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. 2016 Aug;205(4):381-95.
doi: 10.1007/s00430-016-0461-2. Epub 2016 Jun 11.

PA-X-associated early alleviation of the acute lung injury contributes to the attenuation of a highly pathogenic H5N1 avian influenza virus in mice

Jiao Hu  1   2 , Yiqun Mo  1   2 , Zhao Gao  1   2 , Xiaoquan Wang  1   2 , Min Gu  1   2 , Yanyan Liang  1   2 , Xin Cheng  1   2 , Shunlin Hu  1   2 , Wenbo Liu  1   2 , Huimou Liu  1   2 , Sujuan Chen  1   2 , Xiaowen Liu  1   2 , Daxing Peng  1   2 , Xiufan Liu  3   4
Affiliations

PA-X-associated early alleviation of the acute lung injury contributes to the attenuation of a highly pathogenic H5N1 avian influenza virus in mice

Jiao Hu et al. Med Microbiol Immunol. 2016 Aug.

Abstract

PA-X is a novel discovered accessory protein encoded by the PA mRNA. Our previous study demonstrated that PA-X decreases the virulence of a highly pathogenic H5N1 strain A/Chicken/Jiangsu/k0402/2010 in mice. However, the underlying mechanism of virulence attenuation associated with PA-X is still unknown. In this study, we compared two PA-X-deficient mutant viruses and the parental virus in terms of induction of pathology and manipulation of host response in the mouse lung, stimulation of cell death and PA nuclear accumulation. We first found that down-regulated PA-X expression markedly aggravated the acute lung injury of the infected mice early on day 1 post-infection (p.i.). We then determined that loss of PA-X expression induced higher levels of cytokines, chemokines and complement-derived peptides (C3a and C5a) in the lung, especially at early time point's p.i. In addition, in vitro assays showed that the PA-X-deficient viruses enhanced cell death and increased expression of reactive oxygen species (ROS) in mammalian cells. Moreover, we also found that PA nuclear accumulation of the PA-X-null viruses accelerated in MDCK cells. These results demonstrate that PA-X decreases the level of complement components, ROS, cell death and inflammatory response, which may together contribute to the alleviated lung injury and the attenuation of the virulence of H5N1 virus in mice.

Keywords: ALI; Highly pathogenic H5N1 AIV; Mice; PA-X; Pathogenesis.

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Figures

Fig. 1
Fig. 1
Pathogenicity of recombinant viruses in 6-week-old BABL/c mice. a Mean weight loss of mice infected with 105.0 EID50 of r-CK10, CK-PAX5 and CK-PAX3 (n = 5). Mice were humanly killed when they lost ≥25 % of their initial body weight. Error bar represents stand deviation (SD). b Survival rate of mice infected with the indicated viruses or mock PBS (n = 5)
Fig. 2
Fig. 2
Representative histopathological changes in H&E (hematoxylin and eosin)-stained lung tissues on day 1 p.i. and viral replication in the mouse lung a CK-PAX3-infected mouse lung. Severe interstitial pneumonia and bronchopneumonia characterized by serious thickened alveolar wall, disappeared alveolar structure, severe infiltration of necrotic alveolar epithelial cells and inflammatory cells in the alveolar interstitial (shown as asterisk) and severe necrosis and desquamation of the bronchial epithelial cells (shown as). b CK-PAX5-infected mouse lung. Moderate interstitial pneumonia and bronchopneumonia with mild thickened alveolar wall (shown as triangle), necrosis and desquamation of the bronchial epithelial cells and accompany by infiltration of inflammatory cells (shown as black arrow) and inflammatory cells infiltration around the bronchus (shown as white arrow). c r-CK10-infected mouse lung. Mild bronchopneumonia characterized by necrosis and desquamation of the bronchial epithelial cells and a small quantity of inflammatory cells infiltration (shown as black arrow). d Mock control. No obvious histopathology was observed in the PBS-infected mouse lung. Bar = 100 μm. e Histological changes (expressed as scores). Value shown are the mean ± SD of the results from six individuals (*p < 0.05). f Viral replication in the mouse lung. Value shown are the mean ± SD of the results from six individuals (*p < 0.05). Asterisk indicates significant difference between the PA-X-deficient virus (CK-PAX3 or CK-PAX5) and the parental virus
Fig. 3
Fig. 3
Cytokines and chemokines expression in the mouse lung. Groups of 18 6-week-old female BABL/c mice were intranasally infected with 105.0 EID50 of the indicated virus strain or received PBS intranasally (Control). On day 1, 2 and 3 p.i., six mice of each group were euthanized; the lungs were collected for homogenate preparation. Lung concentrations of cytokines were measured by ELISA. Cytokine or chemokine expression was expressed as the mean concentration ± SEM. *p < 0.05 and **p < 0.01, asterisk or double asterisk indicates significant difference between the PA-X-deficient virus (CK-PAX3 or CK-PAX5) and the parental virus
Fig. 4
Fig. 4
C3a and C5a expression in the mouse lung. Groups of 18 6-week-old female BABL/c mice were intranasally infected with 105.0 EID50 of the indicated virus strain or received PBS intranasally (Mock). On day 1, 2 and 3 p.i., six mice of each group were euthanized; the lungs were collected for homogenate preparation. ELISA Kit was used to analyze the expression of the C3a and C5a complements. Data are presented as mean ± SEM. *p < 0.05 and **p < 0.01, when compared to the indicated group
Fig. 5
Fig. 5
Apoptosis and necrosis detected by annexin V and PI double staining in MDCK and A549 cells. At 12 and 24 h p.i., both infected and non-infected MDCK cells (a) and A549 cells (d) were harvested and stained with FITC annexin V and PI and then subjected to flow cytometry. Apoptotic cells are those with high annexin V and low PI staining, whereas necrotic cells are highly stained with both annexin V and PI. Values shown are the proportion of dead cells among the total number of cells analyzed. b–e Quantitative analysis of apoptosis and necrosis induced by the recombinants by flow cytometry in MDCK cells (b and c) and A549 cells (e and f). Values shown are the average proportion of apoptotic or necrotic cells of the total number of cells ± SD of the results from three independent experiments. *p < 0.05 and **p < 0.01, when compared to the indicated group
Fig. 6
Fig. 6
ROS expression in MDCK cells. ROS levels were determined using the fluorescent marker 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) according to the manufacturer’s instructions. Fluorescence intensity was analyzed with a FACSAria flow cytometer with the FACSDiva software. Values shown are the average expression levels ± SD of the results from three independent experiments. *p < 0.05 and **p < 0.01, when compared to the indicated group
Fig. 7
Fig. 7
PA nuclear accumulation of the recombinants in MDCK cells. MDCK cells were infected with r-CK10 (a), CK-PAX5 (b) or CK-PAX3 (c) at a MOI of 2; cell cultures were fixed and processed for immunofluorescence observation at the indicated times. Cell nuclei were stained with DAPI. d The PA nuclear accumulation in the infected cells was determined as the ratio of cells showing red fluorescence in the nucleus to the total number of cells counted. The values shown are mean ± SD of results for three independent experiments (*p < 0.05). Asterisk indicates significant difference between the PA-X-deficient virus CK-PAX3 or CK-PAX5 and the parental virus
Fig. 8
Fig. 8
Function and effects of the influenza A virus PA-X protein. This picture illustrates that the PA-X protein inhibits the influenza viral replication, virus-induced inflammation (including the cytokine and complement response, and inflammatory cells infiltration), cell death and ROS expression. The pleiotropic effects of PA-X protein contribute to the alleviation of the acute lung injury induced by influenza virus and the subsequently reduced mortality of the infected mouse
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