Functional & Integrative Genomics

, Volume 18, Issue 4, pp 411–424 | Cite as

Integrated analysis of mRNA-seq and miRNA-seq for host susceptibilities to influenza A (H7N9) infection in inbred mouse lines

  • Suying Bao
  • Lilong Jia
  • Xueya Zhou
  • Zhi-Gang Zhang
  • Hazel Wai Lan Wu
  • Zhe Yu
  • Gordon Ng
  • Yanhui Fan
  • Dana S. M. Wong
  • Shishu Huang
  • Kelvin Kai Wang To
  • Kwok-Yung Yuen
  • Man Lung Yeung
  • You-Qiang Song
Original Article


Host genetic factors play an important role in diverse host outcomes after influenza A (H7N9) infection. Studying differential responses of inbred mouse lines with distinct genetic backgrounds to influenza virus infection could substantially increase our understanding of the contributory roles of host genetic factors to disease severity. Here, we utilized an integrated approach of mRNA-seq and miRNA-seq to investigate the transcriptome expression and regulation of host genes in C57BL/6J and DBA/2J mouse strains during influenza virus infection. The differential pathogenicity of influenza virus in C57BL/6J and DBA/2J has been fully demonstrated through immunohistochemical staining, histopathological analyses, and viral replication assessment. A transcriptional molecular signature correlates to differential host response to infection has been uncovered. With the introduction of temporal expression pattern analysis, we demonstrated that host factors responsible for influenza virus replication and host–virus interaction were significantly enriched in genes exhibiting distinct temporal dynamics between different inbred mouse lines. A combination of time-series expression analysis and temporal expression pattern analysis has provided a list of promising candidate genes for future studies. An integrated miRNA regulatory network from both mRNA-seq and miRNA-seq revealed several regulatory modules responsible for regulating host susceptibilities and disease severity. Overall, a comprehensive framework for analyzing host susceptibilities to influenza infection was established by integrating mRNA-seq and miRNA-seq data of inbred mouse lines. This work suggests novel putative molecular targets for therapeutic interventions in seasonal and pandemic influenza.


Influenza (H7N9) virus Host susceptibilities mRNA-seq miRNA-seq Inbred mouse lines Temporal dynamics 



non-negative matrix factorization


principle component analysis


differentially expressed genes


dynamic time warping maximal information coefficient


miRNA-mRNA interaction weight

GGIW matrix

gene–gene interaction weight


Authors’ contributions

Y.Q.S, M.L.Y., K.Y.Y., K.K.W.T., S.H., and S.B. designed research; L.J., Z.G.Z., H.W.L.W., Z.Y., NG., and D.SM.W. performed experiments; S.B., X.Z, and Y.F. analyzed results; and Y.Q.S, M.L.Y., S.B., and X.Z wrote the paper. All authors read and approved the final manuscript.


This work was supported by grants from the Health and Medical Research Fund of Hong Kong Government (01121726, RRG-08). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Compliance with ethical standards

Ethics approval and consent to participate

This study was approved by the Committee of the Use of Live Animals in Teaching and Research (CULATR 3275-14).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Supplementary material

