the current methods for monitoring resistant mutants are potentially flawed because no tissue culture system adequately reflects the receptor specificity of human respiratory tract epithelium.

Amino Acid Substitution↗
PubMed1998

Characterization of influenza A/HongKong/156/97 (H5N1) virus in a mouse model and protective effect of zanamivir on H5N1 infection in mice.

A recent outbreak of influenza in Hong Kong was caused by a highly virulent virus of avian origin. Concern that the appearance of such a virus in the human population may be a harbinger of a new pandemic has brought increased attention to the issue of antivirals available for treatment of influenza. A/HongKong/156/97 (H5N1), the first virus of H5N1 subtype isolated from a human host, is highly virulent in the mouse model and can infect mouse lungs without requiring adaptation. High mortality and evidence of systemic disease, including spread to the brain after intranasal inoculation, are observed. Zanamivir, a novel neuraminidase inhibitor, is effective at decreasing replication of the virus in vitro. In a model of lethal challenge in mice, zanamivir reduces lung titers of the virus and decreases morbidity and mortality.

Animals↗
PubMed1998

Immunogenicity and protective efficacy in mice of influenza B virus vaccines grown in mammalian cells or embryonated chicken eggs.

The immunogenicity and protective efficacy of formalin-inactivated influenza B/Memphis/1/93 virus vaccines propagated exclusively in Vero cells, MDCK cells, or embryonated chicken eggs (hereafter referred to as eggs) were investigated. Mammalian cell-grown viruses differ from the egg-grown variant at amino acid position 198 (Pro/Thr) in the hemagglutinin gene. The level of neuraminidase activity was highest in egg-grown virus, while MDCK and Vero cell-derived viruses possessed 70 and 90% less activity, respectively. After boosting, each of the vaccines induced high levels of hemagglutinin-inhibiting, neuraminidase-inhibiting, and neutralizing antibodies that provided complete protection from MDCK-grown virus challenge. Mammalian cell-derived virus vaccines induced serum antibodies that were more cross-reactive, while those induced by egg-grown virus vaccines were more specific to the homologous antigen. Enzyme-linked immunospot analysis indicated that cell-grown virus vaccines induced high frequencies of immunoglobulin G (IgG)-producing cells directed against both cell- and egg-grown virus antigens, whereas egg-grown virus vaccine induced higher frequencies of IgG- and IgM-producing cells reacting with homologous antigen and low levels of IgG-producing cells reactive with cell-grown viruses. These studies indicate that influenza B virus variants selected in different host systems can elicit different immune responses, but these alterations had no detectable influence on the protective efficacy of the vaccines with the immunization protocol used in this study.

Animals↗
PubMed1998

Molecular basis for the generation in pigs of influenza A viruses with pandemic potential.

Genetic and biologic observations suggest that pigs may serve as "mixing vessels" for the generation of human-avian influenza A virus reassortants, similar to those responsible for the 1957 and 1968 pandemics. Here we demonstrate a structural basis for this hypothesis. Cell surface receptors for both human and avian influenza viruses were identified in the pig trachea, providing a milieu conducive to viral replication and genetic reassortment. Surprisingly, with continued replication, some avian-like swine viruses acquired the ability to recognize human virus receptors, raising the possibility of their direct transmission to human populations. These findings help to explain the emergence of pandemic influenza viruses and support the need for continued surveillance of swine for viruses carrying avian virus genes.

Adaptation, Biological↗
PubMed1997

Continued evolution of H1N1 and H3N2 influenza viruses in pigs in Italy.

Swine influenza viruses possessing avian genes were first detected in Europe in 1979 (Scholtissek et al., 1983, Virology, 129, 521-523) and continue to circulate in pigs in that region of the world. To characterize the molecular epidemiology of swine influenza viruses currently circulating in Europe, we used dot-blot hybridization and sequence analysis to determine the origin of the genes encoding the nonsurface proteins ("internal" genes) of 10 H1N1 and 11 H3N2 swine influenza viruses isolated in Italy between 1992 and 1995. All of the 126 genes examined were of avian origin; thus the currently circulating H3N2 strains which possess A/Port Chalmers/1/73-like surface glycoproteins appear to be descendants of the reassortant human-avian viruses that emerged between 1983 and 1985 in Italy. Sequence analysis of matrix (M), nonstructural, and nucleoprotein genes, as well as phylogenetic analysis of M gene showed that the H1N1 and H3N2 viruses from the pigs were closely related to recent isolates of the avian-like swine H1N1 influenza strain currently circulating in northern Europe and were distinguishable from the genes of viruses isolated from European swine in 1979. To evaluate the frequency of transmission of swine H1N1 and H3N2 viruses to man, we tested 123 human sera for hemagglutination-inhibiting antibodies against avian and mammalian H1N1 and H3N2 virus strains. Our findings indicate that swine influenza viruses possessing A/Port Chalmers/1/73-like hemagglutinin may have transmitted to approximately 20% of young persons under 20 years of age who had contact with pigs. Thus, H3N2 swine viruses, possibly possessing avian-derived internal genes, may be entering humans more often than was previously thought. We strongly recommend that pigs be regularly monitored as a potential early warning system for detection of future pandemic strains.

