Comparative Transcriptomic Analysis of Rhinovirus and Influenza Virus Infection

Thrimendra Kaushika Dissanayake, Sascha Schäuble, Mohammad Hassan Mirhakkak, Wai-Lan Wu, Anthony Chin-Ki Ng, Cyril C Y Yip, Albert García López, Thomas Wolf, Man-Lung Yeung, Kwok-Hung Chan, Kwok-Yung Yuen, Gianni Panagiotou, Kelvin Kai-Wang To, Thrimendra Kaushika Dissanayake, Sascha Schäuble, Mohammad Hassan Mirhakkak, Wai-Lan Wu, Anthony Chin-Ki Ng, Cyril C Y Yip, Albert García López, Thomas Wolf, Man-Lung Yeung, Kwok-Hung Chan, Kwok-Yung Yuen, Gianni Panagiotou, Kelvin Kai-Wang To

Abstract

Rhinovirus (RV) and influenza virus are the most frequently detected respiratory viruses among adult patients with community acquired pneumonia. Previous clinical studies have identified major differences in the clinical presentations and inflammatory or immune response during these infections. A systematic transcriptomic analysis directly comparing influenza and RV is lacking. Here, we sought to compare the transcriptomic response to these viral infections. Human airway epithelial Calu-3 cells were infected with contemporary clinical isolates of RV, influenza A virus (IAV), or influenza B virus (IBV). Host gene expression was determined using RNA-seq. Differentially expressed genes (DEGs) with respect to mock-infected cells were identified using the overlapping gene-set of four different statistical models. Transcriptomic analysis showed that RV-infected cells have a more blunted host response with fewer DEGs than IAV or IBV-infected cells. IFNL1 and CXCL10 were among the most upregulated DEGs during RV, IAV, and IBV infection. Other DEGs that were highly expressed for all 3 viruses were mainly genes related to type I or type III interferons (RSAD2, IDO1) and chemokines (CXCL11). Notably, ICAM5, a known receptor for enterovirus D68, was highly expressed during RV infection only. Gene Set Enrichment Analysis (GSEA) confirmed that pathways associated with interferon response, innate immunity, or regulation of inflammatory response, were most perturbed for all three viruses. Network analysis showed that steroid-related pathways were enriched. Taken together, our data using contemporary virus strains suggests that genes related to interferon and chemokine predominated the host response associated with RV, IAV, and IBV infection. Several highly expressed genes, especially ICAM5 which is preferentially-induced during RV infection, deserve further investigation.

Keywords: ICAM5; cytokines; influenza; interferons; rhinovirus; transcriptomics analysis.

Copyright © 2020 Dissanayake, Schäuble, Mirhakkak, Wu, Ng, Yip, López, Yeung, Chan, Yuen, Panagiotou and To.

Figures

FIGURE 1
FIGURE 1
Infection of influenza A virus (IAV), influenza B virus (IBV) or rhinovirus (RV) in Calu-3 cells. (A) Calu-3 cells were infected with IAV, IBV, or RV at 1 MOI. IAV, IBV, and RV antigen expression was determined at 24 h post infection. Antigen expression was determined using fluorescein-tagged murine monoclonal antibodies against IAV, IBV, or RV. Mock-infected cells stained with respective monoclonal antibodies against IAV, IBV, or RV are shown in the bottom row. White scale bar = 50 μm. (B) Multicycle growth assay. Calu-3 cells were infected with IAV, IBV, or RV at 1 MOI. Viral load was determined using real-time RT-qPCR. (C) Cytokine and chemokine expression of Calu-3 cells infected with IAV, IBV, or RV at 1 MOI. Cytokine expression was determined using real time RT-PCR. GAPDH was used for normalization of gene expression. (D) Cytokine and chemokine protein expression of Calu-3 cells infected with IAV, IBV, or RV at 1 MOI. Protein expression was determined using ELISA. Bars (B,C) represent means (error bars show standard error of mean) of duplicates in two independent experiments. Bars (D) represent means (error bars show standard error of mean) of triplicates in one independent experiment. Statistical significance (for B–D) was calculated with two-way ANOVA. (∗P< 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ****P < 0.0001). hpi, hours post infection; MOI, multiplicity of infection.
FIGURE 2
FIGURE 2
Hierarchical clustering of gene expression for (A) 12 hpi time point and (B) 24 hpi time point showing a distinct gene expression for IAV, IBV, and RV. (C) Principal component analysis for 6, 12, and 24 hpi.
FIGURE 3
FIGURE 3
(A) Heatmap for top-20 differentially expressed genes with an absolute log2 fold change ≥ 2 across all virus infections with significant infection annotated. (B) ICAM5 expression. Monoplex ICAM5 specific real time RT-PCR was performed for IAV, IBV, and RV-infected cells. **P < 0.01; ***P <0.001.
FIGURE 4
FIGURE 4
Heatmap for differentially expressed genes with an absolute log2 fold change ≥ 2 across all virus infections. Three subclusters as determined by hierarchical clustering are shown and GSEA was performed for each individual sub-cluster and the major significantly enriched pathways are indicated.
FIGURE 5
FIGURE 5
Network visualization of GSEA derived categories related to steroid biosynthesis. Red: up-regulated, Blue: down-regulated; Size: log2 fold change. Green edges: gene is shared among all categories; magenta: genes are only shared among response to corticosteroid and response to glucocorticoid categories.