10142_2018_602_MOESM1_ESM.doc (2.5 mb)
Additional File 1 Supplementary figures. Supplementary Fig. S1 showed error bars of virus load in each strain at each day, with mean values and standard deviations calculated from the virus load of all biological replicates at the same day. Supplementary Fig. S2 showed the differential expression levels (log2-fold change) of 10 interferon-stimulated genes between infected and mock-treated mice at day 3. All transcripts for the same gene were presented. Different strains were represented by different colours as shown in the legend. Supplementary Fig. S3 showed the characteristics of genes differentially expressed between C57BL/6J and DBA/2J over time. (A) Heatmap of genes differentially expressed between strains (strain-specific DEGs) showed distinct expression patterns between C57BL/6J and DBA/2J mice. (B) PCA of all samples based on normalized counts of strain-specific DEGs. PC2 revealed distinct transcriptome profile of these DEGs for the two strains due to their genetic background explaining around 26% of the expression variation. Supplementary Fig. S4 demonstrated that genes responsible for host resistance were enriched in DTW-MIC similarities between 0.2-0.25. (A) Histogram of DTW-MIC similarity scores showed two similarity peaks as: between 0.2-0.25 and between 0.7-0.75. (B) PCA of all samples based on normalized counts of genes within different DTW-MIC similarity ranges. The expression variation across samples based on genes with similarity scores between 0.2-0.25 mainly explained the difference from host genetic background, whereas the expression variation based on genes with similarity scores higher than 0.25 were mainly attributed to host response to influenza infection. Supplementary Fig. S5-S6 showed the temporal dynamics of representative genes enriched in cell cycle and ribosome in C57BL/6J and DBA/2J. Supplementary Fig. S7 demonstrated the differential characteristics of time course strain-specific DEGs with high and low temporal similarities between C57BL/6J and DBA/2J. (A) DTW-MIC distribution of all strain-specific DEGs over time. (B) PCA of all samples based on the transcription profiles of strain-specific DEGs with DTW-MIC scores lower than 0.25 (left) and higher than 0.7 (right) respectively C). The same transcription profiles used in B), but presented as heatmaps (DOC 2565 kb)
10142_2018_602_MOESM2_ESM.xls (3.8 mb)
Additional File 2 Supplementary Tables. Supplementary Table S1: Strain-specific DEGs over time. Supplementary Table S2: The DTW-MIC similarity scores and permutation p-values of all genes. Supplementary Table S3: Summary statistics of DTW-MIC similarities. Supplementary Table S4: Host factors responsible for influenza virus replication show significantly differential DTW-MIC similarities between C57BL/6J and DBA/2J. Supplementary Table S5: Host factors interacting with influenza virus show significantly differential DTW-MIC similarities between C57BL/6J and DBA/2J. Supplementary Table S6: The distribution of DTW-MIC similarity scores of host factors either responsible for influenza virus replication or interact with virus. Supplementary Table S7: The list of candidate genes with both significant differential expression levels and distinct temporal expression patterns between C57BL/6J and DBA/2J. Supplementary Table S8: Functional enrichment analysis of candidate genes with both significant differential expression level and distinct temporal expression pattern between C57BL/6J and DBA/2J. Supplementary Table S9: The list of candidate genes which show distinct temporal expression patterns between C57BL/6J and DBA/2J but have no significant differential expression levels between the two mouse strains. Supplementary Table S10: Four clusters of sig.low.simi genes according to the differential expression levels between infected and mock-treated mice over time in both C57BL/6J and DBA/2J strains (XLS 3855 kb)
10142_2018_602_MOESM3_ESM.xlsx (508 kb)
Additional File 3 The main functional modules in the interaction network of sig.low.simi genes from clusters 1, 2 and 3 (XLSX 508 kb)
10142_2018_602_MOESM4_ESM.xlsx (35 kb)
Additional File 4 The modules in the integrated miRNA regulatory network (XLSX 35 kb)


