Biomedical subjects

Robert G Webster

Publications and source records attributed to Robert G Webster.

At least 37 records · Page 2Linked to original sources

PubMed2005

The genesis of a pandemic influenza virus.

Pandemic influenza viruses pose a significant threat to public health worldwide. In a recent Nature paper, Taubenberger et al. (2005) now report remarkable similarities between the polymerase genes of the influenza virus that caused the 1918 Spanish influenza pandemic and those of avian influenza viruses. Meanwhile, Tumpey et al. (2005) reporting in Science show that the reconstructed 1918 Spanish influenza virus kills mice faster than any other influenza virus so far tested.

Animals↗

PubMed2005

Detection of amantadine-resistant variants among avian influenza viruses isolated in North America and Asia.

Here, we report the diversity of amantadine-resistant mutants among avian influenza A viruses with pandemic potential (H5, H6, H7, and H9 hemagglutinin subtypes). Drug-resistant variants were not detected among 1979--83 isolates, whereas 31.1% of H5 and 10.6% of H9 strains from Southeast Asia isolated in 2000-04 carried mutations in M2 protein. In North America, resistant variants occurred among H7 viruses only (16.4% of those tested). H6 viruses were amantadine-sensitive. These findings prompt concern regarding the control of pandemic influenza, the possibility that the next pandemic virus will be amantadine-resistant and the need to monitor the use of the drug in poultry.

Amantadine↗

PubMed2005

Role of specific hemagglutinin amino acids in the immunogenicity and protection of H5N1 influenza virus vaccines.

If H5N1 influenza viruses become transmissible among humans, vaccination will offer the most effective option to limit their spread. Two human vaccine candidates recently generated by reverse genetics are based on antigenically different hemagglutinin (HA) glycoproteins derived from the A/HK/213/03 (H5N1) and A/Vietnam/1203/04 (H5N1) viruses. Their HA1 amino acid sequences differ at 10 positions, one of which (N154) introduces a potential glycosylation site in A/Vietnam/1203/04 (H5N1). To assess the impact of five amino acids in the putative antigenic sites on immunogenicity and immune protection, we generated a series of whole-virus vaccines that differed only in one or two HA amino acids. Sera from ferrets vaccinated with these inactivated preparations had high virus neutralization titers, but their hemagglutination inhibition (HI) titers were usually low. Interestingly, a recombinant virus in which the HA amino acid S223 (characteristic of 2004 viruses) was converted to N223 (as in A/HK/213/03) resulted in higher HI titers. This observation indicates that specific HA residues, such as N223, increase the sensitivity of the HI assay by altering receptor specificity and/or antibody-antigen binding. Ferrets vaccinated with mutant vaccine viruses were protected against lethal challenge with wild-type A/Vietnam/1203/04 virus. Our results suggest that inclusion of the N223 residue in the HA glycoproteins of diagnostic reference viruses may facilitate the evaluation of vaccine efficacy in humans.

Amino Acid Sequence↗

PubMed2005

Virulence may determine the necessary duration and dosage of oseltamivir treatment for highly pathogenic A/Vietnam/1203/04 influenza virus in mice.

BACKGROUND: Control of highly pathogenic avian H5N1 influenza viruses is a major public-health concern. Antiviral drugs could be the only option early in the pandemic.METHODS. BALB/c mice were given oseltamivir (0.1, 1, or 10 mg/kg/day) twice daily by oral gavage; the first dose was given 4 h before inoculation with H5N1 A/Vietnam/1203/04 (VN1203/04) virus. Five- and 8-day regimens were evaluated.RESULTS. Oseltamivir produced a dose-dependent antiviral effect against VN1203/04 in vivo (P<.01). The 5-day regimen at 10 mg/kg/day protected 50% of mice; deaths in this treatment group were delayed and indicated the replication of residual virus after the completion of treatment. Eight-day regimens improved oseltamivir efficacy, and dosages of 1 and 10 mg/kg/day significantly reduced virus titers in organs and provided 60% and 80% survival rates, respectively (P<.05). Overall, the efficacy of the 5- and 8-day regimens differed significantly (death hazard ratio, 2.658; P<.01). The new H5N1 antigenic variant VN1203/04 was more pathogenic in mice than was A/HK/156/97 virus, and a prolonged and higher-dose oseltamivir regimen may be required for the most beneficial antiviral effect.CONCLUSIONS. Oseltamivir prophylaxis is efficacious against lethal challenge with VN1203/04 virus in mice. Viral virulence may affect the antiviral treatment schedule.

