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1.
J Virol ; 97(1): e0143122, 2023 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-36541801

RESUMO

Since 2013, H7N9 avian influenza viruses (AIVs) have caused more than 1,500 human infections and the culling of millions of poultry. Despite large-scale poultry vaccination, H7N9 AIVs continue to circulate among poultry in China and pose a threat to human health. Previously, we isolated and generated four monoclonal antibodies (mAbs) derived from humans naturally infected with H7N9 AIV. Here, we investigated the hemagglutinin (HA) epitopes of H7N9 AIV targeted by these mAbs (L3A-44, K9B-122, L4A-14, and L4B-18) using immune escape studies. Our results revealed four key antigenic epitopes at HA amino acid positions 125, 133, 149, and 217. The mutant H7N9 viruses representing escape mutations containing an alanine-to-threonine substitution at residue 125 (A125T), a glycine-to-glutamic acid substitution at residue 133 (G133E), an asparagine-to-aspartic acid substitution at residue 149 (N149D), or a leucine-to-glutamine substitution at residue 217 (L217Q) showed reduced or completely abolished cross-reactivity with the mAbs, as measured by a hemagglutination inhibition (HI) assay. We further assessed the potential risk of these mutants to humans should they emerge following mAb treatment by measuring the impact of these HA mutations on virus fitness and evasion of host adaptive immunity. Here, we showed that the L4A-14 mAb had broad neutralizing capabilities, and its escape mutant N149D had reduced viral stability and human receptor binding and could be neutralized by both postinfection and antigen-induced sera. Therefore, the L4A-14 mAb could be a therapeutic candidate for H7N9 AIV infection in humans and warrants further investigation for therapeutic applications. IMPORTANCE Avian influenza virus (AIV) H7N9 continues to circulate and evolve in birds, posing a credible threat to humans. Antiviral drugs have proven useful for the treatment of severe influenza infections in humans; however, concerns have been raised as antiviral-resistant mutants have emerged. Monoclonal antibodies (mAbs) have been studied for both prophylactic and therapeutic applications in infectious disease control and have demonstrated great potential. For example, mAb treatment has significantly reduced the risk of people developing severe disease with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In addition to the protection efficiency, we should also consider the potential risk of the escape mutants generated by mAb treatment to public health by assessing their viral fitness and potential to compromise host adaptive immunity. Considering these parameters, we assessed four human mAbs derived from humans naturally infected with H7N9 AIV and showed that the mAb L4A-14 displayed potential as a therapeutic candidate.


Assuntos
Subtipo H7N9 do Vírus da Influenza A , Influenza Humana , Animais , Humanos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/uso terapêutico , Epitopos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Subtipo H7N9 do Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Influenza Humana/terapia , Evasão da Resposta Imune/genética , Mutação
2.
J Virol ; 96(22): e0129022, 2022 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-36342296

RESUMO

H9N2 avian influenza viruses (AIVs) have donated internal gene segments during the emergence of zoonotic AIVs, including H7N9. We used reverse genetics to generate A/Anhui/1/13 (H7N9) and three reassortant viruses (2:6 H7N9) which contained the hemagglutinin and neuraminidase from Anhui/13 (H7N9) and the six internal gene segments from H9N2 AIVs belonging to (i) G1 subgroup 2, (ii) G1 subgroup 3, or (iii) BJ94 lineages, enzootic in different regions throughout Asia. Infection of chickens with the 2:6 H7N9 containing G1-like H9N2 internal genes conferred attenuation in vivo, with reduced shedding and transmission to contact chickens. However, possession of BJ94-like H9N2 internal genes resulted in more rapid transmission and significantly elevated cloacal shedding compared to the parental Anhui/13 H7N9. In vitro analysis showed that the 2:6 H7N9 with BJ94-like internal genes had significantly increased replication compared to the Anhui/13 H7N9 in chicken cells. In vivo coinfection experiments followed, where chickens were coinfected with pairs of Anhui/13 H7N9 and a 2:6 H7N9 reassortant. During ensuing transmission events, the Anhui/13 H7N9 virus outcompeted 2:6 H7N9 AIVs with internal gene segments of BJ94-like or G1-like H9N2 viruses. Coinfection did lead to the emergence of novel reassortant genotypes that were transmitted to contact chickens. Some of the reassortant viruses had a greater replication in chicken and human cells compared to the progenitors. We demonstrated that the internal gene cassette determines the transmission fitness of H7N9 viruses in chickens, and the reassortment events can generate novel H7N9 genotypes with increased virulence in chickens and enhanced zoonotic potential. IMPORTANCE H9N2 avian influenza viruses (AIVs) are enzootic in poultry in different geographical regions. The internal genes of these viruses can be exchanged with other zoonotic AIVs, most notably the A/Anhui/1/2013-lineage H7N9, which can give rise to new virus genotypes with increased veterinary, economic and public health threats to both poultry and humans. We investigated the propensity of the internal genes of H9N2 viruses (G1 or BJ94) in the generation of novel reassortant H7N9 AIVs. We observed that the internal genes of H7N9 which were derivative of BJ94-like H9N2 virus have a fitness advantage compared to those from the G1-like H9N2 viruses for efficient transmission among chickens. We also observed the generation of novel reassortant viruses during chicken transmission which infected and replicated efficiently in human cells. Therefore, such emergent reassortant genotypes may pose an elevated zoonotic threat.


