RESUMEN
H5N1 influenza viruses transmitted from poultry to humans in Asia cause high mortality and pose a pandemic threat. Viral genes important for cell tropism and replication efficiency must be identified to elucidate and target virulence factors. We applied reverse genetics to generate H5N1 reassortants combining genes of lethal A/Vietnam/1203/04 (VN1203), a fatal human case isolate, and nonlethal A/chicken/Vietnam/C58/04 (CH58) and tested their pathogenicity in ferrets and mice. The viruses' hemagglutinins have six amino acids differences, identical cleavage sites, and avian-like alpha-(2,3)-linked receptor specificity. Surprisingly, exchanging hemagglutinin and neuraminidase genes did not alter pathogenicity, but substituting CH58 polymerase genes completely attenuated VN1203 virulence and reduced viral polymerase activity. CH58's NS gene partially attenuated VN1203 in ferrets but not in mice. Our findings suggest that for high virulence in mammalian species an avian H5N1 virus with a cleavable hemagglutinin requires adaptive changes in polymerase genes to overcome the species barrier. Thus, novel antivirals targeting polymerase proteins should be developed.
Asunto(s)
Subtipo H5N1 del Virus de la Influenza A/genética , Gripe Aviar/genética , Gripe Humana/genética , ARN Polimerasa Dependiente del ARN/genética , Factores de Virulencia/genética , Animales , Antivirales/uso terapéutico , Pollos/virología , Inhibidores Enzimáticos/uso terapéutico , Hurones/virología , Variación Genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Gripe Aviar/tratamiento farmacológico , Gripe Aviar/patología , Gripe Humana/tratamiento farmacológico , Gripe Humana/patología , Ratones , Neuraminidasa/genética , Procesamiento Proteico-Postraduccional/genética , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Especificidad de la EspecieRESUMEN
The successful replication of a viral pathogen in a host is a complex process involving many interactions. These interactions develop from the coevolution of pathogen and host and often lead to a species specificity of the virus that can make interspecies transmissions difficult. Nevertheless, viruses do sporadically cross species barriers into other host populations, including humans. In zoonotic infections, many of these interspecies transfer events are dead end, where transmission is confined only to the animal-to-human route but sometimes viruses adapt to enable spread from human to human. A pathogen must overcome many hurdles to replicate successfully in a foreign host. The viral pathogen must enter the host cell, replicate with the assistance of host factors, evade inhibitory host products, exit the first cell and move on to the next, and possibly leave the initial host and transmit to another. Each of these stages may require adaptive changes in the pathogen. Although the factors that influence each stage of the replication and transmission of most agents have not been resolved, the genomics of both hosts and pathogens are now at hand and we have begun to understand some of the molecular changes that enable some viruses to adapt to a new host.
Asunto(s)
Virosis/transmisión , Virus , Zoonosis , Animales , Antivirales , Genoma , Humanos , Modelos Biológicos , Especificidad de la Especie , Vacunas , Virosis/genética , Virosis/prevención & controlRESUMEN
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 antiviral 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.