Acetamides↗

PubMed2005

Efficacy of H5 influenza vaccines produced by reverse genetics in a lethal mouse model.

We studied the efficacy, in mice, of 2 H5 influenza vaccine viruses produced by reverse genetics. Mice were immunized with inactivated viruses and then inoculated with a human H5N1 1997 or 2003 virus or an avian H5N1 2001 virus. Vaccine viruses that we tested raised high levels of hemagglutination-inhibiting (1:160-1:1280) and virus-neutralizing (1:900-1:1900) antibodies on day 21 after a single dose of vaccine and decreased or prevented virus replication in mouse lungs; 54.5%-100% of immunized mice survived, whereas all control mice died. Protection was achieved despite antigenic differences and incomplete matching of the vaccine strain and the challenge virus. Therefore, high levels of cross-protection are predicted in the mouse model.

Animals↗

PubMed2005

Avian influenza viruses in Korean live poultry markets and their pathogenic potential.

We surveyed live-poultry markets in Korea in 2003 and isolated 9 H9N2, 6 H3N2, and 1 H6N1 influenza viruses. Antigenic and phylogenetic analyses showed that all 9 H9N2 isolates were of A/Chicken/Korea/25232-96006/96-like lineage (which caused disease in chickens in Korea in 1996) but were different from H9N2 viruses of southeastern China. They had at least 4 genotypes and replicated in chickens but not in mice. The H3N2 and H6N1 viruses were new to Korea and were probably reassortants of avian influenza viruses from southeastern China and recent Korean H9N2 viruses. All 8 segments of the H3N2 viruses formed a single phylogenetic cluster with 99.1 to 100% homology. The H3N2 viruses replicated in chickens and mice without preadaptation, but the H6N1 virus did not. Our results show an increasingly diverse pool of avian influenza viruses in Korea that are potential pandemic influenza agents.

Amino Acid Sequence↗

PubMed2005

Pathogenesis of Hong Kong H5N1 influenza virus NS gene reassortants in mice: the role of cytokines and B- and T-cell responses.

The severity of disease caused in humans by H5N1 influenza viruses remains unexplained. The NS gene of Hong Kong H5N1/97 viruses was shown to contribute to high pathogenicity of reassortants in a pig model. However, the molecular pathogenesis and host immune response underlying this phenomenon remain unclear. Here, in a mouse model, H1N1 A/Puerto Rico/8/34 (PR/8) reassortants that contained the H5N1/97 NS gene, the H5N1/01 NS gene, or an altered H5N1/97 NS gene encoding a Glu92-->Asp substitution in NS1 was studied. The pathogenicity of reassortant viruses, the induction of cytokines and chemokine CXCL1 (KC) in the lungs and specific B- and T-cell responses was characterized. In mice infected with reassortant virus containing the H5N1/97 NS gene, the mouse lethal dose (50%) and lung virus titres were similar to those of PR/8, which is highly pathogenic to mice. This reassortant virus required two more days than PR/8 to be cleared from the lungs of infected mice. Reassortants containing the altered H5N1/97 NS gene or the H5N1/01 NS gene demonstrated attenuated pathogenicity and lower lung titres in mice. Specific B- and T-cell responses were consistent with viral pathogenicity and did not explain the delayed clearance of the H5N1/97 NS reassortant. The reassortant induced elevated pulmonary concentrations of the inflammatory cytokines IL1alpha, IL1beta, IL6, IFN-gamma and chemokine KC, and decreased concentrations of the anti-inflammatory cytokine IL10. This cytokine imbalance is reminiscent of the clinical findings in two humans who died of H5N1/97 infection and may explain the unusual severity of the disease.

Animals↗

PubMed2005

Restoration of virulence of escape mutants of H5 and H9 influenza viruses by their readaptation to mice.