Assuntos
Coinfecção , Subtipo H7N9 do Vírus da Influenza A , Vírus da Influenza A Subtipo H9N2 , Influenza Aviária , Influenza Humana , Animais , Humanos , Subtipo H7N9 do Vírus da Influenza A/genética , Vírus da Influenza A Subtipo H9N2/genética , Galinhas , Vírus Reordenados/genética , Aves Domésticas , Filogenia
3.
Front Immunol ; 13: 903755, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35757698

RESUMO

The pig is an important agricultural species and powerful biomedical model. We have established the pig, a large natural host animal for influenza with many physiological similarities to humans, as a robust model for testing the therapeutic potential of monoclonal antibodies. Antibodies provide protection through neutralization and recruitment of innate effector functions through the Fc domain. However very little is known about the Fc-mediated functions of porcine IgG subclasses. We have generated 8 subclasses of two porcine monoclonal anti influenza hemagglutinin antibodies. We characterized their ability to activate complement, trigger cytotoxicity and phagocytosis by immune cells and assayed their binding to monocytes, macrophages, and natural killer cells. We show that IgG1, IgG2a, IgG2b, IgG2c and IgG4 bind well to targeted cell types and mediate complement mediated cellular cytotoxicity (CDCC), antibody dependent cellular cytotoxicity (ADCC) and antibody mediated cell phagocytosis (ADCP). IgG5b and IgG5c exhibited weak binding and variable and poor functional activity. Immune complexes of porcine IgG3 did not show any Fc-mediated functions except for binding to monocytes and macrophages and weak binding to NK cells. Interestingly, functionally similar porcine IgG subclasses clustered together in the genome. These novel findings will enhance the utility of the pig model for investigation of therapeutic antibodies.


Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Imunoglobulina G , Animais , Anticorpos Monoclonais , Complexo Antígeno-Anticorpo , Proteínas do Sistema Complemento , Fagocitose , Suínos
4.
J Virol ; 96(5): e0185621, 2022 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-35019727

RESUMO

An H7N9 low-pathogenicity avian influenza virus (LPAIV) emerged in 2013 through genetic reassortment between H9N2 and other LPAIVs circulating in birds in China. This virus causes inapparent clinical disease in chickens, but zoonotic transmission results in severe and fatal disease in humans. To examine a natural reassortment scenario between H7N9 and G1 lineage H9N2 viruses predominant in the Indian subcontinent, we performed an experimental coinfection of chickens with A/Anhui/1/2013/H7N9 (Anhui/13) virus and A/Chicken/Pakistan/UDL-01/2008/H9N2 (UDL/08) virus. Plaque purification and genotyping of the reassortant viruses shed via the oropharynx of contact chickens showed H9N2 and H9N9 as predominant subtypes. The reassortant viruses shed by contact chickens also showed selective enrichment of polymerase genes from H9N2 virus. The viable "6+2" reassortant H9N9 (having nucleoprotein [NP] and neuraminidase [NA] from H7N9 and the remaining genes from H9N2) was successfully shed from the oropharynx of contact chickens, plus it showed an increased replication rate in human A549 cells and a significantly higher receptor binding to α2,6 and α2,3 sialoglycans compared to H9N2. The reassortant H9N9 virus also had a lower fusion pH, replicated in directly infected ferrets at similar levels compared to H7N9 and transmitted via direct contact. Ferrets exposed to H9N9 via aerosol contact were also found to be seropositive, compared to H7N9 aerosol contact ferrets. To the best of our knowledge, this is the first study demonstrating that cocirculation of H7N9 and G1 lineage H9N2 viruses could represent a threat for the generation of novel reassortant H9N9 viruses with greater virulence in poultry and a zoonotic potential. IMPORTANCE We evaluated the consequences of reassortment between the H7N9 and the contemporary H9N2 viruses of the G1 lineage that are enzootic in poultry across the Indian subcontinent and the Middle East. Coinfection of chickens with these viruses resulted in the emergence of novel reassortant H9N9 viruses with genes derived from both H9N2 and H7N9 viruses. The "6+2" reassortant H9N9 (having NP and NA from H7N9) virus was shed from contact chickens in a significantly higher proportion compared to most of the reassortant viruses, showed significantly increased replication fitness in human A549 cells, receptor binding toward human (α2,6) and avian (α2,3) sialic acid receptor analogues, and the potential to transmit via contact among ferrets. This study demonstrated the ability of viruses that already exist in nature to exchange genetic material, highlighting the potential emergence of viruses from these subtypes with zoonotic potential.


