Immune Escape Adaptive Mutations in the H7N9 Avian Influenza Hemagglutinin Protein Increase Virus Replication Fitness and Decrease Pandemic Potential

ABSTRACT 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.

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Immune Escape Variants of H9N2 Influenza Viruses Containing Deletions at the Hemagglutinin Receptor Binding Site Retain Fitness In Vivo and Display Enhanced Zoonotic Characteristics

Journal of Virology ◽

10.1128/jvi.00218-17 ◽

2017 ◽

Vol 91 (14) ◽

Cited By ~ 16

Author(s):

Thomas P. Peacock ◽

Donald J. Benton ◽

Joe James ◽

Jean-Remy Sadeyen ◽

Pengxiang Chang ◽

...

Keyword(s):

Avian Influenza ◽

Receptor Binding ◽

Immune Escape ◽

Influenza Pandemic ◽

Influenza Viruses ◽

Viral Fitness ◽

Antigenic Drift ◽

Zoonotic Infection ◽

Avian Influenza Viruses ◽

The Impact

ABSTRACT H9N2 avian influenza viruses are enzootic in poultry across Asia and North Africa, where they pose a threat to human health as both zoonotic agents and potential pandemic candidates. Poultry vaccination against H9N2 viruses has been employed in many regions; however, vaccine effectiveness is frequently compromised due to antigenic drift arising from amino acid substitutions in the major influenza virus antigen hemagglutinin (HA). Using selection with HA-specific monoclonal antibodies, we previously identified H9N2 antibody escape mutants that contained deletions of amino acids in the 220 loop of the HA receptor binding sites (RBSs). Here we analyzed the impact of these deletions on virus zoonotic infection characteristics and fitness. We demonstrated that mutant viruses with RBS deletions are able to escape polyclonal antiserum binding and are able to infect and be transmitted between chickens. We showed that the deletion mutants have increased binding to human-like receptors and greater replication in primary human airway cells; however, the mutant HAs also displayed reduced pH and thermal stability. In summary, we infer that variant influenza viruses with deletions in the 220 loop could arise in the field due to immune selection pressure; however, due to reduced HA stability, we conclude that these viruses are unlikely to be transmitted from human to human by the airborne route, a prerequisite for pandemic emergence. Our findings underscore the complex interplay between antigenic drift and viral fitness for avian influenza viruses as well as the challenges of predicting which viral variants may pose the greatest threats for zoonotic and pandemic emergence. IMPORTANCE Avian influenza viruses, such as H9N2, cause disease in poultry as well as occasionally infecting humans and are therefore considered viruses with pandemic potential. Many countries have introduced vaccination of poultry to try to control the disease burden; however, influenza viruses are able to rapidly evolve to escape immune pressure in a process known as “antigenic drift.” Previously, we experimentally generated antigenic-drift variants in the laboratory, and here, we test our “drifted” viruses to assess their zoonotic infection characteristics and transmissibility in chickens. We found that the drifted viruses were able to infect and be transmitted between chickens and showed increased binding to human-like receptors. However, the drift mutant viruses displayed reduced stability, and we predict that they are unlikely to be transmitted from human to human and cause an influenza pandemic. These results demonstrate the complex relationship between antigenic drift and the potential of avian influenza viruses to infect humans.

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H3N2 Influenza Viruses with 12- or 16-Amino Acid Deletions in the Receptor-Binding Region of Their Hemagglutinin Protein

mBio ◽

10.1128/mbio.01512-21 ◽

2021 ◽

Author(s):

Huihui Kong ◽

Shufang Fan ◽

Kosuke Takada ◽

Masaki Imai ◽

Gabriele Neumann ◽

...

Keyword(s):

Receptor Binding ◽

Influenza Viruses ◽

Host Immune System ◽

Binding Region ◽

Hemagglutinin Protein ◽

H3n2 Influenza ◽

Ha Protein ◽

Receptor Binding Region ◽

Receptor Binding Protein ◽

Antigenic Epitopes

The hemagglutinin (HA) protein of influenza viruses serves as the receptor-binding protein and is the principal target of the host immune system. The antigenic epitopes in the receptor-binding region are known to tolerate mutations, but here, we show that even deletions of 12 or 16 amino acids in this region can be accommodated.