Asunto(s)
Citocinas/inmunología , Farmacorresistencia Viral Múltiple/inmunología , Subtipo H5N1 del Virus de la Influenza A , Virus de la Influenza A/patogenicidad , Gripe Humana/inmunología , Gripe Humana/virología , Proteínas no Estructurales Virales/metabolismo , Animales , Células Cultivadas , Farmacorresistencia Viral Múltiple/genética , Células Epiteliales/efectos de los fármacos , Células Epiteliales/virología , Humanos , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/genética , Gripe Humana/transmisión , Interferón-alfa/inmunología , Interferón-alfa/farmacología , Interferón gamma/inmunología , Interferón gamma/farmacología , Mutación Puntual , Porcinos , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/farmacología , Proteínas no Estructurales Virales/efectos de los fármacos , Proteínas no Estructurales Virales/genética , Virulencia/genéticaRESUMEN
BACKGROUND: The clinical management of H5N1 influenza virus infection in humans remains unclear. Combination chemotherapy with drugs that target different viral proteins might be more effective than monotherapy. METHODS: BALB/c mice were treated by oral gavage for 5 days with amantadine (1.5, 15 or 30 mg/kg/day) and oseltamivir (1 or 10 mg/kg/day) separately or in combination. Mice were challenged 24 h after initiation of treatment with 10 mouse 50% lethal doses of either amantadine-sensitive (having S31 in the M2 protein) or amantadine-resistant (having N31 in the M2 protein) recombinant A/Vietnam/1203/04 (H5N1) virus. RESULTS: Combination treatment with amantadine (15 or 30 mg/kg/day) and oseltamivir (10 mg/kg/day) provided greater protection (60% and 90%, respectively) against lethal infection with amantadine-sensitive H5N1 virus than did monotherapy. Moreover, spread of the virus to the brain was prevented by both combination regimens. The efficacy of the drug combinations against amantadine-resistant H5N1 virus was comparable to that of oseltamivir alone. Oseltamivir produced a dose-dependent effect against both recombinant H5N1 viruses (P < 0.05) but did not provide complete protection against lethal infection. Importantly, no mutations in the HA, NA and M2 proteins were detected when the two drugs were used in combination. CONCLUSIONS: Combination chemotherapy provided a survival advantage over single-agent treatment of mice inoculated with neurotropic H5N1 influenza virus. This strategy might be an option for the control of pandemic influenza viruses that are sensitive to amantadine. Combinations that include other drugs should be explored.
Asunto(s)
Amantadina/uso terapéutico , Antivirales/uso terapéutico , Subtipo H5N1 del Virus de la Influenza A , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Oseltamivir/uso terapéutico , Administración Oral , Amantadina/administración & dosificación , Amantadina/farmacología , Animales , Antivirales/administración & dosificación , Antivirales/farmacología , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Farmacorresistencia Viral , Quimioterapia Combinada , Femenino , Subtipo H5N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H5N1 del Virus de la Influenza A/genética , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Infecciones por Orthomyxoviridae/virología , Oseltamivir/administración & dosificación , Oseltamivir/farmacologíaRESUMEN
Influenza viruses replicate within the nucleus of infected cells. Viral genomic RNA, three polymerase subunits (PB2, PB1, and PA), and the nucleoprotein (NP) form ribonucleoprotein complexes (RNPs) that are exported from the nucleus late during the infectious cycle. The virus-induced Raf/MEK/ERK (MAPK) signal cascade is crucial for efficient virus replication. Blockade of this pathway retards RNP export and reduces virus titers. Hemagglutinin (HA) accumulation and its tight association with lipid rafts activate ERK and enhance localization of cytoplasmic RNPs. We studied the induction of MAPK signal cascade by two seasonal human influenza A viruses A/HK/218449/06 (H3N2) and A/HK/218847/06 (H1N1) that differed substantially in their replication efficiency in tissue culture. Infection with H3N2 virus, which replicates efficiently, resulted in higher HA expression and its accumulation on the cell membrane, leading to substantially increased activation of MAPK signaling compared to that caused by H1N1 subtype. More H3N2-HAs were expressed and accumulated on the cell membrane than did H1N1-HAs. Viral polymerase genes, particularly H3N2-PB1 and H3N2-PB2, were observed to contribute to increased viral polymerase activity. Applying plasmid-based reverse genetics to analyze the role of PB1 protein in activating HA-induced MAPK cascade showed that recombinant H1N1 virus possessing the H3N2-PB1 (rgH1N1/H3N2-PB1) induced greater ERK activation, resulting in increased nuclear export of the viral genome and higr virus titers. We conclude that enhanced viral polymerase activity promotes the replication and transcription of viral RNA leading to increased accumulation of HA on the cell surface and thereby resulting in an upregulation of the MAPK cascade and more efficient nuclear RNP-export as well as virus production.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/enzimología , Subtipo H3N2 del Virus de la Influenza A/enzimología , Gripe Humana/enzimología , Sistema de Señalización de MAP Quinasas/fisiología , ARN Polimerasa Dependiente del ARN/metabolismo , Animales , Perros , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Hemaglutininas/metabolismo , Humanos , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Subtipo H1N1 del Virus de la Influenza A/fisiología , Subtipo H3N2 del Virus de la Influenza A/metabolismo , Subtipo H3N2 del Virus de la Influenza A/fisiología , Gripe Humana/virología , Quinasas Quinasa Quinasa PAM/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas Virales/metabolismo , Replicación Viral , Quinasas raf/metabolismoRESUMEN
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.
