RESUMO
Broadly neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are sought to curb coronavirus disease 2019 (COVID-19) infections. Here we produced and characterized a set of mouse monoclonal antibodies (mAbs) specific for the ancestral SARS-CoV-2 receptor binding domain (RBD). Two of them, 17A7 and 17B10, were highly potent in microneutralization assay with 50% inhibitory concentration (IC50 ) ≤135 ng/mL against infectious SARS-CoV-2 variants, including G614, Alpha, Beta, Gamma, Delta, Epsilon, Zeta, Kappa, Lambda, B.1.1.298, B.1.222, B.1.5, and R.1. Both mAbs (especially 17A7) also exhibited strong in vivo efficacy in protecting K18-hACE2 transgenic mice from the lethal infection with G614, Alpha, Beta, Gamma, and Delta viruses. Structural analysis indicated that 17A7 and 17B10 target the tip of the receptor binding motif in the RBD-up conformation. A third RBD-reactive mAb (3A6) although escaped by Beta and Gamma, was highly effective in cross-neutralizing Delta and Omicron BA.1 variants in vitro and in vivo. In competition experiments, antibodies targeting epitopes similar to these 3 mAbs were rarely enriched in human COVID-19 convalescent sera or postvaccination sera. These results are helpful to inform new antibody/vaccine design and these mAbs can be useful tools for characterizing SARS-CoV-2 variants and elicited antibody responses.
Assuntos
Anticorpos Monoclonais , COVID-19 , Animais , Camundongos , Humanos , SARS-CoV-2/genética , Soroterapia para COVID-19 , Camundongos Transgênicos , Glicoproteína da Espícula de Coronavírus/genética , Anticorpos Antivirais , Anticorpos Neutralizantes , Testes de NeutralizaçãoRESUMO
The panzootic caused by A/goose/Guangdong/1/96-lineage highly pathogenic avian influenza (HPAI) A(H5) viruses has occurred in multiple waves since 1996. From 2013 onwards, clade 2.3.4.4 viruses of subtypes A(H5N2), A(H5N6), and A(H5N8) emerged to cause panzootic waves of unprecedented magnitude among avian species accompanied by severe losses to the poultry industry around the world. Clade 2.3.4.4 A(H5) viruses have expanded in distinct geographical and evolutionary pathways likely via long distance migratory bird dispersal onto several continents and by poultry trade among neighboring countries. Coupled with regional circulation, the viruses have evolved further by reassorting with local viruses. As of February 2019, there have been 23 cases of humans infected with clade 2.3.4.4 H5N6 viruses, 16 (70%) of which had fatal outcomes. To date, no HPAI A(H5) virus has caused sustainable human-to-human transmission. However, due to the lack of population immunity in humans and ongoing evolution of the virus, there is a continuing risk that clade 2.3.4.4 A(H5) viruses could cause an influenza pandemic if the ability to transmit efficiently among humans was gained. Therefore, multisectoral collaborations among the animal, environmental, and public health sectors are essential to conduct risk assessments and develop countermeasures to prevent disease and to control spread. In this article, we describe an assessment of the likelihood of clade 2.3.4.4 A(H5) viruses gaining human-to-human transmissibility and impact on human health should such human-to-human transmission occur. This structured analysis assessed properties of the virus, attributes of the human population, and ecology and epidemiology of these viruses in animal hosts.
Assuntos
Vírus da Influenza A Subtipo H5N2/fisiologia , Influenza Aviária/transmissão , Influenza Humana/transmissão , Doenças das Aves Domésticas/transmissão , Animais , Humanos , Vírus da Influenza A Subtipo H5N2/genética , Influenza Aviária/epidemiologia , Influenza Aviária/virologia , Influenza Humana/epidemiologia , Influenza Humana/virologia , Pandemias , Aves Domésticas , Doenças das Aves Domésticas/epidemiologia , Doenças das Aves Domésticas/virologiaRESUMO
Baloxavir showed broad-spectrum in vitro replication inhibition of 4 types of influenza viruses (90% effective concentration range 1.2-98.3 nmol/L); susceptibility pattern was influenza A Ë B Ë C Ë D. This drug also inhibited influenza A viruses of avian and swine origin, including viruses that have pandemic potential and those resistant to neuraminidase inhibitors.