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Stability of the Influenza Virus Hemagglutinin Protein Correlates with Evolutionary Dynamics

mSphere ◽

10.1128/mspheredirect.00554-17 ◽

2018 ◽

Vol 3 (1) ◽

Cited By ~ 9

Author(s):

Eili Y. Klein ◽

Deena Blumenkrantz ◽

Adrian Serohijos ◽

Eugene Shakhnovich ◽

Jeong-Mo Choi ◽

...

Keyword(s):

Influenza Virus ◽

Protein Stability ◽

Evolutionary Dynamics ◽

Immune Escape ◽

Antigenic Site ◽

Viral Fitness ◽

Influenza Virus Hemagglutinin ◽

Hemagglutinin Protein ◽

Ha Protein ◽

The Impact

ABSTRACTProtein thermodynamics are an integral determinant of viral fitness and one of the major drivers of protein evolution. Mutations in the influenza A virus (IAV) hemagglutinin (HA) protein can eliminate neutralizing antibody binding to mediate escape from preexisting antiviral immunity. Prior research on the IAV nucleoprotein suggests that protein stability may constrain seasonal IAV evolution; however, the role of stability in shaping the evolutionary dynamics of the HA protein has not been explored. We used the full coding sequence of 9,797 H1N1pdm09 HA sequences and 16,716 human seasonal H3N2 HA sequences to computationally estimate relative changes in the thermal stability of the HA protein between 2009 and 2016. Phylogenetic methods were used to characterize how stability differences impacted the evolutionary dynamics of the virus. We found that pandemic H1N1 IAV strains split into two lineages that had different relative HA protein stabilities and that later variants were descended from the higher-stability lineage. Analysis of the mutations associated with the selective sweep of the higher-stability lineage found that they were characterized by the early appearance of highly stabilizing mutations, the earliest of which was not located in a known antigenic site. Experimental evidence further suggested that H1N1 HA stability may be correlated within vitrovirus production and infection. A similar analysis of H3N2 strains found that surviving lineages were also largely descended from viruses predicted to encode more-stable HA proteins. Our results suggest that HA protein stability likely plays a significant role in the persistence of different IAV lineages.IMPORTANCEOne of the constraints on fast-evolving viruses, such as influenza virus, is protein stability, or how strongly the folded protein holds together. Despite the importance of this protein property, there has been limited investigation of the impact of the stability of the influenza virus hemagglutinin protein—the primary antibody target of the immune system—on its evolution. Using a combination of computational estimates of stability and experiments, our analysis found that viruses with more-stable hemagglutinin proteins were associated with long-term persistence in the population. There are two potential reasons for the observed persistence. One is that more-stable proteins tolerate destabilizing mutations that less-stable proteins could not, thus increasing opportunities for immune escape. The second is that greater stability increases the fitness of the virus through increased production of infectious particles. Further research on the relative importance of these mechanisms could help inform the annual influenza vaccine composition decision process.

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Novel Highly Pathogenic Avian A(H5N2) and A(H5N8) Influenza Viruses of Clade 2.3.4.4 from North America Have Limited Capacity for Replication and Transmission in Mammals

mSphere ◽

10.1128/msphere.00003-16 ◽

2016 ◽

Vol 1 (2) ◽

Cited By ~ 37

Author(s):

Bryan S. Kaplan ◽

Marion Russier ◽

Trushar Jeevan ◽

Bindumadhav Marathe ◽

Elena A. Govorkova ◽

...