Assuntos
Coinfecção , Subtipo H7N9 do Vírus da Influenza A , Vírus da Influenza A Subtipo H9N2 , Influenza Aviária , Vírus Reordenados , Animais , Galinhas , Coinfecção/veterinária , Furões , Humanos , Subtipo H7N9 do Vírus da Influenza A/genética , Vírus da Influenza A Subtipo H9N2/genética , Influenza Aviária/virologia , Influenza Humana , Filogenia , Aves Domésticas , Vírus Reordenados/genética , Vírus Reordenados/patogenicidade
5.
Front Immunol ; 13: 1052297, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36685492

RESUMO

Microbial colonisation is paramount to the normal development of the immune system, particularly at mucosal sites. However, the relationships between the microbiome and the adaptive immune repertoire have mostly been explored in rodents and humans. Here, we report a high-throughput sequencing analysis of the chicken TCRß repertoire and the influences of microbial colonisation on tissue-resident TCRß+ cells. The results reveal that the microbiome is an important driver of TCRß diversity in both intestinal tissues and the bursa of Fabricius, but not in the spleen. Of note, public TCRß sequences (shared across individuals) make a substantial contribution to the repertoire. Additionally, different tissues exhibit biases in terms of their V family and J gene usage, and these effects were influenced by the gut-associated microbiome. TCRß clonal expansions were identified in both colonised and germ-free birds, but differences between the groups were indicative of an influence of the microbiota. Together, these findings provide an insight into the avian adaptive immune system and the influence of the microbiota on the TCRß repertoire.


Assuntos
Galinhas , Sistema Imunitário , Humanos , Animais , Intestinos
6.
Emerg Microbes Infect ; 10(1): 2223-2234, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34753400

RESUMO

Avian influenza virus (AIV) subtypes H5N1 and H9N2 co-circulate in poultry in Bangladesh, causing significant bird morbidity and mortality. Despite their importance to the poultry value chain, the role of farms in spreading and maintaining AIV infections remains poorly understood in most disease-endemic settings. To address this crucial gap, we conducted a cross-sectional study between 2017 and 2019 in the Chattogram Division of Bangladesh in clinically affected and dead chickens in farms with suspected AIV infection. Viral prevalence of each subtype was approximately 10% among farms for which veterinary advice was sought, indicating high levels of virus circulation in chicken farms despite the low number of reported outbreaks. Co-circulation of both subtypes was common in farms, with our findings suggest that in the field, the co-circulation of H5N1 and H9N2 can modulate disease severity, which could facilitate an underestimated level of AIV transmission in the poultry value chain. Finally, using newly generated whole-genome sequences, we investigate the evolutionary history of a small subset of H5N1 and H9N2 viruses. Our analyses revealed that for both subtypes, the sampled viruses were genetically most closely related to other viruses isolated in Bangladesh and represented multiple independent incursions. However, due to lack of longitudinal surveillance in this region, it is difficult to ascertain whether these viruses emerged from endemic strains circulating in Bangladesh or from neighbouring countries. We also show that amino acids at putative antigenic residues underwent a distinct replacement during 2012 which coincides with the use of H5N1 vaccines.


Assuntos
Virus da Influenza A Subtipo H5N1/genética , Vírus da Influenza A Subtipo H9N2/genética , Influenza Aviária/virologia , Doenças das Aves Domésticas/virologia , Animais , Bangladesh/epidemiologia , Galinhas , Estudos Transversais , Surtos de Doenças , Evolução Molecular , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Virus da Influenza A Subtipo H5N1/fisiologia , Vírus da Influenza A Subtipo H9N2/isolamento & purificação , Vírus da Influenza A Subtipo H9N2/fisiologia , Influenza Aviária/epidemiologia , Epidemiologia Molecular , Filogenia , Doenças das Aves Domésticas/epidemiologia , Conformação Proteica
7.
J Gen Virol ; 102(11)2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34726594