Assuntos
Antivirais/farmacologia , Gammainfluenzavirus/efeitos dos fármacos , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza B/efeitos dos fármacos , Oxazinas/farmacologia , Piridinas/farmacologia , Tiepinas/farmacologia , Thogotovirus/efeitos dos fármacos , Triazinas/farmacologia , Animais , Galinhas/virologia , Dibenzotiepinas , Cães , Humanos , Influenza Aviária/tratamento farmacológico , Influenza Aviária/virologia , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , Células Madin Darby de Rim Canino/virologia , Testes de Sensibilidade Microbiana , Morfolinas , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/veterinária , Infecções por Orthomyxoviridae/virologia , Piridonas , Suínos/virologia , Doenças dos Suínos/tratamento farmacológico , Doenças dos Suínos/virologiaRESUMO
Background: Neuraminidase (NA) inhibitors are the recommended antiviral medications for influenza treatment. However, their therapeutic efficacy can be compromised by NA changes that emerge naturally and/or following antiviral treatment. Knowledge of which molecular changes confer drug resistance of influenza A(H7N9) viruses (group 2NA) remains sparse. Methods: Fourteen amino acid substitutions were introduced into the NA of A/Shanghai/2/2013(H7N9). Recombinant N9 (recN9) proteins were expressed in a baculovirus system in insect cells and tested using the Centers for Disease Control and Prevention standardized NA inhibition (NI) assay with oseltamivir, zanamivir, peramivir, and laninamivir. The wild-type N9 crystal structure was determined in complex with oseltamivir, zanamivir, or sialic acid, and structural analysis was performed. Results: All substitutions conferred either reduced or highly reduced inhibition by at least 1 NA inhibitor; half of them caused reduced inhibition or highly reduced inhibition by all NA inhibitors. R292K conferred the highest increase in oseltamivir half-maximal inhibitory concentration (IC50), and E119D conferred the highest zanamivir IC50. Unlike N2 (another group 2NA), H274Y conferred highly reduced inhibition by oseltamivir. Additionally, R152K, a naturally occurring variation at the NA catalytic residue of A(H7N9) viruses, conferred reduced inhibition by laninamivir. Conclusions: The recNA method is a valuable tool for assessing the effect of NA changes on drug susceptibility of emerging influenza viruses.
Assuntos
Antivirais/farmacologia , Farmacorresistência Viral Múltipla/genética , Subtipo H7N9 do Vírus da Influenza A/efeitos dos fármacos , Neuraminidase/antagonistas & inibidores , Proteínas Virais/antagonistas & inibidores , Ácidos Carbocíclicos , Ciclopentanos/farmacologia , Bases de Dados Genéticas , Inibidores Enzimáticos/farmacologia , Guanidinas/farmacologia , Humanos , Subtipo H7N9 do Vírus da Influenza A/genética , Influenza Humana/tratamento farmacológico , Concentração Inibidora 50 , Neuraminidase/genética , Oseltamivir/farmacologia , Conformação Proteica , Piranos , Proteínas Recombinantes/genética , Ácidos Siálicos , Proteínas Virais/genética , Zanamivir/análogos & derivados , Zanamivir/farmacologiaRESUMO
UNLABELLED: Human infections by avian influenza A(H7N9) virus entail substantial morbidity and mortality. Treatment of infected patients with the neuraminidase (NA) inhibitor oseltamivir was associated with emergence of viruses carrying NA substitutions. In the NA inhibition (NI) assay, R292K conferred highly reduced inhibition by oseltamivir, while E119V and I222K each caused reduced inhibition. To facilitate establishment of laboratory correlates of clinically relevant resistance, experiments were conducted in ferrets infected with virus carrying wild-type or variant NA genes recovered from the A/Taiwan/1/2013 isolate. Oseltamivir treatment (5 or 25 mg/kg of body weight/dose) was given 4 h postinfection, followed by twice-daily treatment for 5 days. Treatment of ferrets infected with wild-type virus resulted in a modest dose-dependent reduction (0.7 to 1.5 log10 50% tissue culture infectious dose [TCID50]) in nasal wash viral titers and inflammation response. Conversely, treatment failed to significantly inhibit the replication of R292K or E119V virus. A small reduction of viral titers was detected on day 5 in ferrets infected with the I222K virus. The propensity for oseltamivir resistance emergence was assessed in oseltamivir-treated