Keyword(s):

North America ◽

Avian Influenza ◽

North American ◽

Influenza Viruses ◽

Morbidity And Mortality ◽

Avian Influenza Viruses ◽

Highly Pathogenic ◽

The North ◽

Domestic Poultry ◽

The Impact

ABSTRACT Highly pathogenic H5 influenza viruses have been introduced into North America from Asia, causing extensive morbidity and mortality in domestic poultry. The introduced viruses have reassorted with North American avian influenza viruses, generating viral genotypes not seen on other continents. The experiments and analyses presented here were designed to assess the impact of this genetic diversification on viral phenotypes, particularly as regards mammalian hosts, by comparing the North American viruses with their Eurasian precursor viruses. Highly pathogenic influenza A(H5N8) viruses from clade 2.3.4.4 were introduced to North America by migratory birds in the fall of 2014. Reassortment of A(H5N8) viruses with avian viruses of North American lineage resulted in the generation of novel A(H5N2) viruses with novel genotypes. Through sequencing of recent avian influenza viruses, we identified PB1 and NP gene segments very similar to those in the viruses isolated from North American waterfowl prior to the introduction of A(H5N8) to North America, highlighting these bird species in the origin of reassortant A(H5N2) viruses. While they were highly virulent and transmissible in poultry, we found A(H5N2) viruses to be low pathogenic in mice and ferrets, and replication was limited in both hosts compared with those of recent highly pathogenic avian influenza (HPAI) H5N1 viruses. Molecular characterization of the hemagglutinin protein from A(H5N2) viruses showed that the receptor binding preference, cleavage, and pH of activation were highly adapted for replication in avian species and similar to those of other 2.3.4.4 viruses. In addition, North American and Eurasian clade 2.3.4.4 H5NX viruses replicated to significantly lower titers in differentiated normal human bronchial epithelial cells than did seasonal human A(H1N1) and highly pathogenic A(H5N1) viruses isolated from a human case. Thus, despite their having a high impact on poultry, our findings suggest that the recently emerging North American A(H5N2) viruses are not expected to pose a substantial threat to humans and other mammals without further reassortment and/or adaptation and that reassortment with North American viruses has not had a major impact on viral phenotype. IMPORTANCE Highly pathogenic H5 influenza viruses have been introduced into North America from Asia, causing extensive morbidity and mortality in domestic poultry. The introduced viruses have reassorted with North American avian influenza viruses, generating viral genotypes not seen on other continents. The experiments and analyses presented here were designed to assess the impact of this genetic diversification on viral phenotypes, particularly as regards mammalian hosts, by comparing the North American viruses with their Eurasian precursor viruses.

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Amino Acid Residue 217 in the Hemagglutinin Glycoprotein Is a Key Mediator of Avian Influenza H7N9 Virus Antigenicity

Journal of Virology ◽

10.1128/jvi.01627-18 ◽

2018 ◽

Vol 93 (1) ◽

Cited By ~ 6

Author(s):

Pengxiang Chang ◽

Joshua E. Sealy ◽

Jean-Remy Sadeyen ◽

Munir Iqbal

Keyword(s):

Amino Acid ◽

Avian Influenza ◽

Immune Escape ◽

Influenza Viruses ◽

Single Mutation ◽

H7n9 Virus ◽

Avian Influenza Viruses ◽

Antigenic Analysis ◽

Neutralizing Capacity ◽

High Pathogenicity

ABSTRACTAvian influenza viruses continue to evolve and acquire mutations that facilitate antigenic drift and virulence change. In 2017, low-pathogenicity H7N9 avian influenza viruses evolved to a high-pathogenicity phenotype in China. Comparative antigenic analysis of the low- and high-pathogenicity virus strains showed marked variability. In order to identify residues that may be linked to the antigenic change among the H7N9 viruses, we serially passaged the viruses in the presence of homologous ferret antiserum. Progeny viruses able to overcome the neutralizing capacity of the antiserum were sequenced. The analysis showed that the emergent immune escape viruses contained mutations A125T, A151T, and L217Q in the hemagglutinin (HA) glycoprotein as early as passage 5 and that these mutations persisted until passage 10. The results revealed that a single mutation, L217Q, in the HA of H7N9 virus led to 23- and 8-fold reductions in hemagglutination inhibition (HI) titer with ferret and chicken antisera, respectively. Further analysis showed that this change also contributed to antigenic differences between the low- and high-pathogenicity H7N9 viruses, thus playing a major role in their antigenic diversification. Therefore, evolutionary changes at amino acid position 217 in the H7N9 viruses can serve as a genetic marker for virus antigenic diversity during vaccine seed matching and selection. Thein vitroimmune escape mutant selection method used in this study could also aid in the prediction of emerging antigenic variants in naturally infected or immunized animals.IMPORTANCEAvian influenza H7N9 viruses circulating in poultry and wild birds continue to evolve and acquire important phenotypic changes. Mutations to the virus hemagglutinin (HA) glycoprotein can modulate virus antigenicity and facilitate virus escape from natural or vaccine-induced immunity. The focus of this study was to identify evolutionary markers in the HA of H7N9 that drive escape from antibody-based immunity. To achieve this, we propagated low-pathogenicity H7N9 virus in the presence of polyclonal antiserum derived from ferrets infected with the same strain of virus (homologous antiserum). This selection process was repeated 10 times. The HA gene sequences of viruses recovered after the fifth passage showed that the viruses readily acquired mutations at three different amino acid positions (A125T, A151T, and L217Q). Further functional analysis of these mutations confirmed that the mutation at residue 217 in the HA was responsible for mediating changes to the immunological properties of the H7N9 virus.