Antigenic mapping of the haemagglutinin (HA) molecule of H5 and H9 influenza viruses by selecting escape mutants with monoclonal anti-HA antibodies and subjecting the selected viruses to immunological analysis and sequencing has previously been performed. The viruses used as wild-type strains were mouse-adapted variants of the original H5 and H9 isolates. Phenotypic characterization of the escape mutants revealed that the amino acid change in HA that conferred resistance to a monoclonal antibody was sometimes associated with additional effects, including decreased virulence for mice. In the present study, the low-virulence H5 and H9 escape mutants were readapted to mice. Analysis of the readapted variants revealed that the reacquisition of virulence was not necessarily achieved by reacquisition of the wild-type HA gene sequence, but was also associated either with the removal of a glycosylation site (the one acquired previously by the escape mutant) without the exact restoration of the initial wild-type amino acid sequence, or, for an H5 escape mutant that had no newly acquired glycosylation sites, with an additional amino acid change in a remote part of the HA molecule. The data suggest that such 'compensating' mutations, removing the damaging effects of antibody-selected amino acid changes, may be important in the course of influenza virus evolution.

Adaptation, Physiological↗

PubMed2005

Neuraminidase inhibitor-resistant influenza viruses may differ substantially in fitness and transmissibility.

Mutations of the conserved residues of influenza virus neuraminidase (NA) that are associated with NA inhibitor (NAI) resistance decrease the sialidase activity and/or stability of the NA, thus compromising viral fitness. In fact, clinically derived NAI-resistant variants with different NA mutations have shown different transmissibilities in ferrets (M. L. Herlocher, R. Truscon, S. Elias, H. Yen, N. A. Roberts, S. E. Ohmit, and A. S. Monto, J. Infect. Dis. 190:1627-1630, 2004). Molecular characterization of mutant viruses that have a homogeneous genetic background is required to determine the effect of single mutations at conserved NA residues. We generated recombinant viruses containing either the wild-type NA (RG WT virus) or a single amino acid change at NA residue 119 (RG E119V-NA virus) or 292 (RG R292K-NA virus) in the A/Wuhan/359/95 (H3N2) influenza virus background by reverse genetics. Both mutants showed decreased sensitivity to oseltamivir carboxylate, and the RG R292K-NA virus showed cross-resistance to zanamivir. We also observed differences between the two mutants in NA enzymatic activity and thermostability. The R292K mutation caused greater reduction of sialidase activity and thermostability than the E119V mutation. The NA defect caused by the R292K mutation was associated with compromised growth and transmissibility, whereas the growth and transmissibility of the RG E119V-NA virus were comparable to those of RG WT virus. Our results suggest that NAI-resistant influenza virus variants may differ substantially in fitness and transmissibility, depending on different levels of NA functional loss.

Acetamides↗

PubMed2005

Human infection with an avian H9N2 influenza A virus in Hong Kong in 2003.

Avian H9N2 influenza A virus has caused repeated human infections in Asia since 1998. Here we report that an H9N2 influenza virus infected a 5-year-old child in Hong Kong in 2003. To identify the possible source of the infection, the human isolate and other H9N2 influenza viruses isolated from Hong Kong poultry markets from January to October 2003 were genetically and antigenically characterized. The findings of this study show that the human H9N2 influenza virus, A/Hong Kong/2108/03, is of purely avian origin and is closely related to some viruses circulating in poultry in the markets of Hong Kong. The continued presence of H9N2 influenza viruses in poultry markets in southern China increases the likelihood of avian-to-human interspecies transmission.

Antigens, Viral↗

PubMed2005

Studies of H5N1 influenza virus infection of pigs by using viruses isolated in Vietnam and Thailand in 2004.

To determine whether avian H5N1 influenza viruses associated with human infections in Vietnam had transmitted to pigs, we investigated serologic evidence of exposure to H5N1 influenza virus in Vietnamese pigs in 2004. Of the 3,175 pig sera tested, 8 (0.25%) were positive for avian H5N1 influenza viruses isolated in 2004 by virus neutralization assay and Western blot analysis. Experimental studies of replication and transmissibility of the 2004 Asian H5N1 viruses in pigs revealed that all viruses tested replicated in the swine respiratory tract but none were transmitted to contact pigs. Virus titers from nasal swabs peaked on day 2, and low titers were detected in the liver of two of the four pigs tested. Our findings indicate that pigs can be infected with highly lethal Asian H5N1 viruses but that these viruses are not readily transmitted between pigs under experimental conditions.

Animals↗

PubMed2005

Molecular basis of replication of duck H5N1 influenza viruses in a mammalian mouse model.