RESUMO

Highly pathogenic H5N1 avian influenza viruses cause devastating outbreaks in farmed poultry with serious consequences for animal welfare and economic losses. Zoonotic infection of humans through close contact with H5N1 infected birds is often severe and fatal. England experienced an outbreak of H5N1 in turkeys in 1991 that led to thousands of farmed bird mortalities. Isolation of clonal populations of one such virus from this outbreak uncovered amino acid differences in the virus haemagglutinin (HA) gene whereby the different genotypes could be associated with distinct pathogenic outcomes in chickens; both low pathogenic (LP) and high pathogenic (HP) phenotypes could be observed despite all containing a multi-basic cleavage site (MBCS) in the HA gene. Using reverse genetics, three amino acid substitutions in HA were examined for their ability to affect pathogenesis in the chicken. Restoration of amino acid polymorphisms close to the receptor binding site that are commonly found in H5 viruses only partially improved viral fitness in vitro and in vivo. A third novel substitution in the fusion peptide, HA2G4R, enabled the HP phenotype. HA2G4R decreased the pH stability of HA and increased the pH of HA fusion. The substitutions close to the receptor binding site optimised receptor binding while modulating the pH of HA fusion. Importantly, this study revealed pathogenic determinants beyond the MBCS.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/patogenicidade , Influenza Aviária/virologia , Doenças das Aves Domésticas/virologia , Receptores Virais/metabolismo , Substituição de Aminoácidos , Animais , Fusão Celular , Galinhas , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Concentração de Íons de Hidrogênio , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Virus da Influenza A Subtipo H5N1/metabolismo , Influenza Aviária/genética , Influenza Aviária/metabolismo , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/metabolismo , Ligação Proteica , Receptores Virais/genética , Virulência
8.
PLoS Pathog ; 17(4): e1009530, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33909701

RESUMO

Multi-functional DEAD-box helicase 5 (DDX5), which is important in transcriptional regulation, is hijacked by diverse viruses to facilitate viral replication. However, its regulatory effect in antiviral innate immunity remains unclear. We found that DDX5 interacts with the N6-methyladenosine (m6A) writer METTL3 to regulate methylation of mRNA through affecting the m6A writer METTL3-METTL14 heterodimer complex. Meanwhile, DDX5 promoted the m6A modification and nuclear export of transcripts DHX58, p65, and IKKγ by binding conserved UGCUGCAG element in innate response after viral infection. Stable IKKγ and p65 transcripts underwent YTHDF2-dependent mRNA decay, whereas DHX58 translation was promoted, resulting in inhibited antiviral innate response by DDX5 via blocking the p65 pathway and activating the DHX58-TBK1 pathway after infection with RNA virus. Furthermore, we found that DDX5 suppresses antiviral innate immunity in vivo. Our findings reveal that DDX5 serves as a negative regulator of innate immunity by promoting RNA methylation of antiviral transcripts and consequently facilitating viral propagation.


Assuntos
Adenosina/análogos & derivados , RNA Helicases DEAD-box/fisiologia , Evasão da Resposta Imune/genética , Estabilidade de RNA/genética , Viroses , Adenosina/metabolismo , Animais , Células Cultivadas , Embrião de Galinha , Cricetinae , RNA Helicases DEAD-box/genética , Células HEK293 , Humanos , Imunidade Inata/genética , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/genética , NF-kappa B/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Mensageiro/metabolismo , Viroses/genética , Viroses/imunologia , Viroses/metabolismo , Replicação Viral/genética
9.
J Gen Virol ; 102(3)2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33544070

RESUMO

Influenza A viruses encode several accessory proteins that have host- and strain-specific effects on virulence and replication. The accessory protein PA-X is expressed due to a ribosomal frameshift during translation of the PA gene. Depending on the particular combination of virus strain and host species, PA-X has been described as either acting to reduce or increase virulence and/or virus replication. In this study, we set out to investigate the role PA-X plays in H9N2 avian influenza viruses, focusing on the natural avian host, chickens. We found that the G1 lineage A/chicken/Pakistan/UDL-01/2008 (H9N2) PA-X induced robust host shutoff in both mammalian and avian cells and increased virus replication in mammalian, but not avian cells. We further showed that PA-X affected embryonic lethality in ovo and led to more rapid viral shedding and widespread organ dissemination in vivo in chickens. Overall, we conclude PA-X may act as a virulence factor for H9N2 viruses in chickens, allowing faster replication and wider organ tropism.


Assuntos
Vírus da Influenza A Subtipo H9N2/metabolismo , Influenza Aviária/virologia , Influenza Humana/virologia , Proteínas Repressoras/metabolismo , Proteínas não Estruturais Virais/metabolismo , Fatores de Virulência/metabolismo , Animais , Linhagem Celular , Galinhas , Citocinas/genética , Citocinas/imunologia , Humanos , Vírus da Influenza A Subtipo H9N2/genética , Vírus da Influenza A Subtipo H9N2/patogenicidade , Influenza Aviária/genética , Influenza Aviária/imunologia , Influenza Humana/genética , Influenza Humana/imunologia , Pulmão/imunologia , Pulmão/virologia , Camundongos , Proteínas Repressoras/genética , Proteínas não Estruturais Virais/genética , Fatores de Virulência/genética , Replicação Viral , Eliminação de Partículas Virais
10.
J Virol ; 95(5)2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33268517