Asunto(s)
Citocinas/farmacología , Farmacorresistencia Viral Múltiple , Subtipo H5N1 del Virus de la Influenza A , Virus de la Influenza A/patogenicidad , Gripe Humana/inmunología , Proteínas no Estructurales Virales/genética , Animales , Línea Celular , Citocinas/metabolismo , Farmacorresistencia Viral Múltiple/genética , Humanos , Virus de la Influenza A/genética , Gripe Humana/fisiopatología , Gripe Humana/virología , Interferones/metabolismo , Interferones/farmacología , Mutación , Recombinación Genética , Porcinos/virología , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Because proinflammatory cytokines are markedly elevated during H5N1 influenza virus infection, the "cytokine storm" is hypothesized to be the main cause of mortality. Here, we demonstrate that mice deficient in the hallmark inflammatory cytokines TNF-alpha, IL-6, or CC chemokine ligand 2 succumb to infection with A/Vietnam/1203/04 (H5N1) virus, as do wild-type mice treated with glucocorticoids for suppression of cytokines. Because cytokine inhibition does not protect against death, therapies that target the virus rather than cytokines may be preferable.
Asunto(s)
Citocinas/metabolismo , Subtipo H5N1 del Virus de la Influenza A/fisiología , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/virología , Animales , Citocinas/deficiencia , Citocinas/genética , Glucocorticoides/uso terapéutico , Masculino , Ratones , Ratones Noqueados , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Infecciones por Orthomyxoviridae/prevención & control , Tasa de Supervivencia , Pérdida de PesoRESUMEN
In the present study we determined the antiviral effect of amantadine against influenza A/Netherlands/219/03 (H7N7) virus in cell culture and in a mouse model. Amantadine at concentrations <100 muM failed to inhibit virus replication in Madin-Darby canine kidney (MDCK) cells. When orally administered to mice for 5 days, amantadine at 15 mg kg(-1) day(-1) did not protect animals against lethal challenge with H7N7 infection, and virus titres in mouse organs were not reduced. However, sequence analysis of the M2 protein revealed none of the mutations previously described as being associated with amantadine resistance. We used reverse genetics to generate viruses containing the haemagglutinin (HA) or M gene of A/Netherlands/219/03 virus to investigate the role of these genes in amantadine sensitivity. All recombinant viruses carrying the HA segment of A/Netherlands/219/03 (H7N7) virus were amantadine-resistant, regardless of the origin of their other genes. To study the role of fusion activity in the mechanism of drug resistance, we introduced the Gly(23)-->Cys mutation in the H7 fusion peptide. This substitution resulted in a decrease of the pH of fusion and was also associated with reduced virus replication in both MDCK cells and mice, as compared to that of the wild-type virus. We suggest that H7 HA protein plays a role in amantadine resistance, although all HA amino acids that participate in drug resistance still remain to be characterized. Our finding reveals that sequence analysis of the transmembrane domain of M2 protein may not adequately identify all drug-resistant variants.