  1. Agarwal V, Bell GW, Nam JW, Bartel DP (2015) Predicting effective microRNA target sites in mammalian mRNAs. Elife 4:4. CrossRefGoogle Scholar
  2. Alberts R, Lu L, Williams RW, Schughart K (2011) Genome-wide analysis of the mouse lung transcriptome reveals novel molecular gene interaction networks and cell-specific expression signatures. Respir Res 12:61. CrossRefPubMedPubMedCentralGoogle Scholar
  3. Bagga S, Bouchard MJ (2014) Cell cycle regulation during viral infection Methods. Mol Biol 1170:165–227. Google Scholar
  4. Bao S, Zhou X, Zhang L, Zhou J, To KKW, Wang B, Wang L, Zhang X, Song YQ (2013) Prioritizing genes responsible for host resistance to influenza using network approaches. BMC Genomics 14:816. CrossRefPubMedPubMedCentralGoogle Scholar
  5. Beck MA et al (2001) Selenium deficiency increases the pathology of an influenza virus infection. FASEB J 15:1481–1483CrossRefPubMedGoogle Scholar
  6. Boivin GA, Pothlichet J, Skamene E, Brown EG, Loredo-Osti JC, Sladek R, Vidal SM (2012) Mapping of clinical and expression quantitative trait loci in a sex-dependent effect of host susceptibility to mouse-adapted influenza H3N2/HK/1/68. J Immunol 188:3949–3960. CrossRefPubMedGoogle Scholar
  7. Boon AC et al (2009) Host genetic variation affects resistance to infection with a highly pathogenic H5N1 influenza A virus in mice. J Virol 83:10417–10426. CrossRefPubMedPubMedCentralGoogle Scholar
  8. Brass AL, Huang IC, Benita Y, John SP, Krishnan MN, Feeley EM, Ryan BJ, Weyer JL, van der Weyden L, Fikrig E, Adams DJ, Xavier RJ, Farzan M, Elledge SJ (2009) The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile Virus, and Dengue Virus. Cell 139:1243–1254. CrossRefPubMedPubMedCentralGoogle Scholar
  9. Castro A, Bernis C, Vigneron S, Labbe JC, Lorca T (2005) The anaphase-promoting complex: a key factor in the regulation of cell cycle. Oncogene 24:314–325. CrossRefPubMedGoogle Scholar
  10. Chen CH et al (2010) The essentiality of alpha-2-macroglobulin in human salivary innate immunity against new H1N1 swine origin influenza A virus. Proteomics 10:2396–2401. CrossRefPubMedPubMedCentralGoogle Scholar
  11. Chen EY et al (2013) Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool. BMC Bioinformatics 14:128. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Chen M et al (2016) TRIM14 inhibits cGAS degradation mediated by selective autophagy receptor p62 to promote innate immune responses. Mol Cell 64:105–119. CrossRefPubMedGoogle Scholar
  13. Chen Y et al (2015) Functional variants regulating LGALS1 (galectin 1) expression affect human susceptibility to influenza A(H7N9). Sci Rep 5:8517. CrossRefPubMedPubMedCentralGoogle Scholar
  14. Cheng ZS et al (2015) Identification of TMPRSS2 as a susceptibility gene for severe 2009 pandemic A(H1N1) influenza and A(H7N9) influenza. J Infect Dis 212:1214–1221. CrossRefPubMedGoogle Scholar
  15. Cheung CY et al (2012) H5N1 virus causes significant perturbations in host proteome very early in influenza virus-infected primary human monocyte-derived macrophages. J Infect Dis 206:640–645. CrossRefPubMedPubMedCentralGoogle Scholar
  16. Chou YC, Lai MM, Wu YC, Hsu NC, Jeng KS, Su WC (2015) Variations in genome-wide RNAi screens: lessons from influenza research. J Clin Bioinf 5:2. CrossRefGoogle Scholar
  17. De Santo C et al (2008) Invariant NKT cells reduce the immunosuppressive activity of influenza A virus-induced myeloid-derived suppressor cells in mice and humans. J Clin Invest 118:4036–4048. CrossRefPubMedPubMedCentralGoogle Scholar
