Influenza A virus NS1 gene mutations F103L and M106I increase replication and virulence

<p>Abstract</p> <p>Background</p> <p>To understand the evolutionary steps required for a virus to become virulent in a new host, a human influenza A virus (IAV), A/Hong Kong/1/68(H3N2) (HK-wt), was adapted to increased virulence in the mouse. Among eleven mutations selected in the NS1 gene, two mutations F103L and M106I had been previously detected in the highly virulent human H5N1 isolate, A/HK/156/97, suggesting a role for these mutations in virulence in mice and humans.</p> <p>Results</p> <p>To determine the selective advantage of these mutations, reverse genetics was used to rescue viruses containing each of the NS1 mouse adapted mutations into viruses possessing the HK-wt NS1 gene on the A/PR/8/34 genetic backbone. Both F103L and M106I NS1 mutations significantly enhanced growth <it>in vitro </it>(mouse and canine cells) and <it>in vivo </it>(BALB/c mouse lungs) as well as enhanced virulence in the mouse. Only the M106I NS1 mutation enhanced growth in human cells. Furthermore, these NS1 mutations enhanced early viral protein synthesis in MDCK cells and showed an increased ability to replicate in mouse interferon β (IFN-β) pre-treated mouse cells relative to rPR8-HK-NS-wt NS1. The double mutant, rPR8-HK-NS-F103L + M106I, demonstrated growth attenuation late in infection due to increased IFN-β induction in mouse cells. We then generated a rPR8 virus possessing the A/HK/156/97 NS gene that possesses 103L + 106I, and then rescued the L103F + I106M mutant. The 103L + 106I mutations increased virulence by >10 fold in BALB/c mice. We also inserted the avian A/Ck/Beijing/1/95 NS1 gene (the source lineage of the A/HK/156/97 NS1 gene) that possesses 103L + 106I, onto the A/WSN/33 backbone and then generated the L103F + I106M mutant. None of the H5N1 and H9N2 NS containing viruses resulted in increased IFN-β induction. The rWSN-A/Ck/Beijing/1/95-NS1 gene possessing 103L and 106I demonstrated 100 fold enhanced growth and >10 fold enhanced virulence that was associated with increased tropism for lung alveolar and bronchiolar tissues relative to the corresponding L103F and I106M mutant.</p> <p>Conclusions</p> <p>The F103L and M106I NS1 mutations were adaptive genetic determinants of growth and virulence in both human and avian NS1 genes in the mouse model.</p

Multifunctional Adaptive NS1 Mutations Are Selected upon Human Influenza Virus Evolution in the Mouse

The role of the NS1 protein in modulating influenza A virulence and host range was assessed by adapting A/Hong Kong/1/1968 (H3N2) (HK-wt) to increased virulence in the mouse. Sequencing the NS genome segment of mouse-adapted variants revealed 11 mutations in the NS1 gene and 4 in the overlapping NEP gene. Using the HK-wt virus and reverse genetics to incorporate mutant NS gene segments, we demonstrated that all NS1 mutations were adaptive and enhanced virus replication (up to 100 fold) in mouse cells and/or lungs. All but one NS1 mutant was associated with increased virulence measured by survival and weight loss in the mouse. Ten of twelve NS1 mutants significantly enhanced IFN-β antagonism to reduce the level of IFN β production relative to HK-wt in infected mouse lungs at 1 day post infection, where 9 mutants induced viral yields in the lung that were equivalent to or significantly greater than HK-wt (up to 16 fold increase). Eight of 12 NS1 mutants had reduced or lost the ability to bind the 30 kDa cleavage and polyadenylation specificity factor (CPSF30) thus demonstrating a lack of correlation with reduced IFN β production. Mutant NS1 genes resulted in increased viral mRNA transcription (10 of 12 mutants), and protein production (6 of 12 mutants) in mouse cells. Increased transcription activity was demonstrated in the influenza mini-genome assay for 7 of 11 NS1 mutants. Although we have shown gain-of-function properties for all mutant NS genes, the contribution of the NEP mutations to phenotypic changes remains to be assessed. This study demonstrates that NS1 is a multifunctional virulence factor subject to adaptive evolution

The development of large-scale de-identified biomedical databases in the age of genomics—principles and challenges

Abstract Contemporary biomedical databases include a wide range of information types from various observational and instrumental sources. Among the most important features that unite biomedical databases across the field are high volume of information and high potential to cause damage through data corruption, loss of performance, and loss of patient privacy. Thus, issues of data governance and privacy protection are essential for the construction of data depositories for biomedical research and healthcare. In this paper, we discuss various challenges of data governance in the context of population genome projects. The various challenges along with best practices and current research efforts are discussed through the steps of data collection, storage, sharing, analysis, and knowledge dissemination

Adaptive mutation in influenza A virus non-structural gene is linked to host switching and induces a novel protein by alternative splicing