Asunto(s)
Amantadina/farmacología , Glicoproteínas Hemaglutininas del Virus de la Influenza/fisiología , Subtipo H7N7 del Virus de la Influenza A/efectos de los fármacos , Subtipo H7N7 del Virus de la Influenza A/fisiología , Amantadina/uso terapéutico , Sustitución de Aminoácidos , Animales , Antivirales/farmacología , Antivirales/uso terapéutico , Sangre/virología , Encéfalo/virología , Línea Celular , Embrión de Pollo , Modelos Animales de Enfermedad , Perros , Farmacorresistencia Viral/genética , Femenino , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Pulmón/virología , Ratones , Ratones Endogámicos BALB C , Mutagénesis Sitio-Dirigida , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Infecciones por Orthomyxoviridae/virología , Bazo/virología , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/fisiologíaRESUMEN
Effective antiviral drugs are essential for early control of an influenza pandemic. It is therefore crucial to evaluate the possible threat posed by neuraminidase (NA) inhibitor-resistant influenza viruses with pandemic potential. Four NA mutations (E119G, H274Y, R292K, and N294S) that have been reported to confer resistance to NA inhibitors were each introduced into recombinant A/Vietnam/1203/04 (VN1203) H5N1 influenza virus. For comparison, the same mutations were introduced into recombinant A/Puerto Rico/8/34 (PR8) H1N1 influenza virus. The E119G and R292K mutations significantly compromised viral growth in vitro, but the H274Y and N294S mutations were stably maintained in VN1203 and PR8 viruses. In both backgrounds, the H274Y and N294S mutations conferred resistance to oseltamivir carboxylate (50% inhibitory concentration [IC(50)] increases, >250-fold and >20-fold, respectively), and the N294S mutation reduced susceptibility to zanamivir (IC(50) increase, >3.0-fold). Although the H274Y and N294S mutations did not compromise the replication efficiency of VN1203 or PR8 viruses in vitro, these mutations slightly reduced the lethality of PR8 virus in mice. However, the VN1203 virus carrying either the H274Y or N294S mutation exhibited lethality similar to that of the wild-type VN1203 virus. The different enzyme kinetic parameters (V(max) and K(m)) of avian-like VN1203 NA and human-like PR8 NA suggest that resistance-associated NA mutations can cause different levels of functional loss in NA glycoproteins of the same subtype. Our results suggest that NA inhibitor-resistant H5N1 variants may retain the high pathogenicity of the wild-type virus in mammalian species. Patients receiving NA inhibitors for H5N1 influenza virus infection should be closely monitored for the emergence of resistant variants.
Asunto(s)
Farmacorresistencia Viral/genética , Subtipo H5N1 del Virus de la Influenza A/enzimología , Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Gripe Humana/virología , Neuraminidasa/genética , Proteínas Virales/genética , Animales , Antivirales/farmacología , Inhibidores Enzimáticos/farmacología , Humanos , Subtipo H5N1 del Virus de la Influenza A/genética , Cinética , Ratones , Ratones Endogámicos BALB C , Neuraminidasa/antagonistas & inhibidores , Neuraminidasa/química , Oseltamivir/farmacología , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/química , Virulencia/genética , Replicación Viral/genéticaRESUMEN
We investigated the importance of the host Mx1 gene in protection against highly pathogenic H5N1 avian influenza virus. Mice expressing the Mx1 gene survived infection with the lethal human H5N1 isolate A/Vietnam/1203/04 and with reassortants combining its genes with those of the non-lethal virus A/chicken/Vietnam/C58/04, while all Mx1-/- mice succumbed. Mx1-expressing mice showed lower organ virus titers, fewer lesions, and less pulmonary inflammation. Our data support the hypothesis that Mx1 expression protects mice against the high pathogenicity of H5N1 virus through inhibition of viral polymerase activity ultimately resulting in reduced viral growth and spread. Drugs that mimic this mechanism may be protective in humans.