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The Single E627K Amino Acid Substitution in PB2 Enhances the Pathogenicity of Wild-Bird-Origin H6N6 Subtype Avian Influenza Virus in Mice

Austin Journal of Infectious Diseases ◽

10.26420/austinjinfectdis.2020.1041 ◽

2020 ◽

Vol 7 (1) ◽

Author(s):

Miura H ◽

◽

Ozeki Y ◽

Omatsu T ◽

Katayama Y ◽

...

Keyword(s):

Amino Acid ◽

Avian Influenza ◽

Wild Bird ◽

Virus Transmission ◽

Influenza Viruses ◽

Whole Genome Sequence ◽

Species Barrier ◽

The Impact ◽

Original Virus ◽

Wild Waterfowl

Avian Influenza Viruses (AIVs) are harbored by wild waterfowl as a natural host, and there is a species barrier restricting virus transmission from birds to mammals, including humans. However, it has been reported that, through genetic mutations, AIVs occasionally infect mammals and acquire high pathogenicity. The Amino Acid (aa) substitution of glutamic acid to lysine at position 627 (E627K) in polymerase basic protein 2 (PB2) is one of the wellknown factors underlying mammalian adaptation. Although this substitution was previously observed in mammalian-adapted H5, H7, and H9 AIV subtypes, the impact of this mutation on the mammalian adaptation of other AIV subtypes is not fully verified. Here, we isolated the low pathogenic AIV subtype H6N6 from a wild bird fecal sample in Tokachi Subprefecture, Hokkaido, Japan. We passaged this H6N6 subtype in BALB/c mice four times and acquired the mouse-adapted virus. Whole-genome sequence analysis showed that the adapted virus had only one aa substitution (E627K) in PB2. The adapted virus-inoculated mice tended to show increased weight loss and mortality compared with the original virus-inoculated mice. The viral titer in the lungs of the adapted virus-inoculated mice was significantly higher than that of the original virus-inoculated mice. Additionally, the virus isolated from the lung of the original virus-inoculated mice with serious symptoms harbored the E627K substitution. Our findings indicate the possibility that the PB2 E627K substitution in H6N6 subtype AIV rapidly appears in mammalian hosts and contributes to the enhanced pathogenicity of this virus.

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Correction: Genetics, Receptor Binding Property, and Transmissibility in Mammals of Naturally Isolated H9N2 Avian Influenza Viruses

PLoS Pathogens ◽

10.1371/journal.ppat.1008284 ◽

2020 ◽

Vol 16 (1) ◽

pp. e1008284

Author(s):

Xuyong Li ◽

Jianzhong Shi ◽

Jing Guo ◽

Guohua Deng ◽

Qianyi Zhang ◽

...