  18. Dhillon IS, Sra S (2005) Generalized nonnegative matrix approximations with Bregman divergences.
  19. Dierkes R, Warnking K, Liedmann S, Seyer R, Ludwig S, Ehrhardt C (2014) The Rac1 inhibitor NSC23766 exerts anti-influenza virus properties by affecting the viral polymerase complex activity. PloS One 9:e88520. CrossRefPubMedPubMedCentralGoogle Scholar
  20. Dufner A, Thomas G (1999) Ribosomal S6 kinase signaling and the control of translation. Exp Cell Res 253:100–109. CrossRefPubMedGoogle Scholar
  21. Ehrhardt C et al (2007) Influenza A virus NS1 protein activates the PI3K/Akt pathway to mediate antiapoptotic signaling responses. J Virol 81:3058–3067. CrossRefPubMedPubMedCentralGoogle Scholar
  22. Fan N, Wang J (2016) MicroRNA 34a contributes to virus-mediated apoptosis through binding to its target gene Bax in influenza A virus infection. Biomed Pharmacother 83:1464–1470. CrossRefPubMedGoogle Scholar
  23. Fan Y et al (2017) Cell cycle-independent role of cyclin D3 in host restriction of influenza virus infection. J Biol Chem 292:5070–5088. CrossRefPubMedPubMedCentralGoogle Scholar
  24. Ferris MT, Aylor DL, Bottomly D, Whitmore AC, Aicher LD, Bell TA, Bradel-Tretheway B, Bryan JT, Buus RJ, Gralinski LE, Haagmans BL, McMillan L, Miller DR, Rosenzweig E, Valdar W, Wang J, Churchill GA, Threadgill DW, McWeeney SK, Katze MG, Pardo-Manuel de Villena F, Baric RS, Heise MT (2013) Modeling host genetic regulation of influenza pathogenesis in the collaborative cross. PLoS Pathogens 9:e1003196. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Fujioka Y, Tsuda M, Hattori T, Sasaki J, Sasaki T, Miyazaki T, Ohba Y (2011) The Ras-PI3K signaling pathway is involved in clathrin-independent endocytosis and the internalization of influenza viruses. PloS One 6:e16324. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Fujioka Y et al (2013) A Ca(2+)-dependent signalling circuit regulates influenza A virus internalization and infection. Nat Commun 4:2763. CrossRefPubMedGoogle Scholar
  27. Gaur P, Munjhal A, Lal SK (2011) Influenza virus and cell signaling pathways. Med Sci Monit 17:RA148–RA154CrossRefPubMedPubMedCentralGoogle Scholar
  28. Geiss GK, An MC, Bumgarner RE, Hammersmark E, Cunningham D, Katze MG (2001) Global impact of influenza virus on cellular pathways is mediated by both replication-dependent and -independent events. J Virol 75:4321–4331. CrossRefPubMedPubMedCentralGoogle Scholar
  29. Han J et al (2013) Clinical presentation and sequence analyses of HA and NA antigens of the novel H7N9 viruses. Emerg Microbes Infect 2:e23. CrossRefPubMedPubMedCentralGoogle Scholar
  30. Hao LH et al (2008) Drosophila RNAi screen identifies host genes important for influenza virus replication. Nature 454:890–U846. CrossRefPubMedPubMedCentralGoogle Scholar
  31. He Y et al (2010) Influenza A virus replication induces cell cycle arrest in G0/G1 phase. J Virol 84:12832–12840. CrossRefPubMedPubMedCentralGoogle Scholar
  32. Heilman DW, Green MR, Teodoro JG (2005) The anaphase promoting complex: a critical target for viral proteins and anti-cancer drugs. Cell Cycle 4:560–563CrossRefPubMedGoogle Scholar
  33. Jiang W, Wang Q, Chen S, Gao S, Song L, Liu P, Huang W (2013) Influenza A virus NS1 induces G0/G1 cell cycle arrest by inhibiting the expression and activity of RhoA protein. J Virol 87:3039–3052. CrossRefPubMedPubMedCentralGoogle Scholar
  34. Karlas A et al (2010) Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication. Nature 463:818–U132. CrossRefPubMedGoogle Scholar
