scholarly journals Identification of PB2 Mutations Responsible for the Efficient Replication of H5N1 Influenza Viruses in Human Lung Epithelial Cells

2015 ◽  
Vol 89 (7) ◽  
pp. 3947-3956 ◽  
Author(s):  
Reina Yamaji ◽  
Shinya Yamada ◽  
Mai Q. Le ◽  
Chengjun Li ◽  
Hualan Chen ◽  
...  
Keyword(s):  
Amino Acid ◽  
H5n1 Virus ◽  
Human Lung ◽  
A549 Cells ◽  
Influenza Viruses ◽  
H5n1 Influenza ◽  
H5n1 Avian Influenza ◽  
Lung Epithelial ◽  
Efficient Replication ◽  
Amino Acid Mutations

ABSTRACTHighly pathogenic H5N1 avian influenza viruses have caused outbreaks among poultry worldwide, resulting in sporadic infections in humans with approximately 60% mortality. However, efficient transmission of H5N1 viruses among humans has yet to occur, suggesting that further adaptation of H5N1 viruses to humans is required for their efficient transmission among humans. The viral determinants for efficient replication in humans are currently poorly understood. Here, we report that the polymerase PB2 protein of an H5N1 influenza virus isolated from a human in Vietnam (A/Vietnam/UT36285/2010, virus 36285) increased the growth ability of an avian H5N1 virus (A/wild bird/Anhui/82/2005, virus Wb/AH82) in human lung epithelial A549 cells (however, the reassortant virus did not replicate more efficiently than human 36285 virus). Furthermore, we demonstrate that the amino acid residues at positions 249, 309, and 339 of the PB2 protein from this human isolate were responsible for its efficient replication in A549 cells. PB2 residues 249G and 339M, which are found in the human H5N1 virus, are rare in H5N1 viruses from both human and avian sources. Interestingly, PB2-249G is found in over 30% of human seasonal H3N2 viruses, which suggests that H5N1 viruses may replicate well in human cells when they acquire this mutation. Our data are of value to H5N1 virus surveillance.IMPORTANCEHighly pathogenic H5N1 avian influenza viruses must acquire mutations to overcome the species barrier between avian species and humans. When H5N1 viruses replicate in human respiratory cells, they can acquire amino acid mutations that allow them to adapt to humans through continuous selective pressure. Several amino acid mutations have been shown to be advantageous for virus adaptation to mammalian hosts. Here, we found that amino acid changes at positions 249, 309, and 339 of PB2 contribute to efficient replication of avian H5N1 viruses in human lung cells. These findings are beneficial for evaluating the pandemic risk of circulating avian viruses and for further functional analysis of PB2.

scholarly journals Mammalian Adaptive Mutations of the PA Protein of Highly Pathogenic Avian H5N1 Influenza Virus

2015 ◽  
Vol 89 (8) ◽  
pp. 4117-4125 ◽  
Author(s):  
Reina Yamaji ◽  
Shinya Yamada ◽  
Mai Q. Le ◽  
Mutsumi Ito ◽  
Yuko Sakai-Tagawa ◽  
...  
Keyword(s):  
Amino Acid ◽  
Mammalian Cells ◽  
H5n1 Virus ◽  
Human Lung ◽  
A549 Cells ◽  
Amino Acid Substitutions ◽  
Highly Pathogenic ◽  
Adaptive Mutations ◽  
Human Lung Cells ◽  
H5n1 Influenza

ABSTRACTHighly pathogenic H5N1 influenza A viruses continue to circulate among avian species and cause sporadic cases of human infection. Therefore, the threat of a pandemic persists. However, the human cases of H5N1 infection have been limited mainly to individuals in close contact with infected poultry. These findings suggest that the H5N1 viruses need to acquire adaptive mutations to gain a replicative advantage in mammalian cells to break through the species barrier. Many amino acid mutations of the polymerase complex have been reported to enhance H5N1 virus growth in mammalian cells; however, the mechanism for H5N1 virus of adaptation to humans remains unclear. Here, we propose that the PA of an H5N1 influenza virus isolated from a human in Vietnam (A/Vietnam/UT36285/2010 [36285]) increased the ability of an avian H5N1 virus (A/chicken/Vietnam/TY31/2005 [Ck/TY31]) to grow in human lung epithelial A549 cells. The five PA amino acid substitutions V44I, V127A, C241Y, A343T, and I573V, which are rare in H5N1 viruses from human and avian sources, enhanced the growth capability of this virus in A549 cells. Moreover, these mutations increased the pathogenicity of the virus in mice, suggesting that they contribute to adaptation to mammalian hosts. Intriguingly, PA-241Y, which 36285 encodes, is conserved in more than 90% of human seasonal H1N1 viruses, suggesting that PA-241Y contributes to virus adaptation to human lung cells and mammalian hosts.IMPORTANCEMany amino acid substitutions in highly pathogenic H5N1 avian influenza viruses have been shown to contribute to adaptation to mammalian hosts. However, no naturally isolated H5N1 virus has caused extensive human-to-human transmission, suggesting that additional, as-yet unidentified amino acid mutations are needed for adaptation to humans. Here, we report that five amino acid substitutions in PA (V44I, V127A, C241Y, A343T, and I573V) contribute to the replicative efficiency of H5N1 viruses in human lung cells and to high virulence in mice. These results are helpful for assessing the pandemic risk of isolates and further our understanding of the mechanism of H5N1 virus adaptation to mammalian hosts.