animals infected with wild-type virus; emergence of R292K virus was detected in 3 of 6 ferrets within 5 to 7 days postinfection. Collectively, we demonstrate that R292K, E119V, and I222K reduced the inhibitory activity of oseltamivir, not only in the NI assay, but also in infected ferrets, judged particularly by viral loads in nasal washes, and may signal the need for alternative therapeutics. Thus, these clinical outcomes measured in the ferret model may correlate with clinically relevant oseltamivir resistance in humans. IMPORTANCE: This report provides more evidence for using the ferret model to assess the susceptibility of influenza A(H7N9) viruses to oseltamivir, the most prescribed anti-influenza virus drug. The information gained can be used to assist in the establishment of laboratory correlates of human disease and drug therapy. The rapid emergence of viruses with R292K in treated ferrets correlates well with the multiple reports on this NA variant in treated human patients. Our findings highlight the importance of the discovery and characterization of new antiviral drugs with different mechanisms of action and the use of combination treatment strategies against emerging viruses with pandemic potential, such as avian H7N9 virus, particularly against those carrying drug resistance markers.
Assuntos
Antivirais/farmacologia , Subtipo H7N9 do Vírus da Influenza A/efeitos dos fármacos , Subtipo H7N9 do Vírus da Influenza A/genética , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , Neuraminidase/genética , Oseltamivir/farmacologia , Animais , Modelos Animais de Doenças , Farmacorresistência Viral/genética , Inibidores Enzimáticos/farmacologia , Furões , Genes Virais , Humanos , Subtipo H7N9 do Vírus da Influenza A/fisiologia , Masculino , Mutação , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/virologia , Infecções Respiratórias/tratamento farmacológico , Infecções Respiratórias/patologia , Infecções Respiratórias/virologia , Replicação Viral/efeitos dos fármacosRESUMO
UNLABELLED: Influenza A viruses (IAVs) express the PB1-F2 protein from an alternate reading frame within the PB1 gene segment. The roles of PB1-F2 are not well understood but appear to involve modulation of host cell responses. As shown in previous studies, we find that PB1-F2 proteins of mammalian IAVs frequently have premature stop codons that are expected to cause truncations of the protein, whereas avian IAVs usually express a full-length 90-amino-acid PB1-F2. However, in contrast to other avian IAVs, recent isolates of highly pathogenic H5N1 influenza viruses had a high proportion of PB1-F2 truncations (15% since 2010; 61% of isolates in 2013) due to several independent mutations that have persisted and expanded in circulating viruses. One natural H5N1 IAV containing a mutated PB1-F2 start codon (i.e., lacking ATG) was 1,000-fold more virulent for BALB/c mice than a closely related H5N1 containing intact PB1-F2. In vitro, we detected expression of an in-frame protein (C-terminal PB1-F2) from downstream ATGs in PB1-F2 plasmids lacking the well-conserved ATG start codon. Transient expression of full-length PB1-F2, truncated (24-amino-acid) PB1-F2, and PB1-F2 lacking the initiating ATG in mammalian and avian cells had no effect on cell apoptosis or interferon expression in human lung epithelial cells. Full-length and C-terminal PB1-F2 mutants colocalized with mitochondria in A549 cells. Close monitoring of alterations of PB1-F2 and their frequency in contemporary avian H5N1 viruses should continue, as such changes may be markers for mammalian virulence. IMPORTANCE: Although most avian influenza viruses are harmless for humans, some (such as highly pathogenic H5N1 avian influenza viruses) are capable of infecting humans and causing severe disease with a high mortality rate. A number of risk factors potentially associated with adaptation to mammalian infection have been noted. Here we demonstrate that the protein PB1-F2 is frequently truncated in recent isolates of highly pathogenic H5N1 viruses. Truncation of PB1-F2 has been proposed to act as an adaptation to mammalian infection. We show that some forms of truncation of PB1-F2 may be associated with increased virulence in mammals. Our data support the assessment of PB1-F2 truncations for genomic surveillance of influenza viruses.