Asunto(s)
Proteínas de Unión al GTP/inmunología , Subtipo H5N1 del Virus de la Influenza A/metabolismo , Gripe Humana/inmunología , Infecciones por Orthomyxoviridae/inmunología , Proteínas Virales/metabolismo , Animales , Pollos , Proteínas de Unión al GTP/genética , Humanos , Gripe Humana/virología , Hígado/citología , Hígado/metabolismo , Pulmón/citología , Pulmón/metabolismo , Ratones , Ratones Endogámicos , Proteínas de Resistencia a Mixovirus , Infecciones por Orthomyxoviridae/virologíaRESUMEN
The abilities to infect and transmit efficiently among humans are essential for a novel influenza A virus to cause a pandemic. To evaluate the pandemic potential of widely disseminated H5N1 influenza viruses, a ferret contact model using experimental groups comprised of one inoculated ferret and two contact ferrets was used to study the transmissibility of four human H5N1 viruses isolated from 2003 to 2006. The effects of viral pathogenicity and receptor binding specificity (affinity to synthetic sialosaccharides with alpha2,3 or alpha2,6 linkages) on transmissibility were assessed. A/Vietnam/1203/04 and A/Vietnam/JP36-2/05 viruses, which possess "avian-like" alpha2,3-linked sialic acid (SA) receptor specificity, caused neurological symptoms and death in ferrets inoculated with 10(3) 50% tissue culture infectious doses. A/Hong Kong/213/03 and A/Turkey/65-596/06 viruses, which show binding affinity for "human-like" alpha2,6-linked SA receptors in addition to their affinity for alpha2,3-linked SA receptors, caused mild clinical symptoms and were not lethal to the ferrets. No transmission of A/Vietnam/1203/04 or A/Turkey/65-596/06 virus was detected. One contact ferret developed neutralizing antibodies to A/Hong Kong/213/03 but did not exhibit any clinical signs or detectable virus shedding. In two groups, one of two naïve contact ferrets had detectable virus after 6 to 8 days when housed together with the A/Vietnam/JP36-2/05 virus-inoculated ferrets. Infected contact ferrets showed severe clinical signs, although little or no virus was detected in nasal washes. This limited virus shedding explained the absence of secondary transmission from the infected contact ferret to the other naïve ferret that were housed together. Our results suggest that despite their receptor binding affinity, circulating H5N1 viruses retain molecular determinants that restrict their spread among mammalian species.
Asunto(s)
Modelos Animales de Enfermedad , Hurones/virología , Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Infecciones por Orthomyxoviridae/transmisión , Animales , Brotes de Enfermedades , Hurones/inmunología , Humanos , Subtipo H5N1 del Virus de la Influenza A/inmunología , Cavidad Nasal/patología , Cavidad Nasal/virología , Infecciones por Orthomyxoviridae/epidemiología , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/patología , Especificidad de la Especie , Esparcimiento de Virus/inmunologíaRESUMEN
Ferrets were immunized with two 7- mu g doses of hemagglutinin from inactivated whole-virus vaccines containing the hemagglutinin gene of A/Duck/Singapore/3/97(H5N3) then inoculated with a lethal dose of A/Vietnam/1203/04(H5N1) (Viet/1203/04). Serum samples did not react with Viet/1203/04 in hemagglutination-inhibition (HI) or virus-neutralization (VN) tests. All vaccinated ferrets survived the challenge, whereas all mock-immunized ferrets died. Immunized ferrets had significantly lower virus titers in the upper respiratory tract and less-severe disease. Vaccine generated from antigenically different H5 virus protects against infection by a highly pathogenic H5 strain. Neither HI nor VN testing provides correlates of cross-protection in ferrets.