scholarly journals Glycine at Position 622 in PB1 Contributes to the Virulence of H5N1 Avian Influenza Virus in Mice

2015 ◽  
Vol 90 (4) ◽  
pp. 1872-1879 ◽  
Author(s):  
Xiaoxiao Feng ◽  
Zeng Wang ◽  
Jianzhong Shi ◽  
Guohua Deng ◽  
Huihui Kong ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Aspartic Acid ◽  
H5n1 Virus ◽  
Genetic Basis ◽  
Vaccine Development ◽  
Influenza Viruses ◽  
Avian Influenza Viruses ◽  
Live Attenuated Vaccine ◽  
H5n1 Influenza ◽  
H5n1 Avian Influenza

ABSTRACTWe isolated two H5N1 viruses, A/duck/Hunan/S4020/2008 (DK/08) and A/chicken/Guangxi/S2039/2009 (CK/09), from live-bird markets during routine surveillance and found that these two viruses are genetically similar but differ in their replication and virulence in mice. The CK/09 virus is lethal for mice with a 50% mouse lethal dose (MLD50) of 1.6 log1050% egg infectious doses (EID50), whereas the DK/08 virus is nonpathogenic for mice with an MLD50value of 6.2 log10EID50. We explored the genetic basis of the virulence difference of these two viruses by generating a series of reassortant viruses and mutants in the lethal virus CK/09 background and evaluating their virulence in mice. We found that the PB1 gene of the DK/08 virus dramatically attenuated the virulence of the CK/09 virus and that the amino acid at position 622 in PB1 made an important contribution. We further demonstrated that the mutation of glycine (G) to aspartic acid (D) at position 622 in PB1 partially impaired the binding of PB1 to viral RNA, thereby dramatically decreasing the polymerase activity and attenuating H5N1 virus virulence in mice. Our results identify a novel virulence-related marker of H5N1 influenza viruses and provide a new target for live attenuated vaccine development.IMPORTANCEH5N1 avian influenza viruses have caused the deaths of nearly 60% of the humans that they have infected since 1997 and clearly represent a threat to public health. A thorough understanding of the genetic basis of virulence determinants will provide important insights for antiviral drug and live attenuated vaccine development. Several virulence-related markers in the PB2, PA, M1, and NS1 proteins of H5N1 viruses have been identified. In this study, we isolated two H5N1 avian influenza viruses that are genetically similar but differ in their virulence in mice, and we identified a new virulence-related marker in the PB1 gene. We found that the mutation of glycine (G) to aspartic acid (D) at position 622 in PB1 partially impairs the binding of PB1 to viral RNA, thereby attenuating H5N1 virus virulence in mice. This newly identified virulence-related marker could be applied to the development of live attenuated vaccines against H5N1 influenza.

Enhancing Effect of Poly(amino acid)s on Albumin Uptake in Human Lung Epithelial A549 Cells

2013 ◽  
Vol 28 (6) ◽  
pp. 497-503 ◽  
Author(s):  
Ryoko Yumoto ◽  
Sayuri Suzuka ◽  
Saori Nishimoto ◽  
Junya Nagai ◽  
Mikihisa Takano
Keyword(s):  
Amino Acid ◽  
Human Lung ◽  
A549 Cells ◽  
Enhancing Effect ◽  
Lung Epithelial

scholarly journals High Level of Genetic Compatibility between Swine-Origin H1N1 and Highly Pathogenic Avian H5N1 Influenza Viruses

2010 ◽  
Vol 84 (20) ◽  
pp. 10918-10922 ◽  
Author(s):  
Cássio Pontes Octaviani ◽  
Makoto Ozawa ◽  
Shinya Yamada ◽  
Hideo Goto ◽  
Yoshihiro Kawaoka
Keyword(s):  
Influenza Virus ◽  
H5n1 Virus ◽  
Human Lung ◽  
Cultured Cells ◽  
Influenza Viruses ◽  
Compatibility Studies ◽  
Genetic Compatibility ◽  
Highly Pathogenic ◽  
H5n1 Influenza ◽  
High Level