Adult↗
PubMed1997

Different T helper cell types and antibody isotypes generated by saline and gene gun DNA immunization.

Several routes and methods of DNA immunization have been shown to generate Ab, Th cells, and CTL responses. However, few studies have directly compared the immune responses generated by different routes and methods of DNA immunization. Utilizing an influenza hemagglutinin (H1)-expressing plasmid, we compared the immune response produced by saline injection of DNA into skin or muscle, and gene gun immunization of skin or muscle. We found that saline-DNA immunization raised a predominantly Th1 response with mostly IgG2a anti-H1 Ab, while gene gun DNA immunization produced a predominantly Th2 response with mostly IgG1 anti-H1 Abs. These distinct types of immune responses were generated by the method, not the route, of DNA immunization. The initial immunization established the Th cell-type of the immune response. The Th cell-type did not change with further DNA immunizations by the same or the alternate method, or after a viral challenge. The ability to generate different Th types was not due to differences in the doses of DNA used in saline and gene gun DNA immunization. These findings have important implications for vaccine design and studies of the mechanism of Th cell differentiation.

Animals↗
PubMed1997

Influenza virus: transmission between species and relevance to emergence of the next human pandemic.

Although influenza viruses are not spread from human to human through the conventional food chain, this is not necessarily the case for the transmission of the precursors of the human pandemic influenza viruses. Aquatic birds of the world are the reservoirs for all influenza A viruses; the virus is spread by fecal-oral transmission in untreated water. Influenza A viruses are frequently transmitted to domestic poultry and two of the 15 subtypes H5 and H7 can become highly pathogenic and have the capacity to decimate commercial poultry flocks. Less frequently, avian influenza viruses are transmitted between species-to pigs, horses and sea mammals. This transmission involves mutational, reassortant or recombinational events and can occur through fecal contamination of unprocessed avian protein or through the water. The transmission of avian influenza viruses or virus genes to humans is postulated to occur through pigs that act as the intermediate host. This involves either multiple mutational or reassortant events and is believed to occur by airborne transmission. Once avian influenza viruses are established in mammals, they are transmitted from animal to animal by the respiratory airborne route. The transmission of avian influenza virus from their reservoir in wild aquatic birds to domestic poultry and to mammalian species including humans can be prevented by treatment of the water supply and of avian protein sources with disinfectants or by heating. Agricultural authorities have recommended the separation of wild aquatic and domestic poultry and of pig and poultry farming. It is theoretically possible to reduce the possibility of the next pandemic of influenza in humans by changes in agricultural practices so that ducks are separated from pigs and people.

Animals↗
PubMed1997

Combination intravenous ceftazidime and aminoglycosides in the treatment of pseudomonal scleritis.

BACKGROUND: Pseudomonal scleritis is a serious and potentially blinding infection that usually is resistant to medical management. METHODS: Results for three patients with pseudomonal scleritis who were treated with both topical anti-infectives and a combination of intravenous ceftazidime and aminoglycoside are presented in this case series. RESULTS: All three patients had a rapid response to the addition of combination intravenous drug therapy to topical therapy; eradication of the infection and healing of the ocular surface occurred within 8 weeks. Only one patient, in whom cystoid macular edema developed, lost useful vision as a result of the infection. CONCLUSIONS: Combination therapy with intravenous ceftazidime and aminoglycoside may be more effective than single-intravenous agents when used in addition to topical antibiotics and may obviate the need for adjunctive surgical procedures, such ascryotherapy, surgical extirpation, or conjunctival recession.

Administration, Topical↗
PubMed1997

Predictions for future human influenza pandemics.

Will there be another human influenza pandemic? The certainty is that there will be, and the probability is that the virus will emerge from Eurasian aquatic bird reservoirs and involve reassortment between a human and avian strain, with accumulation of mutations or true recombinational events (or both) that will permit spread and pathogenicity among humans. This process will probably occur in pigs because they possess receptors for both avian and human influenza viruses, and emergence may occur in southern China. Prediction of the subtype is impossible, but there is a hypothesis based on seroarcheology that only H1, H2, and H3 subtypes can infect humans; however, it is arguable that H7 or H2 strains might be equally capable of infecting humans.