Assuntos
Virus da Influenza A Subtipo H5N1/isolamento & purificação , Virus da Influenza A Subtipo H5N1/fisiologia , Infecções por Orthomyxoviridae/virologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Animais , Apoptose , Linhagem Celular , Códon sem Sentido , Modelos Animais de Doenças , Células Epiteliais/fisiologia , Células Epiteliais/virologia , Feminino , Humanos , Virus da Influenza A Subtipo H5N1/genética , Interferons/biossíntese , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/patologia , VirulênciaRESUMO
UNLABELLED: In late 2011, an A(H3N8) influenza virus infection resulted in the deaths of 162 New England harbor seals. Virus sequence analysis and virus receptor binding studies highlighted potential markers responsible for mammalian adaptation and a mixed receptor binding preference (S. J. Anthony, J. A. St Leger, K. Pugliares, H. S. Ip, J. M. Chan, Z. W. Carpenter, I. Navarrete-Macias, M. Sanchez-Leon, J. T. Saliki, J. Pedersen, W. Karesh, P. Daszak, R. Rabadan, T. Rowles, W. I. Lipkin, MBio 3:e00166-00112, 2012, http://dx.doi.org/10.1128/mBio.00166-12). Here, we present a detailed structural and biochemical analysis of the surface antigens of the virus. Results obtained with recombinant proteins for both the hemagglutinin and neuraminidase indicate a true avian receptor binding preference. Although the detection of this virus in new species highlights an increased potential for cross-species transmission, our results indicate that the A(H3N8) virus currently poses a low risk to humans. IMPORTANCE: Cross-species transmission of zoonotic influenza viruses increases public health concerns. Here, we report a molecular and structural study of the major surface proteins from an A(H3N8) influenza virus isolated from New England harbor seals. The results improve our understanding of these viruses as they evolve and provide important information to aid ongoing risk assessment analyses as these zoonotic influenza viruses continue to circulate and adapt to new hosts.
Assuntos
Antígenos Virais/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A Subtipo H3N8/fisiologia , Neuraminidase/metabolismo , Infecções por Orthomyxoviridae/veterinária , Phoca/virologia , Proteínas Virais/metabolismo , Ligação Viral , Sequência de Aminoácidos , Animais , Antígenos Virais/química , Cristalografia por Raios X , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Vírus da Influenza A Subtipo H3N8/química , Vírus da Influenza A Subtipo H3N8/isolamento & purificação , Testes de Sensibilidade Microbiana , Modelos Moleculares , Dados de Sequência Molecular , Neuraminidase/química , New England , Infecções por Orthomyxoviridae/virologia , Polissacarídeos/análise , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Proteínas Virais/químicaRESUMO
BACKGROUND: Patients contracting influenza A(H7N9) infection often developed severe disease causing respiratory failure. Neuraminidase (NA) inhibitors (NAIs) are the primary option for treatment, but information on drug-resistance markers for influenza A(H7N9) is limited. METHODS: Four NA variants of A/Taiwan/1/2013(H7N9) virus containing a single substitution (NA-E119V, NA-I222K, NA-I222R, or NA-R292K) recovered from an oseltamivir-treated patient were tested for NAI susceptibility in vitro; their replicative fitness was evaluated in cell culture, mice, and ferrets. RESULTS: NA-R292K led to highly reduced inhibition by oseltamivir and peramivir, while NA-E119V, NA-I222K, and NA-I222R caused reduced inhibition by oseltamivir. Mice infected with any virus showed severe clinical signs with high mortality rates. NA-I222K virus was the most virulent in mice, whereas virus lacking NA change (NA-WT) and NA-R292K virus seemed the least virulent. Sequence analysis suggests that PB2-S714N increased virulence of NA-I222K virus in mice; NS1-K126R, alone or in combination with PB2-V227M, produced contrasting effects in NA-WT and NA-R292K viruses. In ferrets, all viruses replicated to high titers in the upper respiratory tract but produced only mild illness. NA-R292K virus, showed reduced replicative fitness in this animal model. CONCLUSIONS: Our data highlight challenges in assessment of the replicative fitness of H7N9 NA variants that emerged in NAI-treated patients.