Asunto(s)
Subtipo H5N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/uso terapéutico , Gripe Humana/prevención & control , Animales , Anticuerpos Antivirales/biosíntesis , Modelos Animales de Enfermedad , Femenino , Hurones , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Vacunas contra la Influenza/administración & dosificación , MutaciónRESUMEN
Neuraminidase inhibitors (NAIs) are antivirals designed to target conserved residues at the neuraminidase (NA) enzyme active site in influenza A and B viruses. The conserved residues that interact with NAIs are under selective pressure, but only a few have been linked to resistance. In the A/Wuhan/359/95 (H3N2) recombinant virus background, we characterized seven charged, conserved NA residues (R118, R371, E227, R152, R224, E276, and D151) that directly interact with the NAIs but have not been reported to confer resistance to NAIs. These NA residues were replaced with amino acids that possess side chains having similar properties to maintain their original charge. The NA mutations we introduced significantly decreased NA activity compared to that of the A/Wuhan/359/95 recombinant wild-type and R292K (an NA mutation frequently reported to confer resistance) viruses, which were analyzed for comparison. However, the recombinant viruses differed in replication efficiency when we serially passaged them in vitro; the growth of the R118K and E227D viruses was most impaired. The R224K, E276D, and R371K mutations conferred resistance to both zanamivir and oseltamivir, while the D151E mutation reduced susceptibility to oseltamivir only (approximately 10-fold) and the R152K mutation did not alter susceptibility to either drug. Because the R224K mutation was genetically unstable and the emergence of the R371K mutation in the N2 subtype is statistically unlikely, our results suggest that only the E276D mutation is likely to emerge under selective pressure. The results of our study may help to optimize the design of NAIs.
Asunto(s)
Antivirales/farmacología , Sitios de Unión/genética , Farmacorresistencia Viral , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Neuraminidasa/antagonistas & inhibidores , Neuraminidasa/química , Animales , Línea Celular , Humanos , Subtipo H3N2 del Virus de la Influenza A/enzimología , Subtipo H3N2 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/fisiología , Pruebas de Sensibilidad Microbiana/métodos , Mutación , Neuraminidasa/metabolismo , Recombinación Genética , Ensayo de Placa ViralRESUMEN
H5N1 avian influenza viruses are continuing to spread in waterfowl in Eurasia and to threaten the health of avian and mammalian species. The possibility that highly pathogenic (HP) H5N1 avian influenza is now endemic in both domestic and migratory birds in Eurasia makes it unlikely that culling alone will control H5N1 influenza. Because ducks are not uniformly killed by HP H5N1 viruses, they are considered a major contributor to virus spread. Here, we describe a reverse genetics-derived high-growth H5N3 strain containing the modified H5 of A/chicken/Vietnam/C58/04, the N3 of A/duck/Germany/1215/73, and the internal genes of A/PR/8/34. One or two doses of inactivated oil emulsion vaccine containing 0.015 to 1.2 microg of HA protein provide highly efficacious protection against lethal H5N1 challenge in ducks; only the two dose regimen has so far been tested in chickens with high protective efficacy.
Asunto(s)
Pollos/inmunología , Patos/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Gripe Aviar/inmunología , Gripe Aviar/prevención & control , Animales , Pollos/virología , Cloaca/virología , Relación Dosis-Respuesta Inmunológica , Patos/virología , Vacunas contra la Influenza/administración & dosificación , Gripe Aviar/virología , Especificidad de la Especie , Organismos Libres de Patógenos Específicos , Tráquea/virología , Esparcimiento de VirusRESUMEN
The spread of H5N1 avian influenza viruses (AIVs) from China to Europe has raised global concern about their potential to infect humans and cause a pandemic. In spite of their substantial threat to human health, remarkably little AIV whole-genome information is available. We report here a preliminary analysis of the first large-scale sequencing of AIVs, including 2196 AIV genes and 169 complete genomes. We combine this new information with public AIV data to identify new gene alleles, persistent genotypes, compensatory mutations, and a potential virulence determinant.
Asunto(s)
Genes Virales , Subtipo H5N1 del Virus de la Influenza A/genética , Virus de la Influenza A/genética , Proteínas no Estructurales Virales/química , Factores de Virulencia/química , Animales , Aves/virología , Biología Computacional , Genoma Viral , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H2N2 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/genética , Subtipo H3N8 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/química , Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Subtipo H5N2 del Virus de la Influenza A/genética , Subtipo H7N7 del Virus de la Influenza A/genética , Subtipo H9N2 del Virus de la Influenza A/genética , Virus de la Influenza A/química , Virus de la Influenza A/aislamiento & purificación , Virus de la Influenza A/patogenicidad , Gripe Aviar/virología , Gripe Humana/virología , Datos de Secuencia Molecular , Mutación , Filogenia , ARN Viral/genética , Virus Reordenados/genética , Análisis de Secuencia de ADN , Proteínas no Estructurales Virales/genética , Proteínas Virales/química , Proteínas Virales/genética , Factores de Virulencia/genéticaRESUMEN
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.