RESUMO

Receptor recognition and binding is the first step of viral infection and a key determinant of host specificity. The inability of avian influenza viruses to effectively bind human-like sialylated receptors is a major impediment to their efficient transmission in humans and pandemic capacity. Influenza H9N2 viruses are endemic in poultry across Asia and parts of Africa where they occasionally infect humans and are therefore considered viruses with zoonotic potential. We previously described H9N2 viruses, including several isolated from human zoonotic cases, showing a preference for human-like receptors. Here we take a mutagenesis approach, making viruses with single or multiple substitutions in H9 haemagglutinin and test binding to avian and human receptor analogues using biolayer interferometry. We determine the genetic basis of preferences for alternative avian receptors and for human-like receptors, describing amino acid motifs at positions 190, 226 and 227 that play a major role in determining receptor specificity, and several other residues such as 159, 188, 193, 196, 198 and 225 that play a smaller role. Furthermore, we show changes at residues 135, 137, 147, 157, 158, 184, 188, and 192 can also modulate virus receptor avidity and that substitutions that increased or decreased the net positive charge around the haemagglutinin receptor-binding site show increases and decreases in avidity, respectively. The motifs we identify as increasing preference for the human-receptor will help guide future H9N2 surveillance efforts and facilitate our understanding of the emergence of influenza viruses with increased zoonotic potential.IMPORTANCE As of 2020, over 60 infections of humans by H9N2 influenza viruses have been recorded in countries where the virus is endemic. Avian-like cellular receptors are the primary target for these viruses. However, given that human infections have been detected on an almost monthly basis since 2015, there may be a capacity for H9N2 viruses to evolve and gain the ability to target human-like cellular receptors. Here we identify molecular signatures that can cause viruses to bind human-like receptors, and we identify the molecular basis for the distinctive preference for sulphated receptors displayed by the majority of recent H9N2 viruses. This work will help guide future surveillance by providing markers that signify the emergence of viruses with enhanced zoonotic potential as well as improving understanding of the basis of influenza virus receptor-binding.

11.
Emerg Microbes Infect ; 9(1): 2622-2631, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33179567

RESUMO

Influenza viruses have an error-prone polymerase complex that facilitates a mutagenic environment. Antigenic mutants swiftly arise from this environment with the capacity to persist in both humans and economically important livestock even in the face of vaccination. Furthermore, influenza viruses can adjust the antigenicity of the haemagglutinin (HA) protein, the primary influenza immunogen, using one of four molecular mechanisms. Two prominent mechanisms are: (1) enhancing binding avidity of HA toward cellular receptors to outcompete antibody binding and (2) amino acid substitutions that introduce an N-linked glycan on HA that sterically block antibody binding. In this study we investigate the impact that adsorptive mutation and N-linked glycosylation have on receptor-binding, viral fitness, and antigenicity. We utilize the H9N2 A/chicken/Pakistan/SKP-827/16 virus which naturally contains HA residue T180 that we have previously shown to be an adsorptive mutant relative to virus with T180A. We find that the addition of N-linked glycans can be beneficial or deleterious to virus replication depending on the background receptor binding avidity. We also find that in some cases, an N-linked glycan can trump the effect of an avidity enhancing substitution with respect to antigenicity. Taken together these data shed light on a potential route to the generation of a virus which is "fit" and able to overcome vaccine pressure.


Assuntos
Substituição de Aminoácidos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H9N2/fisiologia , Adsorção , Animais , Células Cultivadas , Galinhas , Cães , Aptidão Genética , Glicosilação , Células HEK293 , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Vírus da Influenza A Subtipo H9N2/imunologia , Células Madin Darby de Rim Canino , Mutagênese Sítio-Dirigida , Replicação Viral
12.
J Virol ; 94(19)2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32699084

RESUMO

H7N9 avian influenza viruses (AIVs) continue to evolve and remain a huge threat to human health and the poultry industry. Previously, serially passaging the H7N9 A/Anhui/1/2013 virus in the presence of homologous ferret antiserum resulted in immune escape viruses containing amino acid substitutions alanine to threonine at residues 125 (A125T) and 151 (A151T) and leucine to glutamine at residue 217 (L217Q) in the hemagglutinin (HA) protein. These HA mutations have also been found in field isolates in 2019. To investigate the potential threat of serum escape mutant viruses to humans and poultry, the impact of these HA substitutions, either individually or in combination, on receptor binding, pH of fusion, thermal stability, and virus replication were investigated. Our results showed the serum escape mutant formed large plaques in Madin-Darby canine kidney (MDCK) cells and grew robustly in vitro and in ovo They had a lower pH of fusion and increased thermal stability. Of note, the serum escape mutant completely lost the ability to bind to human-like receptor analogues. Further analysis revealed that N-linked glycosylation, as a result of A125T or A151T substitutions in HA, resulted in reduced receptor-binding avidity toward both human and avian-like receptor analogues, and the A125T+A151T mutations completely abolished human-like receptor binding. The L217Q mutation enhanced the H7N9 acid and thermal stability while the A151T mutation dramatically decreased H7N9 HA thermal stability. To conclude, H7N9 AIVs that contain A125T+A151T+L217Q mutations in the HA protein may pose a reduced pandemic risk but remain a heightened threat for poultry.IMPORTANCE Avian influenza H7N9 viruses have been causing disease outbreaks in poultry and humans. We previously determined that propagation of H7N9 virus in virus-specific antiserum gives rise to mutant viruses carrying mutations A125T+A151T+L217Q in their hemagglutinin protein, enabling the virus to overcome vaccine-induced immunity. As predicted, these immune escape mutations were also observed in the field viruses that likely emerged in the immunized or naturally exposed birds. This study demonstrates that the immune escape mutants also (i) gained greater replication ability in cultured cells and in chicken embryos as well as (ii) increased acid and thermal stability but (iii) lost preferences for binding to human-type receptor while maintaining binding for the avian-like receptor. Therefore, they potentially pose reduced pandemic risk. However, the emergent virus variants containing the indicated mutations remain a significant risk to poultry due to antigenic drift and improved fitness for poultry.