Keyword(s):

Avian Influenza ◽

Receptor Binding ◽

Influenza Viruses ◽

Binding Property ◽

Avian Influenza Viruses

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A Single Mutation N166D in Hemagglutinin Affects Antigenicity and Pathogenesis of H9N2 Avian Influenza Virus

Viruses ◽

10.3390/v11080709 ◽

2019 ◽

Vol 11 (8) ◽

pp. 709 ◽

Cited By ~ 3

Author(s):

Fang Jin ◽

Xiaomei Dong ◽

Zhimin Wan ◽

Dan Ren ◽

Min Liu ◽

...

Keyword(s):

Immune Escape ◽

Mdck Cells ◽

Body Weight Loss ◽

Escape Mutant ◽

Single Mutation ◽

H9n2 Avian Influenza Virus ◽

Viral Shedding ◽

Escape Mutants ◽

The Impact ◽

Darby Canine Kidney

Some immune escape mutants of H9N2 virus and the corresponding mutations in hemagglutinin (HA) have been documented, but little is known about the impact of a single mutation on the antigenicity and pathogenesis of H9N2. In this study, seven critical sites in HA associated with the antigenicity were identified and the effects of a HA mutation (N166D) derived from a H9N2 escape mutant (m3F2) were investigated. Although N166D did not significantly affect viral replication in Madin–Darby canine kidney (MDCK) cells and viral shedding in the larynx and cloaca of chicken, N166D attenuated the pathogenesis of the virus in mice. Compared to the rescued RgPR8-H9_166D, RgPR8-H9_166N caused greater body weight loss and higher viral titers in the lungs of the infected mice. Moreover, hemagglutination inhibition (HI) assay for the sera from the chickens infected with wild type H9N2 and mutant m3F2 showed that N166D mutation could result in weak antibody response in chickens. Considering the field strains of H9N2 with N166D mutation are frequently isolated in the countries with H9N2 vaccination, the findings that the single mutation in HA, N166D, affected both the antigenicity and pathogenesis of H9N2 highlight the significance of surveillance on such mutation that may contribute to the failure of H9N2 vaccination in the field.

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Swine ANP32A Supports Avian Influenza Virus Polymerase

Journal of Virology ◽

10.1128/jvi.00132-20 ◽

2020 ◽

Vol 94 (12) ◽

Cited By ~ 2

Author(s):

Thomas P. Peacock ◽

Olivia C. Swann ◽

Hamish A. Salvesen ◽

Ecco Staller ◽

P. Brian Leung ◽

...

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Avian Influenza Virus ◽

Virus Replication ◽

Mammalian Species ◽

Gene Mutations ◽

Influenza Viruses ◽

Avian Influenza Viruses ◽

2009 H1n1 ◽

Mixing Vessels

ABSTRACT Avian influenza viruses occasionally infect and adapt to mammals, including humans. Swine are often described as “mixing vessels,” being susceptible to both avian- and human-origin viruses, which allows the emergence of novel reassortants, such as the precursor to the 2009 H1N1 pandemic. ANP32 proteins are host factors that act as influenza virus polymerase cofactors. In this study, we describe how swine ANP32A, uniquely among the mammalian ANP32 proteins tested, supports the activity of avian-origin influenza virus polymerases and avian influenza virus replication. We further show that after the swine-origin influenza virus emerged in humans and caused the 2009 pandemic, it evolved polymerase gene mutations that enabled it to more efficiently use human ANP32 proteins. We map the enhanced proviral activity of swine ANP32A to a pair of amino acids, 106 and 156, in the leucine-rich repeat and central domains and show these mutations enhance binding to influenza virus trimeric polymerase. These findings help elucidate the molecular basis for the mixing vessel trait of swine and further our understanding of the evolution and ecology of viruses in this host. IMPORTANCE Avian influenza viruses can jump from wild birds and poultry into mammalian species such as humans or swine, but they only continue to transmit if they accumulate mammalian adapting mutations. Pigs appear uniquely susceptible to both avian and human strains of influenza and are often described as virus “mixing vessels.” In this study, we describe how a host factor responsible for regulating virus replication, ANP32A, is different between swine and humans. Swine ANP32A allows a greater range of influenza viruses, specifically those from birds, to replicate. It does this by binding the virus polymerase more tightly than the human version of the protein. This work helps to explain the unique properties of swine as mixing vessels.

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