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Adaptive mutations of neuraminidase stalk truncation and deglycosylation confer enhanced pathogenicity of influenza A viruses.

ABSTRACT: It has been noticed that neuraminidase (NA) stalk truncation has arisen from evolutionary adaptation of avian influenza A viruses (IAVs) from wild aquatic birds to domestic poultry. We identified this molecular alteration after the adaptation of a 2009 pandemic H1N1 virus (pH1N1) in BALB/c mice. The mouse-adapted pH1N1 lost its eight consecutive amino acids including one potential N-linked glycosite from the NA stalk region. To explore the relationship of NA stalk truncation or deglycosylation with viral pathogenicity changes, we generated NA stalk mutant viruses on the pH1N1 backbone by reverse genetics. Intriguingly, either NA stalk truncation or deglycosylation changed pH1N1 into a lethal virus to mice by resulting in extensive pathologic transformation in the mouse lungs and systemic infection affecting beyond the respiratory organs in mice. The increased pathogenicity of these NA stalk mutants was also reproduced in ferrets. In further investigation using a human-infecting H7N9 avian IAV strain, NA stalk truncation or deglycosylation enhanced the replication property and pathogenicity of H7N9 NA stalk mutant viruses in the same mouse model. Taken together, our results suggest that NA stalk truncation or deglycosylation can be the pathogenic determinants of seasonal influenza viruses associated with the evolutionary adaptation of IAVs.

SUBMITTER: Park S

PROVIDER: S-EPMC5589767 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Adaptive mutations of neuraminidase stalk truncation and deglycosylation confer enhanced pathogenicity of influenza A viruses.

Park Sehee S Il Kim Jin J Lee Ilseob I Bae Joon-Yong JY Yoo Kirim K Nam Misun M Kim Juwon J Sook Park Mee M Song Ki-Joon KJ Song Jin-Won JW Kee Sun-Ho SH Park Man-Seong MS

Scientific reports 20170907 1

It has been noticed that neuraminidase (NA) stalk truncation has arisen from evolutionary adaptation of avian influenza A viruses (IAVs) from wild aquatic birds to domestic poultry. We identified this molecular alteration after the adaptation of a 2009 pandemic H1N1 virus (pH1N1) in BALB/c mice. The mouse-adapted pH1N1 lost its eight consecutive amino acids including one potential N-linked glycosite from the NA stalk region. To explore the relationship of NA stalk truncation or deglycosylation w ...[more]

PMID: 28883554

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Project description:Oligonucleotides corresponding to neuraminidase (NA) stalk motifs that have been associated with enhanced influenza virulence have been identified using surface-enhanced Raman spectroscopy (SERS). 5'-Thiolated ssDNA oligonucleotides were immobilized onto a hexadecyltrimethylammonium bromide (CTAB) coated Au nanoparticles (AuNP). Three synthetic RNA sequences corresponding to specific amino acid deletions in the influenza NA stalk region were attached to the CTAB-modified AuNPs. Two of these sequences were specific to sequences with amino acid deletions associated with increased virulence, and one was a low virulence sequence with no amino acid deletions. Hybridization of synthetic matched and mismatched DNA-RNA complexes were detected based on the intrinsic SERS spectra. In addition, this platform was used to analyze RNA sequences isolated from laboratory grown influenza viruses having the NA stalk motif associated with enhanced virulence, including A/WSN/33/H1N1, A/Anhui/1/2005/H5N, and A/Vietnam/1203/2004/H5N1 strains. Multivariate feature selection methods were employed to determine the specific wavenumbers in the Raman spectra that contributed the most information for class discrimination. A one-way analysis of variance (ANOVA) test identified 884 and 1196 wavenumbers as being highly significant in the high and low virulence spectra, respectively (p < 0.01). A post-hoc Tukey Honestly Significance Difference (HSD) test identified the wavenumbers that played a major role in differentiating the DNA-RNA hybrid classes. An estimate of the spectral variability, based on the Wilcoxon rank sum test, found the major source of variation to be predominately between the different classes, and not within the classes, thus confirming that the spectra reflected real class differences and not sampling artifacts. The multivariate classification methods partial least squares discriminant analysis (PLS-DA) and support vector machine discriminant analysis (SVM-DA) were able to distinguish between different NA stalk-motifs linked to NA-enhanced influenza virus virulence (NA-EIV) with >95% sensitivity and specificity in both synthetic RNA sequences as well as the isolated viral RNA. This study demonstrates the feasibility of SERS for direct identification of influenza NA stalk mutations associated with virulence without sample amplification or labeling.

Project description:Influenza viruses express two surface glycoproteins, the hemagglutinin (HA) and the neuraminidase (NA). Anti-NA antibodies protect from lethal influenza virus challenge in the mouse model and correlate inversely with virus shedding and symptoms in humans. Consequently, the NA is a promising target for influenza virus vaccine design. Current seasonal vaccines, however, poorly induce anti-NA antibodies, partly because of the immunodominance of the HA over the NA when the two glycoproteins are closely associated. To address this issue, here we investigated whether extending the stalk domain of the NA could render it more immunogenic on virus particles. Two recombinant influenza viruses based on the H1N1 strain A/Puerto Rico/8/1934 (PR8) were rescued with NA stalk domains extended by 15 or 30 amino acids. Formalin-inactivated viruses expressing wild-type NA or the stalk-extended NA variants were used to vaccinate mice. The virus with the 30-amino-acid stalk extension induced significantly higher anti-NA IgG responses (characterized by increased in vitro antibody-dependent cellular cytotoxicity [ADCC] activity) than the wild-type PR8 virus, while anti-HA IgG levels were unaffected. Similarly, extending the stalk domain of the NA of a recent H3N2 virus enhanced the induction of anti-NA IgGs in mice. On the basis of these results, we hypothesize that the subdominance of the NA can be modulated if the protein is modified such that its height surpasses that of the HA on the viral membrane. Extending the stalk domain of NA may help to enhance its immunogenicity in influenza virus vaccines without compromising antibody responses to HA.IMPORTANCE The efficacy of influenza virus vaccines could be improved by enhancing the immunogenicity of the NA protein. One of the reasons for its poor immunogenicity is the immunodominance of the HA over the NA in many seasonal influenza virus vaccines. Here we demonstrate that, in the mouse model, extending the stalk domain of the NA protein can enhance its immunogenicity on virus particles and overcome the immunodominance of the HA without affecting antibody responses to the HA. The antibody repertoire is broadened by the extended NA and includes additional ADCC-active antibodies. Our findings may assist in the efforts toward more effective influenza virus vaccines.

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Adaptive mutations of neuraminidase stalk truncation and deglycosylation confer enhanced pathogenicity of influenza A viruses.

Publications

Adaptive mutations of neuraminidase stalk truncation and deglycosylation confer enhanced pathogenicity of influenza A viruses.

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