Differential Biphasic Transcriptional Host Response Associated with Coevolution of Hemagglutinin Quasispecies of Influenza A Virus

Severe influenza associated with strong symptoms and lung inflammation can be caused by intra-host evolution of quasispecies with aspartic acid or glycine in hemagglutinin position 222 (HA-222D/G; H1 numbering). To gain insights into the dynamics of host response to this coevolution and to identify key mechanisms contributing to copathogenesis, the lung transcriptional response of BALB/c mice infected with an A(H1N1)pdm09 isolate consisting HA-222D/G quasispecies was analyzed from days 1 to 12 post infection (p.i). At day 2 p.i. 968 differentially expressed genes (DEGs) were detected. The DEG number declined to 359 at day 4 and reached 1001 at day 7 p.i. prior to recovery. Interestingly, a biphasic expression profile was shown for the majority of these genes. Cytokine assays confirmed these results on protein level exemplarily for two key inflammatory cytokines, interferon gamma and interleukin 6. Using a reverse engineering strategy, a regulatory network was inferred to hypothetically explain the biphasic pattern for selected DEGs. Known regulatory interactions were extracted by Pathway Studio 9.0 and integrated during network inference. The hypothetic gene regulatory network revealed a positive feedback loop of Ifng, Stat1, and Tlr3 gene signaling that was triggered by the HA-G222 variant and correlated with a clinical symptom score indicating disease severity.

Supplementary Figure 6. Six-Gene submodel. Gene-regulatory network prediction from high throughput time series microarray data by NetGenerator 2.0 involving DEGs connected to the positive feedback loop. 'Influenza 1' and 'Influenza 2' represent the two influenza variants (mpJena/5258 HA-D222 and mpJena/5258 HA-G222, respectively). Black edges represent the newly predicted ones, green edges represent edges supported by the prior knowledge and confirmed by the expression data-based network inference and grey dotted edges represent prior knowledge not included in the network prediction based on the measured gene expression profiles. Arrow-head represents activation or positive regulation while bar-head represents repression or negative regulation (that may also represent indirect interaction).

Supplementary Tables
Supplementary Table 1   2) It is involved in viral doublestranded (ds) RNA recognition and the regulation of immune response.
Innate immune response.
Positive regulation of Ifnalpha/-beta production.
Response to exogenous dsRNA.
RIG-I signalling pathway. 2) Also involved in the regulation of signal transduction, apoptosis, cell proliferation and differentiation.
-ve regulation of viral genome replication.
Positive regulation of chemokine n cytokine regulation.
Positive regulation of NFkappa-b signalling. Antigen processing and presentation.

Genes Function GO Category other than "response to virus"
Cluster Association

macrophages.
Positive regulation of IL-6, IL-12 biosynthetic process and T-cell proliferation.
Irf1: IFN regulatory factor 1 1) Activator of IFN alpha and beta transcription and also transcription activators of IFN induced genes like stat1.
2) Plays an important role in immune response directly affecting NK maturation and activity, macrophage production of IL12, Th1 development and maturation of CD8+ T-cells Positive regulation of type 1 IFN & IL-12. IFN-g mediated signalling regulation of Myd88dependent TLR signalling.
4 Irf-7 1) play a role in the transcriptional activation of virus-inducible cellular genes, including Ifnb.
2) Can efficiently activate both the IFN-beta and the IFN-alpha genes and mediate their induction via both the virus-activated, MyD88-independent pathway and the TLR-activated, MyD88-dependent pathway.
Positive regulation of IFNalpha/-beta production.
Regulation of Myd88 dependent TLR signalling pathway.
1 Lcn2: Lipocalin 2 1) Iron trafficking proteins involved in multiple processes such as apoptosis (due to IL3 deprivation), innate immunity and renal development.
Iron ion binding. Regulation of inflammatory response.
2) Mutations in this gene have been associated with host susceptibility to viral infection.
Negative regulation of viral process.
2'-5'-oligoadenylate synthetase activity. 2) Displays antiviral activity against influenza A virus by inhibiting the budding of the virus from the plasma membrane by disturbing the lipid rafts.
Positive regulation of TLR-7 and TLR-9 signalling pathway. 2) plays a key role in many cellular processes like cell growth and apoptosis.
Involved in JAK-STAT cascade.
Negative regulation of cell death & cell proliferation 1 Tlr3: Toll like receptor-3 1) They recognize pathogen associated molecular patterns (PAMPs) and mediates the production of various cytokines.
2) It recognize dsRNA associated with viral infection and induces the activation of NF-kappa-B and production of type I interferons.
Positive regulation of type-I and type-III IFNs.
Positive regulation of NFkappa-B signalling.
1 Tlr7: Toll like receptor-7 1) Plays a role in pathogen recognition and activation of innate immunity.
2) Predominantly expressed in lung, placenta and spleen.
Positive regulation of IL-6, IFN-alpha Defense response to virus.
Positive regulation of chemokine & IL-8 production and IFN-alpha/beta synthesis 1 Tgtp1: T cell specific GTPase 1 1) It performs GTPase activity. Response to IFN-alpha/gamma. cellular response to IFNbeta. GTP catabolic process.