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User name	Nicolas Tchitchek
Email	nicolas.tchitchek@ihes.fr
Phone / Fax number	0033610887604 /
Location	Institut des Hautes Ã‰tudes Scientifiques (Systems Epigenomics Group) - 35, route de Chartres - 91440 Bures-sur-Yvette, FRANCE

Scientific description

From abstract of the published manuscript

While pandemic 2009 H1N1 influenza A viruses were responsible for numerous severe infections in humans, these viruses do not typically cause corresponding severe disease in mammalian models. However, the generation of a virulent 2009 H1N1 virus following serial lung passage in mice has allowed for the modeling of human lung pathology in this species. Genetic determinants of mouse-adapted 2009 H1N1 viral pathogenicity have been identified, but the molecular and signaling characteristics of the host response following infection with this adapted virus have not been described. Here, we compared the gene-expression response following infection of mice with A/CA/04/2009 (CA/04) or the virulent mouse-adapted strain (MA-CA/04). Microarray analysis revealed that increased pathogenicity of MA-CA/04 was associated with: (1) early and sustained inflammatory and interferon response that could be driven in part by interferon regulatory factors (IRFs) and increased NFÎºB activation, as well as inhibition of the negative regulator TRIM24, (2) early and persistent infiltration of immune cells, including inflammatory macrophages, and (3) the absence of activation of lipid metabolism later in infection, that may be mediated by nuclear receptors inhibition, including PPARG, HNF1A and 4A, with pro-inflammatory consequences. Further investigation of these signatures in the host response to other H1N1 viruses of varied pathogenicity confirmed their general relevance for virulence of influenza virus and suggested that lung response to MA-CA/04 virus was similar to that following lethal H1N1 r1918 influenza virus. This study links differential activation of IRFs, nuclear receptors, and macrophage infiltration with influenza virulence in vivo.

Technical description

From the material and methods section of the published manuscript

VIRUSES. 
Viral stocks for A/California/04/2009, mouse-adapted MA1-A/California/04/2009 (generously provided by Richard Webby, St. Judeâ€™s Childrenâ€™s Hospital, Memphis, Tennessee, (16)), A/Mexico/4482/09 (Mex/4482) and A/Brisbane/59/07 (Brisbane/59/07) were propagated in Madin-Darby canine kidney (MDCK) cells for 48 h at 37 oC. A/New Jersey/8/76 (NJ/8/76) was propagated in the allantoic cavity of 10-day-old embryonated hensâ€™ eggs for stock preparation. The supernatants were clarified by centrifugation, aliquoted, and stored at -70oC. Virus titers were determined by standard plaque assay on MDCK cells (55). The identity of virus genes was confirmed by sequence analysis to verify that no inadvertent mutations were present during the generation of virus stocks. 

MOUSE EXPERIMENTS. 
Six-to-eight-week-old female BALB/c mice were purchased from Charles River Laboratories (Wilmington, MA). All 2009 H1N1 in vivo experiments were performed under biosafety level 3 enhanced (BSL3+) containment. Mice were anesthetized with 2,2,2-tribromoethanol in tert-amyl alcohol (Avertin; Sigma-Aldrich, St. Louis, MO) and intranasally (i.n.) inoculated with 50 Î¼l of phosphate-buffered saline (PBS; Mock) or with 106 pfu of virus in a 50 Î¼l volume. Six animals in each group were monitored for morbidity and mortality as measured by weight loss and survival over a period of 16 days. Viral titers were determined in the lungs, spleen and brain on days 1, 3 and 5 post-inoculation in nine animals per group (three animals for each time point). An additional nine animals were used for transcriptional profiling, again three animals per time point. Histopathology was performed on another batch of nine mice for each virus, with three animals used per time point. Animal research was conducted under the guidance of the CDCâ€™s Institutional Animal Care and Use Committee in an animal facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.

MICROARRAY EXPERIMENTS. 
Transcriptional profiles were determined by microarray analysis of RNA isolated from lung tissue from three individual animals per group at days 1, 3 and 5 post-infection (pi). Total RNA extraction from lung samples was performed as previously described (4). Probe labeling and microarray slide hybridization for each biological replicate was performed using Whole Mouse Genome Microarray Kit (G4122-60520; Agilent Technologies) according to manufacturerâ€™s instructions. Raw microarray data have been deposited in NCBI's Gene Expression Omnibus (15) under GEO Series accession number GSE36328 and are also accessible through the Systems Virology website (http://www.systemsvirology.org).

Reference

Implication of inflammatory macrophages, nuclear receptors and interferon regulatory factors in increased virulence of pandemic 2009 H1N1 influenza A virus after host adaptation. J Virol. 2012 Apr 24. Josset L, Belser JA, Pantin-Jackwood MJ, Chang JH, Chang ST, Belisle SE, Tumpey TM, Katze MG.

Pubmed : http://www.ncbi.nlm.nih.gov/pubmed/22532695