Assuntos
Antivirais/uso terapêutico , Farmacorresistência Viral , Subtipo H7N9 do Vírus da Influenza A/efeitos dos fármacos , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , Oseltamivir/uso terapêutico , Animais , Modelos Animais de Doenças , Furões , Humanos , Subtipo H7N9 do Vírus da Influenza A/enzimologia , Subtipo H7N9 do Vírus da Influenza A/genética , Subtipo H7N9 do Vírus da Influenza A/isolamento & purificação , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Proteínas Mutantes/genética , Mutação de Sentido Incorreto , Neuraminidase/genética , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Proteínas Virais/genética , Cultura de Vírus , Replicação ViralRESUMO
Aquatic birds harbor diverse influenza A viruses and are a major viral reservoir in nature. The recent discovery of influenza viruses of a new H17N10 subtype in Central American fruit bats suggests that other New World species may similarly carry divergent influenza viruses. Using consensus degenerate RT-PCR, we identified a novel influenza A virus, designated as H18N11, in a flat-faced fruit bat (Artibeus planirostris) from Peru. Serologic studies with the recombinant H18 protein indicated that several Peruvian bat species were infected by this virus. Phylogenetic analyses demonstrate that, in some gene segments, New World bats harbor more influenza virus genetic diversity than all other mammalian and avian species combined, indicative of a long-standing host-virus association. Structural and functional analyses of the hemagglutinin and neuraminidase indicate that sialic acid is not a ligand for virus attachment nor a substrate for release, suggesting a unique mode of influenza A virus attachment and activation of membrane fusion for entry into host cells. Taken together, these findings indicate that bats constitute a potentially important and likely ancient reservoir for a diverse pool of influenza viruses.
Assuntos
Quirópteros/virologia , Reservatórios de Doenças/virologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A/genética , Infecções por Orthomyxoviridae/genética , Filogenia , Animais , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/veterinária , Peru/epidemiologiaRESUMO
Influenza A virus reservoirs in animals have provided novel genetic elements leading to the emergence of global pandemics in humans. Most influenza A viruses circulate in waterfowl, but those that infect mammalian hosts are thought to pose the greatest risk for zoonotic spread to humans and the generation of pandemic or panzootic viruses. We have identified an influenza A virus from little yellow-shouldered bats captured at two locations in Guatemala. It is significantly divergent from known influenza A viruses. The HA of the bat virus was estimated to have diverged at roughly the same time as the known subtypes of HA and was designated as H17. The neuraminidase (NA) gene is highly divergent from all known influenza NAs, and the internal genes from the bat virus diverged from those of known influenza A viruses before the estimated divergence of the known influenza A internal gene lineages. Attempts to propagate this virus in cell cultures and chicken embryos were unsuccessful, suggesting distinct requirements compared with known influenza viruses. Despite its divergence from known influenza A viruses, the bat virus is compatible for genetic exchange with human influenza viruses in human cells, suggesting the potential capability for reassortment and contributions to new pandemic or panzootic influenza A viruses.
Assuntos
Quirópteros/virologia , Vírus da Influenza A/genética , Filogenia , Animais , RNA Polimerases Dirigidas por DNA/metabolismo , Genes Reporter/genética , Genoma Viral/genética , Geografia , Guatemala , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Dados de Sequência Molecular , Neuraminidase/química , Neuraminidase/genética , Análise de Sequência de DNARESUMO
We assessed drug susceptibilities of 125 avian influenza A(H5N1) viruses isolated from poultry in Vietnam during 2009-2011. Of 25 clade 1.1 viruses, all possessed a marker of resistance to M2 blockers amantadine and rimantadine; 24 were inhibited by neuraminidase inhibitors. One clade 1.1 virus contained the R430W neuraminidase gene and reduced inhibition by oseltamivir, zanamivir, and laninamivir 12-, 73-, and 29-fold, respectively. Three of 30 clade 2.3.4 viruses contained a I223T mutation and showed 7-fold reduced inhibition by oseltamivir. One of 70 clade 2.3.2.1 viruses had the H275Y marker of oseltamivir resistance and exhibited highly reduced inhibition by oseltamivir and peramivir; antiviral agents DAS181 and favipiravir inhibited H275Y mutant virus replication in MDCK-SIAT1 cells. Replicative fitness of the H275Y mutant virus was comparable to that of wildtype virus. These findings highlight the role of drug susceptibility monitoring of H5N1 subtype viruses circulating among birds to inform antiviral stockpiling decisions for pandemic preparedness.