Asunto(s)
Aminoácidos/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Vacunas contra la Influenza/química , Vacunas contra la Influenza/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Orthomyxoviridae/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/inmunología , Línea Celular , Femenino , Hurones , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Masculino , Modelos Moleculares , Mutación/genética , Pruebas de Neutralización , Orthomyxoviridae/química , Orthomyxoviridae/genética , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/virología , Estructura Cuaternaria de Proteína , Análisis de Secuencia de ADNRESUMEN
Serial passage of an initially avirulent influenza B virus, B/Memphis/12/97, resulted in the selection of a variant which was lethal in mice. Virulence correlated with improved growth in vivo and prolonged replication. Sequencing of the complete coding regions of the parent and mouse-adapted viruses revealed 8 amino acid differences. Sequencing and characterization of intermediate passages suggested that one change in the C-terminal domain of the M1 protein, an asparagine to a serine at position 221, was responsible for acquisition of virulence and lethality. Site-directed mutagenesis of the M segment of a different virus, B/Yamanashi/166/98, to change this amino acid residue confirmed its importance by conferring improved growth and virulence in mice. This observation suggests a role for the C domain of the M1 protein in growth and virulence in a mammalian host.
Asunto(s)
Virus de la Influenza B/patogenicidad , Infecciones por Orthomyxoviridae/virología , Proteínas de la Matriz Viral/fisiología , Adaptación Fisiológica , Sustitución de Aminoácidos , Animales , Asparagina , Femenino , Virus de la Influenza B/fisiología , Ratones , Ratones Endogámicos BALB C , Estructura Terciaria de Proteína , Pase Seriado , Serina , Proteínas de la Matriz Viral/genética , Virulencia , Replicación ViralRESUMEN
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.
Asunto(s)
Patos/virología , Subtipo H5N1 del Virus de la Influenza A , Virus de la Influenza A/genética , Virus de la Influenza A/fisiología , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , China , ADN Viral/genética , Femenino , Humanos , Virus de la Influenza A/aislamiento & purificación , Virus de la Influenza A/patogenicidad , Gripe Humana/virología , Ratones , Ratones Endogámicos BALB C , Especificidad de la Especie , Proteínas Virales/genética , Virulencia/genética , Replicación Viral/genéticaRESUMEN
Cold-adapted (ca) B/Ann Arbor/1/66 is the influenza B virus strain master donor virus for FluMist, a live, attenuated, influenza virus vaccine licensed in 2003 in the United States. Each FluMist vaccine strain contains six gene segments of the master donor virus; these master donor gene segments control the vaccine's replication and attenuation. These gene segments also express characteristic biological traits in model systems. Unlike most virulent wild-type (wt) influenza B viruses, ca B/Ann Arbor/1/66 is temperature sensitive (ts) at 37 degrees C and attenuated (att) in the ferret model. In order to define the minimal genetic components of these phenotypes, the amino acid sequences of the internal genes of ca B/Ann Arbor/1/66 were aligned to those of other influenza B viruses. These analyses revealed eight unique amino acids in three proteins: two in the polymerase subunit PA, two in the M1 matrix protein, and four in the nucleoprotein (NP). Using reverse genetics, these eight wt amino acids were engineered into a plasmid-derived recombinant of ca B/Ann Arbor/1/66, and these changes reverted both the ts and the att phenotypes. A detailed mutational analysis revealed that a combination of two sites in NP (A114 and H410) and one in PA (M431) controlled expression of ts, whereas these same changes plus two additional residues in M1 (Q159 and V183) controlled the att phenotype. Transferring this genetic signature to the divergent wt B/Yamanashi/166/98 strain conferred both the ts and the att phenotypes on the recombinant, demonstrating that this small, complex, genetic signature encoded the essential elements for these traits.