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

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.

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

Journal of Virology ◽

10.1128/jvi.03328-14 ◽

2015 ◽

Vol 89 (7) ◽

pp. 3947-3956 ◽

Cited By ~ 17

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.

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

Journal of Virology ◽

10.1128/jvi.01140-10 ◽

2010 ◽

Vol 84 (20) ◽

pp. 10918-10922 ◽

Cited By ~ 79

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.

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Epitope Mapping of the Hemagglutinin Molecule of a Highly Pathogenic H5N1 Influenza Virus by Using Monoclonal Antibodies

Journal of Virology ◽

10.1128/jvi.01522-07 ◽

2007 ◽

Vol 81 (23) ◽

pp. 12911-12917 ◽

Cited By ~ 136

Author(s):

Nikolai V. Kaverin ◽

Irina A. Rudneva ◽

Elena A. Govorkova ◽

Tatyana A. Timofeeva ◽

Aleksandr A. Shilov ◽

...

Keyword(s):

Influenza Virus ◽

Amino Acid ◽

H5n1 Virus ◽

Antigenic Site ◽

H5n1 Influenza Virus ◽

Highly Pathogenic ◽

H5n1 Influenza ◽

Antigenic Sites ◽

Escape Mutants ◽

Pathogenic H5n1

ABSTRACT We mapped the hemagglutinin (HA) antigenic epitopes of a highly pathogenic H5N1 influenza virus on the three-dimensional HA structure by characterizing escape mutants of a recombinant virus containing A/Vietnam/1203/04 (H5N1) ΔHA and neuraminidase genes in the genetic background of A/Puerto Rico/8/34 (H1N1) virus. The mutants were selected with a panel of eight anti-HA monoclonal antibodies (MAbs), seven to A/Vietnam/1203/04 (H5N1) virus and one to A/Chicken/Pennsylvania/8125/83 (H5N2) virus, and the mutants’ HA genes were sequenced. The amino acid changes suggested three MAb groups: four MAbs reacted with the complex epitope comprising parts of the antigenic site B of H3 HA and site Sa of H1 HA, two MAbs reacted with the epitope corresponding to the antigenic site A in H3 HA, and two MAbs displayed unusual behavior: each recognized amino acid changes at two widely separate antigenic sites. Five changes were detected in amino acid residues not previously reported as changed in H5 escape mutants, and four others had substitutions not previously described. The HA antigenic structure differs substantially between A/Vietnam/1203/04 (H5N1) virus and the low-pathogenic A/Mallard/Pennsylvania/10218/84 (H5N2) virus we previously characterized (N. V. Kaverin et al., J. Gen. Virol. 83:2497-2505, 2002). The hemagglutination inhibition reactions of the MAbs with recent highly pathogenic H5N1 viruses were consistent with the antigenic-site amino acid changes but not with clades and subclades based on H5 phylogenetic analysis. These results provide information on the recognition sites of the MAbs widely used to study H5N1 viruses and demonstrate the involvement of the HA antigenic sites in the evolution of highly pathogenic H5N1 viruses, findings that can be critical for characterizing pathogenesis and vaccine design.

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Neurovirulence in Mice of H5N1 Influenza Virus Genotypes Isolated from Hong Kong Poultry in 2001

Journal of Virology ◽

10.1128/jvi.77.6.3816-3823.2003 ◽

2003 ◽

Vol 77 (6) ◽

pp. 3816-3823 ◽

Cited By ~ 53

Author(s):

Aleksandr S. Lipatov ◽

Scott Krauss ◽

Yi Guan ◽

Malik Peiris ◽

Jerold E. Rehg ◽

...