We recently analyzed a series of H5N1 viruses isolated from healthy ducks in southern China since 1999 and found that these viruses had progressively acquired the ability to replicate and cause disease in mice. In the present study, we explored the genetic basis of this change in host range by comparing two of the viruses that are genetically similar but differ in their ability to infect mice and have different pathogenicity in mice. A/duck/Guangxi/22/2001 (DKGX/22) is nonpathogenic in mice, whereas A/duck/Guangxi/35/2001 (DKGX/35) is highly pathogenic. We used reverse genetics to create a series of single-gene recombinants that contained one gene from DKGX/22 and the remaining seven gene segments from DKGX/35. We find that the PA, NA, and NS genes of DKGX/22 could attenuate DKGX/35 virus to some extent, but PB2 of DKGX/22 virus attenuated the DKGX/35 virus dramatically, and an Asn-to-Asp substitution at position 701 of PB2 plays a key role in this function. Conversely, of the recombinant viruses in the DKGX/22 background, only the one that contains the PB2 gene of DKGX/35 was able to replicate in mice. A single amino acid substitution (Asp to Asn) at position 701 of PB2 enabled DKGX/22 to infect and become lethal for mice. These results demonstrate that amino acid Asn 701 of PB2 is one of the important determinants for this avian influenza virus to cross the host species barrier and infect mice, though the replication and lethality of H5N1 influenza viruses involve multiple genes and may result from a constellation of genes. Our findings may help to explain the expansion of the host range and lethality of the H5N1 influenza viruses to humans.

Amino Acid Substitution↗

PubMed2005

Lethality to ferrets of H5N1 influenza viruses isolated from humans and poultry in 2004.

The 2004 outbreaks of H5N1 influenza viruses in Vietnam and Thailand were highly lethal to humans and to poultry; therefore, newly emerging avian influenza A viruses pose a continued threat, not only to avian species but also to humans. We studied the pathogenicity of four human and nine avian H5N1/04 influenza viruses in ferrets (an excellent model for influenza studies). All four human isolates were fatal to intranasally inoculated ferrets. The human isolate A/Vietnam/1203/04 (H5N1) was the most pathogenic isolate; the severity of disease was associated with a broad tissue tropism and high virus titers in multiple organs, including the brain. High fever, weight loss, anorexia, extreme lethargy, and diarrhea were observed. Two avian H5N1/04 isolates were as pathogenic as the human viruses, causing lethal systemic infections in ferrets. Seven of nine H5N1/04 viruses isolated from avian species caused mild infections, with virus replication restricted to the upper respiratory tract. All chicken isolates were nonlethal to ferrets. A sequence analysis revealed polybasic amino acids in the hemagglutinin connecting peptides of all H5N1/04 viruses, indicating that multiple molecular differences in other genes are important for a high level of virulence. Interestingly, the human A/Vietnam/1203/04 isolate had a lysine substitution at position 627 of PB2 and had one to eight amino acid changes in all gene products except that of the M1 gene, unlike the A/chicken/Vietnam/C58/04 and A/quail/Vietnam/36/04 viruses. Our results indicate that viruses that are lethal to mammals are circulating among birds in Asia and suggest that pathogenicity in ferrets, and perhaps humans, reflects a complex combination of different residues rather than a single amino acid difference.

Animals↗

PubMed2005

Neuraminidase inhibitor susceptibility network position statement: antiviral resistance in influenza A/H5N1 viruses.

The emerging epidemic of H5N1 avian influenza virus with spillover into the human population in Asia has provoked intense concern globally about the potential of these particularly pathogenic viruses to evolve with the capacity for human-to-human transmission with a consequent pandemic. The availability of antiviral drugs with activity against influenza A viruses and the recognition of drug-resistant variants to these drugs prompted the following report by a select group of the global experts--members of the Neuraminidase Inhibitor Susceptibility Network--on the best use of the available drugs, both for prophylaxis and treatment. The editors of Antiviral Therapy are pleased to be able to provide this document in an expeditious manner.

Acetamides↗

PubMed2004

No apoptotic deaths and different levels of inductions of inflammatory cytokines in alveolar macrophages infected with influenza viruses.

Influenza viruses are reported to infect mainly the respiratory tract epithelium of hosts. Our studies in a pig model show that influenza A viruses infect alveolar macrophages that constitutively reside in the respiratory tract, without causing apoptosis. Tumor necrosis factor alpha was the inflammatory cytokine most highly induced in these macrophages. In vivo, alveolar macrophages infected with human H3N2 influenza virus showed greater expression of tumor necrosis factor alpha than did alveolar macrophages infected with human H1N1 influenza virus. Induction of specific inflammatory cytokine such as TNF-alpha is a polygenic trait that involves the HA and NA genes. Markedly elevated expression of tumor necrosis factor alpha may be responsible for the high mortality rate caused by H3N2 influenza virus infection in elderly patients.