Assuntos
Antivirais/farmacologia , Virus da Influenza A Subtipo H5N1/efeitos dos fármacos , Influenza Aviária/epidemiologia , Influenza Aviária/virologia , Animais , Linhagem Celular , Farmacorresistência Viral , Furões , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Mutação , Neuraminidase/genética , Neuraminidase/metabolismo , Oseltamivir/farmacologia , Filogenia , Aves Domésticas/virologia , Vigilância em Saúde Pública , Vietnã/epidemiologia , Replicação Viral/efeitos dos fármacosRESUMO
Clade 2.3.4.4b highly pathogenic avian influenza (HPAI) A(H5N1) viruses that are responsible for devastating outbreaks in birds and mammals pose a potential threat to public health. Here, we evaluated their susceptibility to influenza antivirals. Of 1,015 sequences of HPAI A(H5N1) viruses collected in the United States during 2022, eight viruses (â¼0.8%) had a molecular marker of drug resistance to an FDA-approved antiviral: three adamantane-resistant (M2-V27A), four oseltamivir-resistant (NA-H275Y), and one baloxavir-resistant (PA-I38T). Additionally, 31 viruses contained mutations that may reduce susceptibility to inhibitors of neuraminidase (NA) (n = 20) or cap-dependent endonuclease (CEN) (n = 11). A panel of 22 representative viruses was tested phenotypically. Overall, clade 2.3.4.4b A(H5N1) viruses lacking recognized resistance mutations were susceptible to FDA-approved antivirals. Oseltamivir was least potent at inhibiting NA activity, while the investigational NA inhibitor AV5080 was most potent, including against NA mutants. A novel NA substitution T438N conferred 12-fold reduced inhibition by zanamivir, and in combination with the known marker N295S, synergistically affected susceptibility to all five NA inhibitors. In cell culture-based assays HINT and IRINA, the PA-I38T virus displayed 75- to 108-fold and 37- to 78-fold reduced susceptibility to CEN inhibitors, baloxavir and the investigational AV5116, respectively. Viruses with PA-I38M or PA-A37T showed 5- to 10-fold reduced susceptibilities. As HPAI A(H5N1) viruses continue to circulate and evolve, close monitoring of drug susceptibility is needed for risk assessment and to inform decisions regarding antiviral stockpiling.
Assuntos
Virus da Influenza A Subtipo H5N1 , Influenza Aviária , Animais , Estados Unidos/epidemiologia , Antivirais/farmacologia , Oseltamivir/farmacologia , Virus da Influenza A Subtipo H5N1/genética , Influenza Aviária/epidemiologia , Inibidores Enzimáticos/farmacologia , Aves , Mamíferos , Farmacorresistência Viral/genética , NeuraminidaseRESUMO
The NS1 protein of human influenza A viruses binds the 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), a protein required for 3' end processing of cellular pre-mRNAs, thereby inhibiting production of beta interferon (IFN-ß) mRNA. The NS1 proteins of pathogenic 1997 H5N1 viruses contain the CPSF30-binding site but lack the consensus amino acids at positions 103 and 106, F and M, respectively, that are required for the stabilization of CPSF30 binding, resulting in nonoptimal CPSF30 binding in infected cells. Here we have demonstrated that strengthening CPSF30 binding, by changing positions 103 and 106 in the 1997 H5N1 NS1 protein to the consensus amino acids, results in a remarkable 300-fold increase in the lethality of the virus in mice. Unexpectedly, this increase in virulence is not associated with increased lung pathology but rather is characterized by faster systemic spread of the virus, particularly to the brain, where increased replication and severe pathology occur. This increased spread is associated with increased cytokine and chemokine levels in extrapulmonary tissues. We conclude that strengthening CPSF30 binding by the NS1 protein of 1997 H5N1 viruses enhances virulence in mice by increasing the systemic spread of the virus from the lungs, particularly to the brain.