Keyword(s):

Influenza Virus ◽

Amino Acid ◽

H5n1 Virus ◽

Gene Products ◽

H5n1 Influenza Virus ◽

Highly Pathogenic ◽

H5n1 Influenza ◽

B Virus ◽

Virus Genotypes ◽

The Brain

ABSTRACT We studied the pathogenicity of five different genotypes (A to E) of highly pathogenic avian H5N1 viruses, which contained HA genes similar to those of the H5N1 virus A/goose/Guangdong/1/96 and five different combinations of “internal” genes, in a mouse model. Highly pathogenic, neurotropic variants of genotypes A, C, D, and E were isolated from the brain after a single intranasal passage in mice. Genotype B virus was isolated from lungs only. The mouse brain variants had amino acid changes in all gene products except PB1, NP, and NS1 proteins but no common sets of mutations. We conclude that the original H5N1/01 isolates of genotypes A, C, D, and E were heterogeneous and that highly pathogenic neurotropic variants can be rapidly selected in mice.

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

Drug Metabolism and Pharmacokinetics ◽

10.2133/dmpk.dmpk-13-rg-028 ◽

2013 ◽

Vol 28 (6) ◽

pp. 497-503 ◽

Cited By ~ 4

Author(s):

Ryoko Yumoto ◽

Sayuri Suzuka ◽

Saori Nishimoto ◽

Junya Nagai ◽

Mikihisa Takano

Keyword(s):

Amino Acid ◽

Human Lung ◽

A549 Cells ◽

Enhancing Effect ◽

Lung Epithelial

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Mutations in the PA Protein of Avian H5N1 Influenza Viruses Affect Polymerase Activity and Mouse Virulence

Journal of Virology ◽

10.1128/jvi.01557-17 ◽

2017 ◽

pp. JVI.01557-17 ◽

Cited By ~ 5

Author(s):

Gongxun Zhong ◽

Mai Quynh Le ◽

Tiago J.S. Lopes ◽

Peter Halfmann ◽

Masato Hatta ◽

...

Keyword(s):

Amino Acid ◽

Case Fatality ◽

Polymerase Activity ◽

Influenza Viruses ◽

Viral Polymerase ◽

Highly Pathogenic ◽

H5n1 Influenza ◽

Increased Risk ◽

Severe Infections ◽

Viral Determinants

To study the influenza viral determinants of pathogenicity, we characterized two highly pathogenic avian H5N1 influenza viruses isolated in Vietnam in 2012 (A/duck/Vietnam/QT1480/2012; QT1480) and 2013 (A/duck/Vietnam/QT1728/2013; QT1728) and found that the activity of their polymerase complexes differed significantly, even though both viruses were highly pathogenic in mice. Further studies revealed that the PA-S343A/E347D mutations reduced viral polymerase activity and mouse virulence when tested in the genetic background of QT1728 virus. In contrast, the PA-343S/347E mutations increased the polymerase activity of QT1480 and the virulence of a low pathogenic H5N1 influenza virus. The PA-343S residue (which alone increased viral polymerase activity and mouse virulence significantly relative to viral replication complexes encoding PA-343A) is frequently found in H5N1 influenza viruses of several subclades; infection with a virus possessing this amino acid may pose an increased risk to humans.IMPORTANCEH5N1 influenza viruses cause severe infections in humans with a case fatality rate that exceeds 50%. The factors that determine the high virulence of these viruses in humans are not fully understood. Here, we identified two amino acid changes in the viral polymerase PA protein that affect the activity of the viral polymerase complex and virulence in mice. Infection with viruses possessing these amino acid changes may pose an increased risk to humans.

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Plasticity of the Influenza Virus H5 HA Protein

mBio ◽

10.1128/mbio.03324-20 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Huihui Kong ◽

David F. Burke ◽

Tiago Jose da Silva Lopes ◽

Kosuke Takada ◽

Masaki Imai ◽

...