Animals↗

PubMed2004

Interspecies transmission of an H7N3 influenza virus from wild birds to intensively reared domestic poultry in Italy.

Since the "bird flu" incident in Hong Kong SAR in 1997, several studies have highlighted the substantial role of domestic birds, such as turkeys and chickens, in the ecology of influenza A viruses. Even if recent evidence suggests that chickens can maintain several influenza serotypes, avian influenza viruses (AIVs) circulating in domestic species are believed to be introduced each time from the wild bird reservoir. However, so far the direct precursor of influenza viruses from domestic birds has never been identified. In this report, we describe the antigenic and genetic characterization of the surface proteins of H7N3 viruses isolated from wild ducks in Italy in 2001 in comparison to H7N3 strains that circulated in Italian turkeys in 2002-2003. The wild and domestic avian strains appeared strictly related at both phenotypic and genetic level: homology percentages in seven of their genes were comprised between 99.8% (for PB2) and 99.1% (for M), and their NA genes differed mainly because of a 23-aminoacid deletion in the NA stalk. Outside this region of the molecule, the NAs of the two virus groups showed 99% similarity. These findings indicate that turkey H7N3 viruses were derived "in toto" from avian influenza strains circulating in wild waterfowl 1 year earlier, and represent an important step towards the comprehension of the mechanisms leading to interspecies transmission and emergence of potentially pandemic influenza viruses.

Amino Acid Sequence↗

PubMed2004

Wet markets--a continuing source of severe acute respiratory syndrome and influenza?

CONTEXT: Live-animal markets (wet markets) provide a source of vertebrate and invertebrate animals for customers in tropical and subtropical regions of the world. Wet markets sell live poultry, fish, reptiles, and mammals of every kind. Live-poultry markets (mostly chicken, pigeon, quail, ducks, geese, and a wide range of exotic wild-caught and farm-raised fowl) are usually separated from markets selling fish or red-meat animals, but the stalls can be near each other with no physical separation. Despite the widespread availability of affordable refrigeration, many Asian people prefer live animals for fresh produce. Wet markets are widespread in Asian countries and in countries where Asian people have migrated. Live-poultry markets were the source of the H5N1 bird-influenza virus that transmitted to and killed six of 18 people in Hong Kong. STARTING POINT: Yi Guan and colleagues (Science 2003; 302: 276-78) recently reported the isolation of severe acute respiratory syndrome (SARS) coronavirus (CoV) from Himalayan palm civets (Paguna larvata) in wet markets in Shenzen, southern China. These researchers also found serological evidence of infection in raccoon dogs (Nyctereutes procuyoinboides). Serological evidence for SARS CoV in human beings working in these markets, taken together with the earliest cases of SARS in restaurant workers, supports the contention of a potential zoonotic origin for SARS. WHERE NEXT? Will SARS reappear? This question confronts public-health officials worldwide, particularly infectious disease personnel in those regions of the world most affected by the disease and the economic burden of SARS, including China, Taiwan, and Canada. Will the virus re-emerge from wet markets or from laboratories working with SARS CoV, or are asymptomatic infections ongoing in human beings? Similar questions can be asked about a pandemic of influenza that is probably imminent. Knowledge of the ecology of influenza in wet markets can be used as an early-warning system to detect the reappearance of SARS or pandemic influenza.

Animals↗

PubMed2004

The NS1 gene of H5N1 influenza viruses circumvents the host anti-viral cytokine responses.

The H5N1 influenza viruses transmitted to humans in 1997 were highly virulent, but the mechanism of their virulence in humans is largely unknown. Here we show that lethal H5N1 influenza viruses, unlike other human, avian, and swine influenza viruses, are resistant to the anti-viral effects of interferons and tumor necrosis factor alpha The nonstructural (NS) gene of H5N1 viruses is associated with this resistance. Pigs infected with recombinant human H1N1 influenza virus that carried the H5N1 NS gene experienced significantly greater and more prolonged viremia, fever, and weight loss than did pigs infected with wild-type human H1N1 influenza virus. These effects required the presence of glutamic acid at position 92 of the NS1 molecule. These findings may explain the mechanism of the high virulence of H5N1 influenza viruses in humans and provide insight into the virulence of 1918 Spanish influenza.

Animals↗