Animals↗
PubMed1997

DNA immunization for influenza virus: studies using hemagglutinin- and nucleoprotein-expressing DNAs.

DNA-based immunizations have been used to analyze the ability of DNA-expressed hemagglutinin (HA) and nucleoprotein (NP) to protect BALB/c mice against a homologous influenza virus, A/PR/8/34 (H1N1), challenge. The HA DNA, but not the NP DNA, protected mice against the lethal viral challenge. For the HA DNA, single gene gun inoculations of 0.04 microg and boosted inoculations of 0.004 microg of DNA raised complete protection. For the NP DNA, boosted gene gun immunizations of 0.4 microg of DNA and boosted intradermal or intramuscular injections of 50 microg of DNA failed to protect. The protection elicited by the HA DNA vaccine correlated with the titers of neutralizing antibody.

Animals↗
PubMed1997

Single-chain Fv fragments of anti-neuraminidase antibody NC10 containing five- and ten-residue linkers form dimers and with zero-residue linker a trimer.

Single-chain variable fragments (scFvs) of anti-neuraminidase antibody NC10 were constructed by joining the VH and VL domains with 10-residue (Gly4Ser)2 and five-residue (Gly4Ser) linkers; a zero-residue linker scFv was constructed by joining the C-terminal residue of the VH domain to the N-terminus of the VL domain. The scFv with the 10- and five-residue linkers exclusively formed dimeric antibody fragments (M(r) 52000). These were shown to be bivalent and were able to cross-link two neuraminidase tetramers to form a 'sandwich' type complex; each antigen combining site could also bind an anti-idiotype Fab'. The zero-residue linker scFv (M(r) 70000) was shown to form a trimer with three active antigen combining sites, each binding an anti-idiotype Fab' to yield a complex of M(r) 212000. The orientation of the combining sites in the zero-residue linker scFv, however, was such that it could not cross-link tetramers of neuraminidase. BIAcore biosensor experiments showed that the affinity of each individual antigen combining site in both the 10- and five-residue linker scFv dimers and zero-residue linker scFv trimer was essentially the same when the scFvs were immobilized onto the sensor surface. However, when the scFvs were used as the analyte, the dimeric and trimeric scFvs showed an apparent increase in binding affinity due to the avidity of binding the multivalent scFvs.

Amino Acid Sequence↗
PubMed1997

Catalytic and framework mutations in the neuraminidase active site of influenza viruses that are resistant to 4-guanidino-Neu5Ac2en.

Here we report the isolation of influenza virus A/turkey/Minnesota/833/80 (H4N2) with a mutation at the catalytic residue of the neuraminidase (NA) active site, rendering it resistant to the novel NA inhibitor 4-guanidino-Neu5Ac2en (GG167). The resistance of the mutant stems from replacement of one of three invariant arginines (Arg 292-->Lys) that are conserved among all viral and bacterial NAs and participate in the conformational change of sialic acid moiety necessary for substrate catalysis. The Lys292 mutant was selected in vitro after 15 passages at increasing concentrations of GG167 (from 0.1 to 1,000 microM), conditions that earlier gave rise to GG167-resistant mutants with a substitution at the framework residue Glu119. Both types of mutants showed similar degrees of resistance in plaque reduction assays, but the Lys292 mutant was more sensitive to the inhibitor in NA inhibition tests than were mutants bearing a substitution at framework residue 119 (Asp, Ala, or Gly). Cross-resistance to other NA inhibitors (4-amino-Neu5Ac2en and Neu5Ac2en) varied among mutants resistant to GG167, being lowest for Lys292 and highest for Asp119. All GG167-resistant mutants demonstrated markedly reduced NA activity, only 3 to 50% of the parental level, depending on the particular amino acid substitution. The catalytic mutant (Lys292) showed a significant change in pH optimum of NA activity, from 5.9 to 5.3. All of the mutant NAs were less stable than the parental enzyme at low pH. Despite their impaired NA activity, the GG167-resistant mutants grew as well as parental virus in Madin-Darby canine kidney cells or in embryonated chicken eggs. However, the infectivity in mice was 500-fold lower for Lys292 than for the parental virus. These findings demonstrate that amino acid substitution in the NA active site at the catalytic or framework residues, followed by multiple passages in vitro, in the presence of increasing concentrations of the NA inhibitor GG167, generates GG167-resistant viruses with reduced NA activity and decreased infectivity in animals.

Animals↗
PubMed1997

Cross-protection among lethal H5N2 influenza viruses induced by DNA vaccine to the hemagglutinin.