Reassortment is an important mechanism for the evolution of influenza viruses. Here, we coinfected cultured cells with the pandemic swine-origin influenza virus (S-OIV) and a contemporary H5N1 virus and found that these two viruses have high genetic compatibility. Studies of human lung cell lines indicated that some reassortants had better growth kinetics than their parental viruses. We conclude that reassortment between these two viruses can occur and could create pandemic H5N1 viruses.

scholarly journals A Single-Amino-Acid Substitution in the NS1 Protein Changes the Pathogenicity of H5N1 Avian Influenza Viruses in Mice

2007 ◽  
Vol 82 (3) ◽  
pp. 1146-1154 ◽  
Author(s):  
Peirong Jiao ◽  
Guobin Tian ◽  
Yanbing Li ◽  
Guohua Deng ◽  
Yongping Jiang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Avian Influenza ◽  
Host Cell ◽  
Amino Acid Substitution ◽  
Influenza Viruses ◽  
Single Amino Acid ◽  
Ns1 Protein ◽  
Avian Influenza Viruses ◽  
H5n1 Influenza ◽  
H5n1 Avian Influenza

ABSTRACT In this study, we explored the molecular basis determining the virulence of H5N1 avian influenza viruses in mammalian hosts by comparing two viruses, A/Duck/Guangxi/12/03 (DK/12) and A/Duck/Guangxi/27/03 (DK/27), which are genetically similar but differ in their pathogenicities in mice. To assess the genetic basis for this difference in virulence, we used reverse genetics to generate a series of reassortants and mutants of these two viruses. We found that a single-amino-acid substitution of serine for proline at position 42 (P42S) in the NS1 protein dramatically increased the virulence of the DK/12 virus in mice, whereas the substitution of proline for serine at the same position (S42P) completely attenuated the DK/27 virus. We further demonstrated that the amino acid S42 of NS1 is critical for the H5N1 influenza virus to antagonize host cell interferon induction and for the NS1 protein to prevent the double-stranded RNA-mediated activation of the NF-κB pathway and the IRF-3 pathway. Our results indicate that the NS1 protein is critical for the pathogenicity of H5N1 influenza viruses in mammalian hosts and that the amino acid S42 of NS1 plays a key role in undermining the antiviral immune response of the host cell.

scholarly journals Apoptotic and Early Innate Immune Responses to PB1-F2 Protein of Influenza A Viruses Belonging to Different Subtypes in Human Lung Epithelial A549 Cells

2018 ◽  
Vol 2018 ◽  
pp. 1-12
Author(s):  
Gunisha Pasricha ◽  
Sanjay Mukherjee ◽  
Alok K. Chakrabarti
Keyword(s):  
Inflammatory Response ◽  
Influenza A ◽  
H5n1 Virus ◽  
A549 Cells ◽  
Terminal Region ◽  
Influenza Viruses ◽  
Influenza A Viruses ◽  
Highly Pathogenic ◽  
Lung Epithelial ◽  
Pathogenic H5n1

PB1-F2 is a multifunctional protein and contributes to the pathogenicity of influenza A viruses. PB1-F2 is known to have strain and cell specific functions. In this study we have investigated the apoptotic and inflammatory responses of PB1-F2 protein from influenza viruses of diverse pathogenicities in A549 lung epithelial cells. Overexpression of PB1-F2 resulted in apoptosis and heightened inflammatory response in A549 cells. Comparison revealed that the response varied with each subtype. PB1-F2 protein from highly pathogenic H5N1 virus induced least apoptosis but maximum inflammatory response. Results indicated that apoptosis was mediated through death receptor ligands TNFα and TRAIL via Caspase 8 activation. Significant induction of cytokines/chemokines CXCL10, CCL5, CCL2, IFNα, and IL-6 was noted in A549 cells transfected with PB1-F2 gene construct of 2008 West Bengal H5N1 virus (H5N1-WB). On the contrary, PB1-F2 construct from 2007 highly pathogenic H5N1 isolate (H5N1-M) with truncated N-terminal region did not evoke as exuberant inflammatory response as the other H5N1-WB with full length PB1-F2, signifying the importance of N-terminal region of PB1-F2. Sequence analysis revealed that PB1-F2 proteins derived from different influenza viruses varied at multiple amino acid positions. The secondary structure prediction showed each of the PB1-F2 proteins had distinct helix-loop-helix structure. Thus, our data substantiate the notion that the contribution of PB1-F2 to influenza pathogenicity is greatly strain specific and involves multiple host factors. This data demonstrates that PB1-F2 protein of influenza A virus, when expressed independently is minimally apoptotic and strongly influences the early host response in A549 cells.

scholarly journals Rapid Evolution of H5N1 Influenza Viruses in Chickens in Hong Kong