  35. Kuleshov MV et al (2016) Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. Nucleic Acids Res 44:W90–W97. CrossRefPubMedPubMedCentralGoogle Scholar
  36. Lakadamyali M, Rust MJ, Zhuang X (2004) Endocytosis of influenza viruses Microbes and infection / Institut Pasteur. Microbes Infect 6(10):929–936CrossRefPubMedPubMedCentralGoogle Scholar
  37. Lam TT-Y et al. (2013) The genesis and source of the H7N9 influenza viruses causing human infections in China. Nature advance online publication. doi:
  38. Lee ASY, Burdeinick-Kerr R, Whelan SPJ (2013) A ribosome-specialized translation initiation pathway is required for cap-dependent translation of vesicular stomatitis virus mRNAs. Proc Natl Acad Sci USA 110:324–329. CrossRefPubMedGoogle Scholar
  39. Li Y, Liang C, Wong KC, Luo J, Zhang Z (2014) Mirsynergy: detecting synergistic miRNA regulatory modules by overlapping neighbourhood expansion. Bioinformatics 30:2627–2635. CrossRefPubMedGoogle Scholar
  40. Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15:550. CrossRefPubMedPubMedCentralGoogle Scholar
  41. Mainiero F et al (2000) RAC1/P38 MAPK signaling pathway controls beta1 integrin-induced interleukin-8 production in human natural killer cells. Immunity 12:7–16CrossRefPubMedGoogle Scholar
  42. Nadeau JH (2001) Modifier genes in mice and humans. Nat Rev Genet 2:165–174. CrossRefPubMedGoogle Scholar
  43. Nedelko T et al (2012) Distinct gene loci control the host response to influenza H1N1 virus infection in a time-dependent manner. BMC Genomics 13:411. CrossRefPubMedPubMedCentralGoogle Scholar
  44. Nencioni L, de Chiara G, Sgarbanti R, Amatore D, Aquilano K, Marcocci ME, Serafino A, Torcia M, Cozzolino F, Ciriolo MR, Garaci E, Palamara AT (2009) Bcl-2 expression and p38MAPK activity in cells infected with influenza A virus: impact on virally induced apoptosis and viral replication. J Biol Chem 284:16004–16015. CrossRefPubMedPubMedCentralGoogle Scholar
  45. Nencioni L et al (2003) Influenza A virus replication is dependent on an antioxidant pathway that involves GSH and Bcl-2. FASEB J 17:758–760. CrossRefPubMedGoogle Scholar
  46. Olsen CW, Kehren JC, Dybdahl-Sissoko NR, Hinshaw VS (1996) bcl-2 alters influenza virus yield, spread, and hemagglutinin glycosylation. J Virol 70:663–666PubMedPubMedCentralGoogle Scholar
  47. Parnell G, McLean A, Booth D, Huang S, Nalos M, Tang B (2011) Aberrant cell cycle and apoptotic changes characterise severe influenza A infection—a meta-analysis of genomic signatures in circulating leukocytes. PloS One 6:e17186. CrossRefPubMedPubMedCentralGoogle Scholar
  48. Pommerenke C, Wilk E, Srivastava B, Schulze A, Novoselova N, Geffers R, Schughart K (2012) Global transcriptome analysis in influenza-infected mouse lungs reveals the kinetics of innate and adaptive host immune responses. PloS One 7:e41169. CrossRefPubMedPubMedCentralGoogle Scholar
  49. Riccadonna S, Jurman G, Visintainer R, Filosi M, Furlanello C (2016) DTW-MIC coexpression networks from time-course data. PloS One 11:e0152648. CrossRefPubMedPubMedCentralGoogle Scholar
  50. Ryan-Poirier KA, Kawaoka Y (1993) Alpha 2-macroglobulin is the major neutralizing inhibitor of influenza A virus in pig serum. Virology 193:974–976. CrossRefPubMedGoogle Scholar
  51. Shannon P et al (2003) Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 13:2498–2504. CrossRefPubMedPubMedCentralGoogle Scholar
  52. Shapira SD et al (2009) A physical and regulatory map of host-influenza interactions reveals pathways in H1N1 infection. Cell 139:1255–1267. CrossRefPubMedPubMedCentralGoogle Scholar