Inoculation of mice with hemagglutinin (HA)-expressing DNA affords reliable protection against lethal influenza virus infection, while in chickens the same strategy has yielded variable results. Here we show that gene gun delivery of DNA encoding an H5 HA protein confers complete immune protection to chickens challenged with lethal H5 viruses. In tests of the influence of promoter selection on vaccine efficacy, close correlations were obtained between immune responses and the dose of DNA administered, whether a cytomegalovirus (CMV) immediate-early promoter or a chicken beta-actin promoter was used. Perhaps most important, the HA-DNA vaccine conferred 95% cross-protection against challenge with lethal antigenic variants that differed from the primary antigen by 11 to 13% (HA1 amino acid sequence homology). Overall, the high levels of protection seen with gene gun delivery of HA-DNA were as good as, if not better than, those achieved with a conventional whole-virus vaccine, with fewer instances of morbidity and death. The absence of detectable antibody titers after primary immunization, together with the rapid appearance of high titers immediately after challenge, implicates efficient B-cell priming as the principal mechanism of DNA-mediated immune protection. Our results suggest that the efficacy of HA-DNA influenza virus vaccine in mice extends to chickens and probably to other avian species as well. Indeed, the H5 preparation we describe offers an attractive means to protect the domestic poultry industry in the United States from lethal H5N2 viruses, which continue to circulate in Mexico.

Animals↗
PubMed1997

Coinfection of wild ducks by influenza A viruses: distribution patterns and biological significance.

Coinfection of wild birds by influenza A viruses is thought to be an important mechanism for the diversification of viral phenotypes by generation of reassortants. However, it is not known whether coinfection is a random event or follows discernible patterns with biological significance. In the present study, conducted with viruses collected throughout 15 years from a wild-duck population in Alberta, Canada, we identified three discrete distributions of coinfections. In about one-third of the events, which involved subtypes of viruses that appear to be maintained in this duck reservoir, coinfection occurred at rates either close to or significantly lower than one would predict from rates of single-virus infection. Apparently, the better adapted an influenza A virus is to an avian population, the greater is its ability to prevent coinfections. Conversely, poorly adapted, nonmaintained viruses were significantly overrepresented as coinfectants. Rarely encountered subtypes appear to represent viruses whose chances of successfully infiltrating avian reservoirs are increased by coinfection. Mallards (Anas platyrhynchos) and pintails (A. acuta) were significantly more likely to be infected by a single influenza A virus than were the other species sampled, but no species was significantly more likely to be coinfected. These observations provide the first evidence of nonrandom coinfection of wild birds by influenza A viruses, suggesting that reassortment of these viruses in a natural population does not occur randomly. These results suggest that even though infections may occur in a species, all subtypes are not maintained by all avian species. They also suggest that specific influenza A virus subtypes are differentially adapted to different avian hosts and that the fact that a particular subtype is isolated from a particular avian species does not mean that the virus is maintained by that species.

Adaptation, Physiological↗
PubMed1996

Long-term maintenance of B cell immunity to influenza virus hemagglutinin in mice following DNA-based immunization.

This study demonstrates that gene-gun inoculation of mice with DNA encoding the influenza virus hemagglutinin (HA) results in the life-long maintenance of protective B cell responses. Using a sensitive single-cell enzyme-linked immunospot assay, we show that all of the HA-specific plasma cells are localized in the bone marrow and spleen 1 year postimmunization. As a consequence of prior virus challenge, only a small population of antibody-forming cells was found in the lymphoid tissues associated with the respiratory tract. The tissue distribution of HA-specific plasma cells in these mice was identical to the profile in infected controls. Complete protection against live virus challenge in the aged vaccinated mice did not require prior exposure to virus. Thus, immunization with the DNA, vaccine provides long-term protective immunity against otherwise lethal infection.

Animals↗
PubMed1996

Different hemagglutinin cleavage site variants of H7N7 in an influenza outbreak in chickens in Leipzig, Germany.

The hemagglutinin (HA) genes from four avian H7N7 influenza A isolates, from a single outbreak, were shown to possess different cleavage sites that contain varying numbers of basic amino acid residues (KKKKR, KRKKR, KKRKKR, KKKKKKR). All four variants are highly pathogenic in chickens and share an immediate common ancestral HA with A/tern/Potsdam/342-6/79 (H7N7) and A/swan/Potsdam/63-6/81 (H7N7). These viruses are nonpathogenic and contain no extra basic amino acids at the cleavage site of their HA. During evolution a common precursor virus acquired different sequences at the cleavage site of the HA and became highly pathogenic in chickens. In vitro assays revealed that the HA from A/chicken/Leipzig/79 with KKKKR at the cleavage site was only partially cleaved (41%), compared to 93-100% cleavage of the other HAs. Since all four viruses were highly pathogenic in chickens, these findings confirm that the degree of pathogenicity in vivo is not exclusively determined by the degree of HA cleavability.

Amino Acid Sequence↗