Phospholipid analysis of two influenza A virus-infected cell lines differing in their viral replication kinetics.

"using this analysis with our rna preparations positive signals were obtained with 7681 and 7924 probes in mock- or iav-infected a549 cells and h292 cells respectively (supplementary data 1).; four hundred microliters of the cell suspension was added to 600 ul of distilled water as well as10 ul of a mixture of internal standard for lc-ms/ms analysis containing standard lysolipids and diacyllipids (as shown in supplementary data 2) and 3 7 ml of a chloroform-methanol mixture (1:2 v/v).; ms/ms parameters of each lysolipid and diacyllipid are shown in supplementary data 2 a549 or h292 cells (10 5 cells/well in 48-well plates) were infected with the a/h3n2/udorn/72 strain at an moi of 3.; iav- and mock-infected h292 or a549 cells at 0 4 8 and 12 hpi were subjected to lipid profile analysis (supplementary data 3).; microarray analysis showed that 7681 genes were significantly expressed in iav-infected and mock-infected a549 cells and that 7924 genes were expressed in h292 cells (supplementary data 1).; table 2 number of genes with expression levels that were altered by at least 4-fold during iav infection h292 cells4 hpi 8 hpi 4 hpi or 8 hpi 4 hpi and 8 hpi total 99 131 181 49 upregulated 44 28 68 4 downregulated 55 103 113 45 a549 cells total 157 311 426 42 upregulated 113 251 346 18 downregulated 44 60 80 24 mrna levels were compared between iav-infected and mock-infected cells using microarray analysis (supplementary data 1).; s) as well as interferon-inducible genes including mx1 mx2 and 2'-5'-oligoadenylate synthetase 1 2 and 3 were upregulated in iav-infected a549 cells at 8 hpi but most of these genes were not significantly affected by iav infection in h292 cells (supplementary data 4 and 5).; table 3 genes related to pathogen recognition with altered levels of expression during iav infection gene description h292 a549 accession no 4 hpi 8 hpi 4 hpi 8 hpi tlrs tlr3 toll-like receptor 3 0 34 0 62 0 17 4 15 nm_003265 myd88 myeloid differentiation primary response gene 88 0 31 0 83 0 36 2 48 nm_002468 rlrs trim21 tripartite motif containing 21 0 02 1 77 0 72 3 74 nm_003141 rig-i dead (asp-glu-ala-asp) box polypeptide 58 0 26 1 02 1 05 4 62 nm_014314 (ddx58) mavs mitochondrial antiviral signaling protein -3 05 -2 74 0 08 0 13 nm_020746 nlrs nlrc5 nlr family card domain containing 5 0 18 0 08 0 44 3 91 nm_032206 nod2 nucleotide-binding oligomerization domain containing 2 0 17 0 50 -0 08 2 61 nm_022162 changes in gene expression of 4-fold or more (shown as log 2 values) as indicated by microarray analysis (supplementary data 1) are shown in bold mrna expression levels were compared between iav-infected cells and their corresponding mock-infected cells at 4 and 8 hpi.; in this study we used cells that differ in their sensitivity to iav infection a549 cells can recognize the invasion of pathogens and induce an antiviral state or express interferon-related genes [ ] (table 2 supplementary data 4) h292 cells do not recognize iav and therefore virus replication disturbance of cellular metabolism and cell death occur more rapidly in these cells.; expression of genes encoding enzymes involved in phospholipid metabolism including sphingomyelinase ceramide synthetase and diacylglycerol lipase did not change significantly in the early stage of virus replication (supplementary data 6) but the relative levels of cer and mag increased with time as iav infection progressed."

"Declarations Conflict of interestThe authors declare that they have no competing interests. Conflict of interest The authors declare that they have no competing interests."

"Funding This work was supported in part by a grant from Yasuda Women’s University."