Assuntos
Virus da Influenza A Subtipo H5N1/patogenicidade , Modelos Animais , Proteínas não Estruturais Virais/fisiologia , Animais , Linhagem Celular , Cães , Feminino , Citometria de Fluxo , Humanos , Virus da Influenza A Subtipo H5N1/fisiologia , Interferon beta/genética , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , Proteínas não Estruturais Virais/genética , Virulência , Replicação ViralRESUMO
We identified avian influenza virus A (H5N1) infection in a child in Bangladesh in 2008 by routine influenza surveillance. The virus was of the same clade and phylogenetic subgroup as that circulating among poultry during the period. This case illustrates the value of routine surveillance for detection of novel influenza virus.
Assuntos
Doenças Transmissíveis Emergentes/virologia , Virus da Influenza A Subtipo H5N1 , Influenza Humana/virologia , Animais , Bangladesh , Galinhas/virologia , Doenças Transmissíveis Emergentes/transmissão , Humanos , Lactente , Virus da Influenza A Subtipo H5N1/classificação , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Humana/transmissão , Masculino , Filogenia , Vigilância da PopulaçãoRESUMO
Recent advances in instrumentation and data analysis in field flow fractionation and multi-angle light scattering (FFF-MALS) have enabled greater use of this technique to characterize and quantitate viruses. In this study, the FFF-MALS technique was applied to the characterization and quantitation of type A influenza virus particles to assess its usefulness for vaccine preparation. The use of FFF-MALS for quantitation and measurement of control particles provided data accurate to within 5% of known values, reproducible with a coefficient of variation of 1.9%. The methods, sensitivity and limit of detection were established by analyzing different volumes of purified virus, which produced a linear regression with fitting value R2 of 0.99. FFF-MALS was further applied to detect and quantitate influenza virus in the supernatant of infected MDCK cells and allantoic fluids of infected eggs. FFF fractograms of the virus present in these different fluids revealed similar distribution of monomeric and oligomeric virions. However, the monomer fraction of cell grown virus had greater size variety. Notably, ß-propialactone (BPL) inactivation of influenza viruses did not influence any of the FFF-MALS measurements. Quantitation analysis by FFF-MALS was compared to infectivity assays and real-time RT-PCR (qRT-PCR) and the limitations of each assay were discussed.
Assuntos
Fracionamento por Campo e Fluxo/métodos , Vírus da Influenza A/isolamento & purificação , Luz , Carga Viral/métodos , Animais , Linhagem Celular , Embrião de Galinha , Cães , Reprodutibilidade dos TestesRESUMO
Antigenic variation among circulating H5N1 highly pathogenic avian influenza A viruses mandates the continuous production of strain-specific pre-pandemic vaccine candidates and represents a significant challenge for pandemic preparedness. Here we assessed the structural, antigenic and receptor-binding properties of three H5N1 HPAI virus hemagglutinins, which were recently selected by the WHO as vaccine candidates [A/Egypt/N03072/2010 (Egypt10, clade 2.2.1), A/Hubei/1/2010 (Hubei10, clade 2.3.2.1) and A/Anhui/1/2005 (Anhui05, clade 2.3.4)]. These analyses revealed that antigenic diversity among these three isolates was restricted to changes in the size and charge of amino acid side chains at a handful of positions, spatially equivalent to the antigenic sites identified in H1 subtype viruses circulating among humans. All three of the H5N1 viruses analyzed in this study were responsible for fatal human infections, with the most recently-isolated strains, Hubei10 and Egypt10, containing multiple residues in the receptor-binding site of the HA, which were suspected to enhance mammalian transmission. However, glycan-binding analyses demonstrated a lack of binding to human α2-6-linked sialic acid receptor analogs for all three HAs, reinforcing the notion that receptor-binding specificity contributes only partially to transmissibility and pathogenesis of HPAI viruses and suggesting that changes in host specificity must be interpreted in the context of the host and environmental factors, as well as the virus as a whole. Together, our data reveal structural linkages with phylogenetic and antigenic analyses of recently emerged H5N1 virus clades and should assist in interpreting the significance of future changes in antigenic and receptor-binding properties.
