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.2011 Jul;85(14):7048-58.
doi: 10.1128/JVI.00417-11. Epub 2011 May 18.

The virulence of 1997 H5N1 influenza viruses in the mouse model is increased by correcting a defect in their NS1 proteins

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The virulence of 1997 H5N1 influenza viruses in the mouse model is increased by correcting a defect in their NS1 proteins

April Spesock et al. J Virol.2011 Jul.

Abstract

The NS1 protein of human influenza A viruses binds the 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), a protein required for 3' end processing of cellular pre-mRNAs, thereby inhibiting production of beta interferon (IFN-β) mRNA. The NS1 proteins of pathogenic 1997 H5N1 viruses contain the CPSF30-binding site but lack the consensus amino acids at positions 103 and 106, F and M, respectively, that are required for the stabilization of CPSF30 binding, resulting in nonoptimal CPSF30 binding in infected cells. Here we have demonstrated that strengthening CPSF30 binding, by changing positions 103 and 106 in the 1997 H5N1 NS1 protein to the consensus amino acids, results in a remarkable 300-fold increase in the lethality of the virus in mice. Unexpectedly, this increase in virulence is not associated with increased lung pathology but rather is characterized by faster systemic spread of the virus, particularly to the brain, where increased replication and severe pathology occur. This increased spread is associated with increased cytokine and chemokine levels in extrapulmonary tissues. We conclude that strengthening CPSF30 binding by the NS1 protein of 1997 H5N1 viruses enhances virulence in mice by increasing the systemic spread of the virus from the lungs, particularly to the brain.

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Figures

Fig. 1.
Fig. 1.
Survival of mice infected with wt HK97 or mutant HK97G2+ virus. BALB/c mice were infected in groups of 5 with 100 PFU (A), 10 PFU (B), 1 PFU (C), or 0.1 PFU (D) of indicated virus and monitored daily. Values ofP were calculated by log-rank (Mantel-Cox) test. Data represent 3 experiments with similar results.
Fig. 2.
Fig. 2.
Replication kinetics of wt HK97 and HK97G2+ viruses in mice. BALB/c mice were infected intranasally with the indicated dose of virus. *,P < 0.05; **,P < 0.01; ***,P < 0.001. For panels A (n = 4), C (n = 3) and D (n = 3), tissue was harvested at the indicated times and plaque assay was performed. For panel E (n = 3), mice were infected with the indicated PFU and tissue was harvested 4 days later for plaque assay. For panels A and C to E, bars indicate means with SD,P values were calculated by Bonferroni's posttest of two-way ANOVA, and data represent 3 experiments with similar results. +,n = 2 due to early mortality. The dotted line indicates the level of detection of the assay in cases where virus was not detected in one or more samples; for data below the detection level of the assay, theP value could not be determined. For panel B, blood from 5 mice was pooled 24 h after infection, plasma was separated, and the EID50 was determined. Bars indicate geometric means with SD for 4 pools (20 mice) per virus.P values were calculated by Student'st test of the log-transformed values.
Fig. 3.
Fig. 3.
Histopathology after infection of mice with wt HK97 and HK97G2+. (A) Hemotoxylin-and-eosin staining of lung sections 6 days after infection with 100 PFU of indicated virus. Magnification, ×10. (B) Caspase-3 staining of lung sections 4 days after infection with 100 PFU of indicated virus. Magnification, ×20. (C) Hemotoxylin-and-eosin staining of brain sections 6 days after infection with 10 PFU of indicated virus. Magnification, ×20.
Fig. 4.
Fig. 4.
Identification of cells in lungs by flow cytometry. Lung cells were isolated, stained, and analyzed by flow cytometry. Using the FlowJo software program, data were gated to remove doublets and debris smaller than cells (not shown). Surface marker staining was then used to identify immune cell subsets. (A) Polymorphonuclear neutrophils (PMN) were gated as Ly6-G+ Ly6-C+. Nonneutrophils were then gated by CD11c and major histocompatibility complex class II (MHC II) expression to identify double-negative cells (DN), double-intermediate cells (DI), macrophages (Mac), and dendritic cells (DC). DN cells were further gated by CD11b and Ly6-C expression to identify inflammatory monocytes (InMo) and constitutive monocytes (ConMo). Macrophages were further gated by CD11b expression to identify alveolar macrophages (AM) and exudate macrophages (exMac). (B) Cells were gated as CD11b negative to exclude myeloid lineage cells also expressing lymphocyte markers. Then, natural killer (NK), B cells, and CD4+ and CD8+ T cells were identified as indicated.
Fig. 5.
Fig. 5.
Inflammatory cells recruited to the lungs of mice after infection with wt HK97 and HK97G2+. BALB/c mice in groups of 5 were infected with 100 PFU of virus, including a group of uninfected mice as a control. Cell populations in the lung were identified as described in the legend for Fig. 4. The absolute numbers of different immune cell subsets found in the lungs are shown. Bars indicate means with SD. *,P < 0.05;P < 0.01; ***,P < 0.001 by unpaired two-tailed Student'st test of results for wt HK97 compared to those for HK97G2+ at indicated times. Similar numbers of cells were found in uninfected mice and in mice 1 day after infection, except for alveolar macrophages (F), where the mean number of cells found in uninfected mice is indicated by the dotted line.
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