Assuntos
Hemaglutininas Virais/genética , Hemaglutininas Virais/imunologia , Subtipo H7N9 do Vírus da Influenza A/genética , Subtipo H7N9 do Vírus da Influenza A/imunologia , Mutação , Pandemias , Replicação Viral/fisiologia , Substituição de Aminoácidos , Animais , Cães , Hemaglutininas Virais/química , Concentração de Íons de Hidrogênio , Influenza Aviária/virologia , Células Madin Darby de Rim Canino , Modelos Moleculares , Aves Domésticas , Ligação Proteica , Conformação Proteica , Estabilidade Proteica
13.
J Virol ; 94(20)2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32727875

RESUMO

H9N2 avian influenza viruses (AIVs) circulate in poultry throughout much of Asia, the Middle East, and Africa. These viruses cause huge economic damage to poultry production systems and pose a zoonotic threat both in their own right and in the generation of novel zoonotic viruses, for example, H7N9. In recent years, it has been observed that H9N2 viruses have further adapted to gallinaceous poultry, becoming more highly transmissible and causing higher morbidity and mortality. Here, we investigate the molecular basis for this increased virulence, comparing a virus from the 1990s and a contemporary field strain. The modern virus replicated to higher titers in various systems, and this difference mapped to a single amino acid polymorphism at position 26 of the endonuclease domain shared by the PA and PA-X proteins. This change was responsible for increased replication and higher morbidity and mortality rates along with extended tissue tropism seen in chickens. Although the PA K26E change correlated with increased host cell shutoff activity of the PA-X protein in vitro, it could not be overridden by frameshift site mutations that block PA-X expression and therefore increased PA-X activity could not explain the differences in replication phenotype. Instead, this indicates that these differences are due to subtle effects on PA function. This work gives insight into the ongoing evolution and poultry adaptation of H9N2 and other avian influenza viruses and helps us understand the striking morbidity and mortality rates in the field, as well as the rapidly expanding geographical range seen in these viruses.IMPORTANCE Avian influenza viruses, such as H9N2, cause huge economic damage to poultry production worldwide and are additionally considered potential pandemic threats. Understanding how these viruses evolve in their natural hosts is key to effective control strategies. In the Middle East and South Asia, an older H9N2 virus strain has been replaced by a new reassortant strain with greater fitness. Here, we take representative viruses and investigate the genetic basis for this "fitness." A single mutation in the virus was responsible for greater fitness, enabling high growth of the contemporary H9N2 virus in cells, as well as in chickens. The genetic mutation that modulates this change is within the viral PA protein, a part of the virus polymerase gene that contributes to viral replication as well as to virus accessory functions-however, we find that the fitness effect is specifically due to changes in the protein polymerase activity.


Assuntos
Vírus da Influenza A Subtipo H9N2 , Influenza Aviária , Doenças das Aves Domésticas , RNA Polimerase Dependente de RNA , Proteínas Virais , Tropismo Viral , Animais , Galinhas , Cães , Células HEK293 , Humanos , Vírus da Influenza A Subtipo H9N2/patogenicidade , Vírus da Influenza A Subtipo H9N2/fisiologia , Influenza Aviária/genética , Influenza Aviária/metabolismo , Influenza Aviária/patologia , Células Madin Darby de Rim Canino , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/metabolismo , Doenças das Aves Domésticas/patologia , Doenças das Aves Domésticas/virologia , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
14.
Viruses ; 12(6)2020 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-32580412