Keyword(s):

Influenza Virus ◽

Amino Acid ◽

Mammalian Cells ◽

Influenza A ◽

Viral Antigen ◽

Influenza Viruses ◽

Influenza A Viruses ◽

Highly Pathogenic ◽

Antigenic Properties ◽

Ha Protein

ABSTRACT Since the emergence of highly pathogenic avian influenza viruses of the H5 subtype, the major viral antigen, hemagglutinin (HA), has undergone constant evolution, resulting in numerous genetic and antigenic (sub)clades. To explore the consequences of amino acid changes at sites that may affect the antigenicity of H5 viruses, we simultaneously mutated 17 amino acid positions of an H5 HA by using a synthetic gene library that, theoretically, encodes all combinations of the 20 amino acids at the 17 positions. All 251 mutant viruses sequenced possessed ≥13 amino acid substitutions in HA, demonstrating that the targeted sites can accommodate a substantial number of mutations. Selection with ferret sera raised against H5 viruses of different clades resulted in the isolation of 39 genotypes. Further analysis of seven variants demonstrated that they were antigenically different from the parental virus and replicated efficiently in mammalian cells. Our data demonstrate the substantial plasticity of the influenza virus H5 HA protein, which may lead to novel antigenic variants. IMPORTANCE The HA protein of influenza A viruses is the major viral antigen. In this study, we simultaneously introduced mutations at 17 amino acid positions of an H5 HA expected to affect antigenicity. Viruses with ≥13 amino acid changes in HA were viable, and some had altered antigenic properties. H5 HA can therefore accommodate many mutations in regions that affect antigenicity. The substantial plasticity of H5 HA may facilitate the emergence of novel antigenic variants.

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Three amino acid substitutions in the NS1 protein change the virus replication of H5N1 influenza virus in human cells

Virology ◽

10.1016/j.virol.2018.04.004 ◽

2018 ◽

Vol 519 ◽

pp. 64-73 ◽

Cited By ~ 2

Author(s):

Jing Li ◽

Kun Zhang ◽

Quanjiao Chen ◽

Xiaoshuang Zhang ◽

Yeping Sun ◽

...

Keyword(s):

Influenza Virus ◽

Amino Acid ◽

Virus Replication ◽

Human Cells ◽

Amino Acid Substitutions ◽

Ns1 Protein ◽

H5n1 Influenza Virus ◽

H5n1 Influenza ◽

Protein Change

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The Hag Protein of Moraxella catarrhalis Strain O35E Is Associated with Adherence to Human Lung and Middle Ear Cells

Infection and Immunity ◽

10.1128/iai.71.9.4977-4984.2003 ◽

2003 ◽

Vol 71 (9) ◽

pp. 4977-4984 ◽

Cited By ~ 55

Author(s):

Melissa M. Holm ◽

Serena L. Vanlerberg ◽

Darren D. Sledjeski ◽

Eric R. Lafontaine

Keyword(s):

Cell Lines ◽

Middle Ear ◽

Moraxella Catarrhalis ◽

Human Lung ◽

Primary Cultures ◽

A549 Cells ◽

Pcr Analysis ◽

Nucleotide Sequence Data ◽

Human Lung Cells ◽

Conjunctival Cells

ABSTRACT Previous studies have demonstrated that the Moraxella catarrhalis surface antigen UspA1 is an adhesin for Chang human conjunctival cells. The present report demonstrates that lack of UspA1 expression does not affect the adherence of strain O35E to A549 human lung cells or primary cultures of human middle ear epithelial (HMEE) cells. These results imply that another molecule mediates the adherence of M. catarrhalis to these two cell lines. To identify this adhesin, strain O35E was mutagenized with a transposon and 1,000 mutants were screened in a microcolony formation assay using A549 cells. Nine independent isolates exhibited an 8- to 19-fold reduction in adherence and contained a transposon in the same locus. Nucleotide sequence data and PCR analysis indicated that the transposons were inserted in different locations in the gene encoding the surface protein Hag. Quantitative assays using one representative transposon mutant, O35E.TN2, showed considerably decreased binding to A549 as well as HMEE cells. However, this mutant adhered at wild-type levels to Chang conjunctival cells. These findings suggest that the M. catarrhalis Hag protein is an adhesin for cell lines derived from human lung and middle ear tissues.

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