Little is known about the processes that enable influenza A viruses to jump into new host species. Here we show that the non-structural protein1 nucleotide substitution, A374G, encoding the D125G(GAT→GGT) mutation, which evolved during the adaptation of a human virus within a mouse host, activates a novel donor splice site in the non-structural gene, hence producing a novel influenza A viral protein, NS3. Using synonymous 125G mutations that do not activate the novel donor splice site, NS3 was shown to provide replicative gain-of-function. The protein sequence of NS3 is similar to NS1 protein but with an internal deletion of a motif comprised of three antiparallel β-strands spanning codons 126 to 168 in NS1. The NS1-125G(GGT) codon was also found in 33 natural influenza A viruses that were strongly associated with switching from avian to mammalian hosts, including human, swine and canine populations. In addition to the experimental human to mouse switch, the NS1-125G(GGT) codon was selected on avian to human transmission of the 1997 H5N1 and 1999 H9N2 lineages, as well as the avian to swine jump of 1979 H1N1 Eurasian swine influenza viruses, linking the NS1 125G(GGT) codon with host adaptation and switching among multiple species

PB2 and Hemagglutinin Mutations Are Major Determinants of Host Range and Virulence in Mouse-Adapted Influenza A Virus▿

Serial mouse lung passage of a human influenza A virus, A/Hong Kong/1/68 (H3N2) (HK-wt), produced a mouse-adapted variant, MA, with nine mutations that was >103.8-fold more virulent. In this study, we demonstrate that MA mutations of the PB2 (D701N) and hemagglutinin (HA) (G218W in HA1 and T156N in HA2) genes were the most adaptive genetic determinants for increased growth and virulence in the mouse model. Recombinant viruses expressing each of the mutated MA genome segments on the HK-wt backbone showed significantly increased disease severity, whereas only the mouse-adapted PB2 gene increased virulence, as determined by the 50% lethal dose ([LD50] >101.4-fold). The converse comparisons of recombinant MA viruses expressing each of the HK-wt genome segments showed the greatest decrease in virulence due to the HA gene (102-fold), with lesser decreases due to the M1, NS1, NA, and PB1 genes (100.3- to 100.8-fold), and undetectable effects on the LD50 for the PB2 and NP genes. The HK PB2 gene did, however, attenuate MA infection, as measured by weight loss and time to death. Replication of adaptive mutations in vivo and in vitro showed both viral gene backbone and host range effects. Minigenome transcription assays showed that PB1 and PB2 mutations increased polymerase activity and that the PB2 D701N mutation was comparable in effect to the mammalian adaptive PB2 E627K mutation. Our results demonstrate that host range and virulence are controlled by multiple genes, with major roles for mutations in PB2 and HA

Influenza A/Hong Kong/156/1997(H5N1) virus NS1 gene mutations F103L and M106I both increase IFN antagonism, virulence and cytoplasmic localization but differ in binding to RIG-I and CPSF30

The genetic basis for avian to mammalian host switching in influenza A virus is largely unknown. The human A/HK/156/1997 (H5N1) virus that transmitted from poultry possesses NS1 gene mutations F103L + M106I that are virulence determinants in the mouse model of pneumonia; however their individual roles have not been determined. The emergent A/Shanghai/patient1/2013(H7N9)-like viruses also possess these mutations which may contribute to their virulence and ability to switch species

Mouse-adapted NS1 mutations affect IFN-β production and viral yield in the mouse lung.

<p>Groups of 6–12 female CD-1 mice were infected intranasally with 1×10⁵ PFU dose of rHK NS mutants or rHK-wt or mock-infected with PBS. Lungs were collected 1 and 3 days pi, homogenized, and quantified for IFN β concentration using a commercial murine IFN β ELISA kit (left panel), and for viral titre by plaque assay on MDCK cells (right panel) Data represent the means ± SD (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; two-tailed unequal variance student's t-test).</p

Model of adaptation of human HK-wt influenza virus to the mouse lung to enhance IFN-β antagonism.

<p>Mouse adaptation involves the selection of NS1 mutants with enhanced IFN-β antagonism, so that upon infection of mouse lung cells <i>in vivo</i> the mutant NS1 gene reduces IFN-β gene (in yellow) activation in the nucleus and thus IFN-β protein (green) production and release. Therefore adaptive mutations prevent the IFN-β induction of an antiviral state that is mediated through binding of to the IFN-α/β receptor (IFNAR). This model proposes that reduced IFN-β production leads to a reduction in the antiviral state and greater virus yield.</p

Adaptive properties of rHK viruses expressing mouse-adapted NS mutations⁵.

1<p>Assessed at respective dpi with maximum weight loss for each respective virus.</p>2<p>Fold change values of lung IFN-β levels 1 day pi.</p>3<p>Relative maximum titre obtained 72 hpi (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031839#pone-0031839-g006" target="_blank">Fig. 6</a>).</p>4<p>Fold change of overall mRNA expression (NP + M1 + NS1; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031839#pone-0031839-g007" target="_blank">Fig. 7</a>) or overall viral protein expression (M1 + NS1; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031839#pone-0031839-g009" target="_blank">Fig. 9c–d</a>), respectively.</p>5<p>All fold values are relative to rHK-wt phenotype; where 0 represents below the limit of detection by the respective assay, with the excetion of % mortality, which is expressed as values independent to rHK-wt. (nd, not determined).</p