RESUMO

Avian influenza viruses of the subtype H6Nx are being detected globally with increasing frequency. Some H6Nx lineages are becoming enzootic in Asian poultry and sporadic incursions into European poultry are occurring more frequently. H6Nx viruses that contain mammalian adaptation motifs pose a zoonotic threat and have caused human cases. Although currently understudied globally, H6Nx avian influenza viruses pose a substantial threat to both poultry and human health. In this review we examine the current state of knowledge of H6Nx viruses including their global distribution, tropism, transmission routes and human health risk.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A/genética , Influenza Aviária/epidemiologia , Influenza Humana/epidemiologia , Doenças das Aves Domésticas/epidemiologia , Animais , Humanos , Vírus da Influenza A/classificação , Influenza Aviária/virologia , Influenza Humana/virologia , Aves Domésticas/virologia , Doenças das Aves Domésticas/virologia
15.
Virol J ; 17(1): 82, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576218

RESUMO

BACKGROUND: Reverse genetics is used in many laboratories around the world and enables the creation of tailor-made influenza viruses with a desired genotype or phenotype. However, the process is not flawless, and difficulties remain during cloning of influenza gene segments into reverse genetics vectors (pHW2000, pHH21, pCAGGS). Reverse genetics begins with making cDNA copies of influenza gene segments and cloning them into bi-directional (pHW2000) or uni-directional plasmids (pHH21, pCAGGS) followed by transfection of the recombinant plasmid(s) to HEK-293 T or any other suitable cells which are permissive to transfection. However, the presence of internal restriction sites in the gene segments of many field isolates of avian influenza viruses makes the cloning process difficult, if employing conventional methods. Further, the genetic instability of influenza gene-containing plasmids in bacteria (especially Polymerase Basic 2 and Polymerase Basic 1 genes; PB2 and PB1) also leads to erroneous incorporation of bacterial genomic sequences into the influenza gene of interest. METHODS: Herein, we report an easy and efficient ligation and restriction enzyme independent (LREI) cloning method for cloning influenza gene segments into pHW2000 vector. The method involves amplification of megaprimers followed by PCR amplification of megaprimers using a bait plasmid, DpnI digestion and transformation. RESULTS: Hard-to-clone genes: PB2 of A/chicken/Bangladesh/23527/2014 (H9N2) and PB1 of A/chicken/Bangladesh/23527/2014 (H9N2), A/chicken/Jiangxi/02.05YGYXG023-P/2015 (H5N6) and A/Chicken/Vietnam/H7F-14-BN4-315/2014 (H9N2) were cloned into pHW2000 using our LREI method and recombinant viruses were subsequently rescued. CONCLUSION: The LREI cloning procedure represents an alternative strategy for cloning influenza gene segments which have internal restriction sites for the enzymes used in reverse genetics. Further, the problem of genetic instability in bacteria can be alleviated by growing recombinant bacterial cultures at a lower temperature. This technique can be applied to clone any influenza gene segment using universal primers, which would help in rapid generation of influenza viruses and facilitate influenza research and vaccine development.


Assuntos
Primers do DNA/genética , Vetores Genéticos , Orthomyxoviridae/genética , Plasmídeos/genética , Recombinação Genética , Genética Reversa/métodos , Animais , Galinhas/virologia , Clonagem Molecular/métodos , Enzimas de Restrição do DNA , Genoma Bacteriano , Células HEK293 , Humanos , Reação em Cadeia da Polimerase , Proteínas Virais/genética
16.
Vaccines (Basel) ; 8(1)2020 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-32138253

RESUMO

Passive immunisation with neutralising antibodies can be a potent therapeutic strategy if used pre- or post-exposure to a variety of pathogens. Herein, we investigated whether recombinant monoclonal antibodies (mAbs) could be used to protect chickens against avian influenza. Avian influenza viruses impose a significant economic burden on the poultry industry and pose a zoonotic infection risk for public health worldwide. Traditional control measures including vaccination do not provide rapid protection from disease, highlighting the need for alternative disease mitigation measures. In this study, previously generated neutralizing anti-H9N2 virus monoclonal antibodies were converted to single-chain variable fragment antibodies (scFvs). These recombinant scFv antibodies were produced in insect cell cultures and the preparations retained neutralization capacity against an H9N2 virus in vitro. To evaluate recombinant scFv antibody efficacy in vivo, chickens were passively immunized with scFvs one day before, and for seven days after virus challenge. Groups receiving scFv treatment showed partial virus load reductions measured by plaque assays and decreased disease manifestation. These results indicate that antibody therapy could reduce clinical disease and shedding of avian influenza virus in infected chicken flocks.

17.
Vaccines (Basel) ; 7(4)2019 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-31766655

RESUMO

Avian influenza viruses (AIVs) are highly contagious and have caused huge economical loss to the poultry industry. AIV vaccines remain one of the most effective methods of controlling this disease. Turkey herpesvirus (HVT) is a commonly used live attenuated vaccine against Marek's disease; it has also been used as a viral vector for recombinant AIV vaccine development. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system is a gene editing tool which, in vaccinology, has facilitated the development of recombinant DNA viral-vectored vaccines. Here, we utilize homology-directed repair (HDR) for the generation of a HVT-H7N9 HA bivalent vaccine; a H7N9 HA expression cassette was inserted into the intergenic region between UL45 and UL46 of HVT. To optimize the selection efficiency of our bivalent vaccine, we combined CRISPR/Cas9 with erythrocyte binding to rapidly generate recombinant HVT-H7HA candidate vaccines.