Assuntos
Variação Antigênica/genética , Hemaglutininas Virais/química , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/genética , Modelos Moleculares , Sequência de Bases , Clonagem Molecular , Biologia Computacional , Cristalização , Epitopos , Hemaglutininas Virais/genética , Humanos , Virus da Influenza A Subtipo H5N1/química , Filogenia , Conformação Proteica , Alinhamento de SequênciaRESUMO
Acquisition of α2-6 sialoside receptor specificity by α2-3 specific highly-pathogenic avian influenza viruses (H5N1) is thought to be a prerequisite for efficient transmission in humans. By in vitro selection for binding α2-6 sialosides, we identified four variant viruses with amino acid substitutions in the hemagglutinin (S227N, D187G, E190G, and Q196R) that revealed modestly increased α2-6 and minimally decreased α2-3 binding by glycan array analysis. However, a mutant virus combining Q196R with mutations from previous pandemic viruses (Q226L and G228S) revealed predominantly α2-6 binding. Unlike the wild type H5N1, this mutant virus was transmitted by direct contact in the ferret model although not by airborne respiratory droplets. However, a reassortant virus with the mutant hemagglutinin, a human N2 neuraminidase and internal genes from an H5N1 virus was partially transmitted via respiratory droplets. The complex changes required for airborne transmissibility in ferrets suggest that extensive evolution is needed for H5N1 transmissibility in humans.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/fisiologia , Polissacarídeos/metabolismo , Receptores Virais/metabolismo , Substituição de Aminoácidos , Animais , Sequência de Bases , Galinhas , Evolução Molecular , Furões , Células HEK293 , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Humanos , Virus da Influenza A Subtipo H5N1/patogenicidade , Influenza Aviária/transmissão , Influenza Aviária/virologia , Influenza Humana/transmissão , Influenza Humana/virologia , Mutação , Neuraminidase/genética , Análise de Sequência com Séries de Oligonucleotídeos , Vírus Reordenados/genética , Vírus Reordenados/fisiologia , Receptores Virais/genética , Análise de Sequência de RNARESUMO
Wild type human influenza viruses do not usually grow well in embryonated hens' eggs, the substrate of choice for the production of inactivated influenza vaccine, and vaccine viruses need to be developed specifically for this purpose. In the event of a pandemic of influenza, vaccine viruses need to be created with utmost speed. At the onset of the current A(H1N1) pandemic in April 2009, a network of laboratories began a race against time to develop suitable candidate vaccine viruses. Two approaches were followed, the classical reassortment approach and the more recent reverse genetics approach. This report describes the development and the characteristics of current pandemic H1N1 candidate vaccine viruses.
Assuntos
Descoberta de Drogas/métodos , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/uso terapêutico , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Pandemias/prevenção & controle , Animais , Linhagem Celular , Cães , Furões , Humanos , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vacinas contra Influenza/síntese química , Vacinas contra Influenza/imunologiaRESUMO
A type A avian influenza (AI) virus was isolated from dead or severely ill red-winged tinamous (Rhynchotus rufescens) found in a hunting ground in April 2008 in Argentina. The subtype of A/red-winged tinamou/Argentina/MP1/2008 was determined as H1N1 by sequence analysis. The cleavage site of the viral hemagglutinin corresponded to a low pathogenic influenza virus, although the clinical presentation and pathological studies suggest that the virus was pathogenic for red-winged tinamous. Phylogenetic analysis of the viral genome suggested that while the hemagglutinin and neuraminidase genes were related to AIV from North America, the internal genes were most closely related to other South American isolates. These findings support the postulated South American phylogenetic lineage for AIV PB2, PB1, PA, M and NS genes, and suggest that the evolutionary pathways of HA and NA genes involve exchanges between the Northern and Southern hemispheres.
Assuntos
Aves/virologia , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Animais , Argentina , Sequência de Bases , Testes de Inibição da Hemaglutinação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H1N1/classificação , Influenza Aviária/diagnóstico , Influenza Aviária/patologia , Dados de Sequência Molecular , Neuraminidase/genética , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Genetic characterization of a human cerebrospinal fluid West Nile virus isolate from Beaumont, Texas, revealed several nucleotide changes and amino acid substitutions that differentiated it from all other North American strains isolated to date, suggesting that isolates from the Texas Gulf Coast may form a unique genetic group among North American strains.