  53. Sieczkarski SB, Whittaker GR (2003) Differential requirements of Rab5 and Rab7 for endocytosis of influenza and other enveloped viruses. Traffic 4:333–343CrossRefPubMedGoogle Scholar
  54. Srivastava B, Błażejewska P, Heßmann M, Bruder D, Geffers R, Mauel S, Gruber AD, Schughart K (2009) Host genetic background strongly influences the response to influenza a virus infections. PloS One 4:e4857. CrossRefPubMedPubMedCentralGoogle Scholar
  55. Staring J et al (2017) PLA2G16 represents a switch between entry and clearance of Picornaviridae. Nature 541:412-+. CrossRefPubMedGoogle Scholar
  56. Szklarczyk D et al (2015) STRING v10: protein-protein interaction networks, integrated over the tree of life. Nucleic Acids Res 43:D447–D452. CrossRefPubMedGoogle Scholar
  57. Tandon R, Sra S (2010) Sparse nonnegative matrix approximation: new formulations and algorithms.[0].pdf
  58. Toth LA, Williams RW (1999) A quantitative genetic analysis of slow-wave sleep in influenza-infected CXB recombinant inbred mice. Behav Genet 29:339–348CrossRefPubMedGoogle Scholar
  59. Tsuno A, Miyoshi K, Tsujii R, Miyakawa T, Mizuta K (2000) RRS1, a conserved essential gene, encodes a novel regulatory protein required for ribosome biogenesis in Saccharomyces cerevisiae. Mol Cell Biol 20:2066–2074. CrossRefPubMedPubMedCentralGoogle Scholar
  60. Walsh D, Mohr I (2011) Viral subversion of the host protein synthesis machinery. Nat Rev Microbiol 9:860–875. CrossRefPubMedGoogle Scholar
  61. Wang ZF et al (2014) Early hypercytokinemia is associated with interferon-induced transmembrane protein-3 dysfunction and predictive of fatal H7N9 infection. Proc Natl Acad Sci USA 111:769–774. CrossRefPubMedGoogle Scholar
  62. Yeung M-L et al (2016) MERS coronavirus induces apoptosis in kidney and lung by upregulating Smad7 and FGF2. Nature Microbiol 1:16004. CrossRefGoogle Scholar
  63. Yu L, Sun L, Nan Y, Zhu LY (2011) Protection from H1N1 influenza virus infections in mice by supplementation with selenium: a comparison with selenium-deficient mice. Biol Trace Elem Res 141:254–261. CrossRefPubMedGoogle Scholar
  64. Zhao XF et al (2014) PI3K/Akt signaling pathway modulates influenza virus induced mouse alveolar macrophage polarization to M1/M2b. PloS One 9:e104506. CrossRefPubMedPubMedCentralGoogle Scholar
  65. Zhou J et al. (2013) Biological features of novel avian influenza A (H7N9) virus. Nature advance online publication doi:
  66. Zhu H et al (2013) Infectivity, transmission, and pathology of human-isolated H7N9 influenza virus in ferrets and pigs. Science 341:183–186. CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Suying Bao
    • 1
  • Lilong Jia
    • 2
  • Xueya Zhou
    • 3
  • Zhi-Gang Zhang
    • 1
  • Hazel Wai Lan Wu
    • 2
  • Zhe Yu
    • 1
  • Gordon Ng
    • 1
  • Yanhui Fan
    • 1
  • Dana S. M. Wong
    • 1
  • Shishu Huang
    • 4
  • Kelvin Kai Wang To
    • 2
  • Kwok-Yung Yuen
    • 2
  • Man Lung Yeung
    • 2
  • You-Qiang Song
    • 1
    • 3
    • 5
    • 6
  1. 1.Schoolof Biomedical SciencesThe University of Hong KongHong KongChina
  2. 2.Department of MicrobiologyThe University of Hong KongHong KongChina
  3. 3.Department of PsychiatryThe University of Hong KongHong KongChina
  4. 4.Department of Orthopedic Surgery, West China HospitalSichuan UniversityChengduChina
  5. 5.HKU-SIRI/ZIRIThe University of Hong KongHong KongChina
  6. 6.HKU-SUSTech Joint Laboratories of Matrix Biology and DiseasesThe University of Hong KongHong KongChina

Personalised recommendations