18.
Int J Biol Macromol ; 140: 1226-1238, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31445153

RESUMO

Bovine herpesvirus 1 (BoHV-1) is a major pathogen of infectious bovine rhinotracheitis in bovine. Previously, we generated the aptamer IBRV A4 using systemic evolution of ligands by exponential enrichment. This aptamer inhibited infectivity of BoHV-1 by blocking viral particle absorption onto cell membranes. In this study, we found that the major tegument protein VP8 of BoHV-1 was involved in inhibition of infectious virus production by IBRV A4. We improved the affinity of IBRV A4 for VP8 by optimizing aptamer's structure and repeat conformation. An optimized aptamer, IBRV A4.7, was constructed with quadruple binding sites and a new stem-loop structure, which had a stronger binding affinity for VP8 or BoHV-1 than raw aptamer IBRV A4. IBRV A4.7 bound to VP8 with a dissociation constant (Kd) value of 0.2054 ±â€¯0.03948 nM and bound to BoHV-1 with a Kd value of 0.3637 ±â€¯0.05452 nM. Crucially, IBRV A4.7 had improved antiviral activity compared to IBRV A4, with a half-maximal inhibitory concentration of 1.16 ±â€¯0.042 µM. Our results also revealed IBRV A4.7 inhibited BoHV-1 production in MDBK cells through blocking nucleocytoplasmic shuttling of viral VP8 in BoHV-1-infected MDBK cells. In conclusion, the aptamer IBRV A4.7 may have potency in preventing outbreaks in herds due to reactivation of latency.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Núcleo Celular/metabolismo , Herpesvirus Bovino 1/metabolismo , Proteínas Virais/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Aptâmeros de Nucleotídeos/química , Sequência de Bases , Sítios de Ligação , Bovinos , Linhagem Celular , Camundongos , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Secundária de Proteína , Replicação Viral
19.
Viruses ; 11(7)2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31284485

RESUMO

H9N2 avian influenza viruses have become globally widespread in poultry over the last two decades and represent a genuine threat both to the global poultry industry but also humans through their high rates of zoonotic infection and pandemic potential. H9N2 viruses are generally hyperendemic in affected countries and have been found in poultry in many new regions in recent years. In this review, we examine the current global spread of H9N2 avian influenza viruses as well as their host range, tropism, transmission routes and the risk posed by these viruses to human health.


Assuntos
Vírus da Influenza A Subtipo H9N2/patogenicidade , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/virologia , Zoonoses/epidemiologia , Zoonoses/virologia , Animais , Humanos , Vírus da Influenza A Subtipo H9N2/genética , Influenza Humana/epidemiologia , Influenza Humana/prevenção & controle , Influenza Humana/transmissão , Influenza Humana/virologia , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/transmissão , Aves Domésticas/virologia , Prevalência , Vírus Reordenados/genética , Vírus Reordenados/patogenicidade , Vacinação , Zoonoses/prevenção & controle , Zoonoses/transmissão
20.
Transbound Emerg Dis ; 66(6): 2507-2516, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31357255

RESUMO

Vietnamese poultry are host to co-circulating subtypes of avian influenza viruses, including H5N1 and H9N2, which pose a great risk to poultry productivity and to human health. AIVs circulate throughout the poultry trade network in Vietnam, with live bird markets being an integral component to this network. Traders at LBMs exhibit a variety of trading practices, which may influence the transmission of AIVs. We identified trading practices that impacted on AIV prevalence in chickens marketed in northern Vietnamese LBMs. We generated sequencing data for 31 H9N2 and two H5N6 viruses. Viruses isolated in the same LBM or from chickens sourced from the same province were genetically closer than viruses isolated in different LBMs or from chickens sourced in different provinces. The position of a vendor in the trading network impacted on their odds of having AIV-infected chickens. Being a retailer and purchasing chickens from middlemen was associated with increased odds of infection, whereas odds decreased if vendors purchased chickens directly from large farms. Odds of infection were also higher for vendors having a greater volume of ducks unsold per day. These results indicate how the spread of AIVs is influenced by the structure of the live poultry trading network.


Assuntos
Comércio , Influenza Aviária/epidemiologia , Produtos Avícolas , Aves Domésticas , Animais , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Vírus da Influenza A Subtipo H9N2/genética , Vírus da Influenza A Subtipo H9N2/isolamento & purificação , Reação em Cadeia da Polimerase , Prevalência , RNA Viral/análise , Fatores de Risco , Vietnã/epidemiologia
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