1999 ◽  
Vol 73 (4) ◽  
pp. 3366-3374 ◽  
Author(s):  
Nan Nan Zhou ◽  
Kennedy F. Shortridge ◽  
Eric C. J. Claas ◽  
Scott L. Krauss ◽  
Robert G. Webster
Keyword(s):  
Amino Acids ◽  
Hong Kong ◽  
Amino Acid ◽  
Rapid Evolution ◽  
Amino Acid Sequences ◽  
Influenza Viruses ◽  
H5n1 Avian Influenza Virus ◽  
H5n1 Influenza ◽  
H5n1 Avian Influenza ◽  
Phylogenetic Lineages

ABSTRACT The H5N1 avian influenza virus that killed 6 of 18 persons infected in Hong Kong in 1997 was transmitted directly from poultry to humans. Viral isolates from this outbreak may provide molecular clues to zoonotic transfer. Here we demonstrate that the H5N1 viruses circulating in poultry comprised two distinguishable phylogenetic lineages in all genes that were in very rapid evolution. When introduced into new hosts, influenza viruses usually undergo rapid alteration of their surface glycoproteins, especially in the hemagglutinin (HA). Surprisingly, these H5N1 isolates had a large proportion of amino acid changes in all gene products except in the HA. These viruses maybe reassortants each of whose HA gene is well adapted to domestic poultry while the rest of the genome arises from a different source. The consensus amino acid sequences of “internal” virion proteins reveal amino acids previously found in human strains. These human-specific amino acids may be important factors in zoonotic transmission.

scholarly journals Predicting potentially permissive substitutions that improve the fitness of A(H1N1)pdm09 viruses bearing the H275Y NA substitution

2021 ◽  
Author(s):  
Rubaiyea Farrukee ◽  
Vithiagaran Gunalan ◽  
Sebastian Maurer-Stroh ◽  
Patrick C Reading ◽  
Aeron C Hurt
Keyword(s):  
Amino Acid ◽  
A549 Cells ◽  
Computational Approach ◽  
Influenza Viruses ◽  
Single Amino Acid ◽  
Oseltamivir Resistance ◽  
Amino Acid Mutations ◽  
H275y Mutation ◽  
Key Residues

Oseltamivir-resistant influenza viruses arise due to amino-acid mutations in key residues, but these changes often reduce their replicative and transmission fitness. Widespread oseltamivir-resistance has not yet been observed in A(H1N1)pdm09 viruses. However, it is known that permissive mutations in the neuraminidase (NA) of former seasonal A(H1N1) viruses from 2007-2009 buffered the detrimental effect of the NA H275Y mutation, resulting in fit oseltamivir-resistant viruses that circulated widely. This study explored two approaches to predict permissive mutations that may enable a fit H275Y A(H1N1)pdm09 variant to arise. A computational approach used phylogenetic and in silico protein stability analyses to predict potentially permissive mutations, which were then evaluated by in vitro NA enzyme activity and expression analysis, followed by in vitro replication. The second approach involved the generation of a virus library which encompassed all possible individual 2.9 x 104 codon mutations in the NA whilst keeping H275Y fixed. To select for variant viruses with the greatest fitness, the virus library was serially passaged in ferrets (via contact and aerosol transmission) and resultant viruses were deep sequenced. The computational approach predicted three NA permissive mutations, and even though they only offset the in vitro impact of H275Y on NA enzyme expression by 10%, they could restore replication fitness of the H275Y variant in A549 cells. In our experimental approach, a diverse virus library (97% of 8911 possible single amino-acid substitutions were sampled) was successfully transmitted through ferrets, and sequence analysis of resulting virus pools in nasal washes identified three mutations that improved virus transmissibility. Of these, one NA mutation, I188T, has been increasing in frequency since 2017 and is now present in 90% of all circulating A(H1N1)pdm09 viruses. Overall, this study provides valuable insights into the evolution of the influenza NA protein and identified several mutations that may potentially facilitate the emergence of a fit H275Y A(H1N1)pdm09 variant.

scholarly journals Deep sequencing of primary human lung epithelial cells challenged with H5N1 influenza virus reveals a proviral role for CEACAM1

2018 ◽  
Author(s):  
Siying Ye ◽  
Chris Cowled ◽  
Cheng-Hon Yap ◽  
John Stambas
Keyword(s):  
Influenza Virus ◽  
Deep Sequencing ◽  
H5n1 Virus ◽  
Influenza Viruses ◽  
Lung Epithelial Cells ◽  
Chemokine Production ◽  
H5n1 Infection ◽  
Alveolar Epithelial ◽  
H5n1 Influenza ◽  
Lung Epithelial

ABSTRACTCurrent prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly up-regulated, including the cell adhesion moleculeCEACAM1. The up-regulation ofCEACAM1was blocked in the presence of the reactive oxygen species inhibitor, apocynin. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to low pathogenic PR8 H1N1 virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenousCEACAM1resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies.

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