References

    1. Aevermann B. D., Pickett B. E., Kumar S., Klem E. B., Agnihothram S., Askovich P. S., et al. (2014). A comprehensive collection of systems biology data characterizing the host response to viral infection. Sci Data 1:140033.
    1. Anders S., Huber W. (2010). Differential expression analysis for sequence count data. Genome Biol. 11:R106.
    1. Chan W. M., Wong L. H., So C. F., Chen L. L., Wu W. L., Ip J. D., et al. (2019). Development and evaluation of a conventional RT-PCR for differentiating emerging influenza B/Victoria lineage viruses with hemagglutinin amino acid deletion from B/Yamagata lineage viruses. J. Med. Virol. 92 382–385. 10.1002/jmv.25607
    1. Contoli M., Message S. D., Laza-Stanca V., Edwards M. R., Wark P. A., Bartlett N. W., et al. (2006). Role of deficient type III interferon-lambda production in asthma exacerbations. Nat. Med. 12 1023–1026. 10.1038/nm1462
    1. Davidson S., McCabe T. M., Crotta S., Gad H. H., Hessel E. M., Beinke S., et al. (2016). IFNlambda is a potent anti-influenza therapeutic without the inflammatory side effects of IFNalpha treatment. EMBO Mol. Med. 8 1099–1112. 10.15252/emmm.201606413
    1. Duschene K. S., Broderick J. B. (2012). Viperin: a radical response to viral infection. Biomol. Concepts 3 255–266. 10.1515/bmc-2011-0057
    1. Egli A., Santer D. M., O’Shea D., Barakat K., Syedbasha M., Vollmer M., et al. (2014). IL-28B is a key regulator of B- and T-cell vaccine responses against influenza. PLoS Pathog. 10:e1004556. 10.1371/journal.ppat.1004556
    1. Gaelings L., Soderholm S., Bugai A., Fu Y., Nandania J., Schepens B., et al. (2017). Regulation of kynurenine biosynthesis during influenza virus infection. FEBS J. 284 222–236. 10.1111/febs.13966
    1. Hillyer P., Shepard R., Uehling M., Krenz M., Sheikh F., Thayer K. R., et al. (2018). Differential responses by human respiratory epithelial cell lines to respiratory syncytial virus reflect distinct patterns of infection control. J. Virol. 92:e02202-17.
    1. Hu Y., Wang J., Yang B., Zheng N., Qin M., Ji Y., et al. (2011). Guanylate binding protein 4 negatively regulates virus-induced type I IFN and antiviral response by targeting IFN regulatory factor 7. J. Immunol. 187 6456–6462. 10.4049/jimmunol.1003691
    1. Hung I. F., Zhang A. J., To K. K., Chan J. F., Zhu S. H., Zhang R., et al. (2017). Unexpectedly higher morbidity and mortality of hospitalized elderly patients associated with rhinovirus compared with influenza virus respiratory tract infection. Int. J. Mol. Sci. 18:259. 10.3390/ijms18020259
    1. Ieven M., Coenen S., Loens K., Lammens C., Coenjaerts F., Vanderstraeten A., et al. (2018). Aetiology of lower respiratory tract infection in adults in primary care: a prospective study in 11 European countries. Clin. Microbiol. Infect 24 1158–1163. 10.1016/j.cmi.2018.02.004
    1. Iuliano A. D., Roguski K. M., Chang H. H., Muscatello D. J., Palekar R., Tempia S., et al. (2018). Estimates of global seasonal influenza-associated respiratory mortality: a modelling study. Lancet 391 1285–1300.
    1. Jain S., Self W. H., Wunderink R. G., Fakhran S., Balk R., Bramley A. M., et al. (2015). Community-Acquired Pneumonia Requiring Hospitalization among U.S. Adults. N. Engl. J. Med. 373 415–427.
    1. Kim T. K., Bheda-Malge A., Lin Y., Sreekrishna K., Adams R., Robinson M. K., et al. (2015). A systems approach to understanding human rhinovirus and influenza virus infection. Virology 486 146–157. 10.1016/j.virol.2015.08.014
    1. Kitada S., Kayama H., Okuzaki D., Koga R., Kobayashi M., Arima Y., et al. (2017). BATF2 inhibits immunopathological Th17 responses by suppressing Il23a expression during Trypanosoma cruzi infection. J. Exp. Med. 214 1313–1331. 10.1084/jem.20161076
    1. Landry M. L., Foxman E. F. (2018). Antiviral response in the nasopharynx identifies patients with respiratory virus infection. J. Infect. Dis. 217 897–905. 10.1093/infdis/jix648
    1. Lee A. C. Y., To K. K. W., Zhu H., Chu H., Li C., Mak W. W. N., et al. (2017). Avian influenza virus A H7N9 infects multiple mononuclear cell types in peripheral blood and induces dysregulated cytokine responses and apoptosis in infected monocytes. J. Gen. Virol. 98 922–934. 10.1099/jgv.0.000751
    1. Love M. I., Huber W., Anders S. (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15:550.
    1. Marcone D. N., Carballal G., Iraneta M., Rubies Y., Vidaurreta S. M., Echavarria M. (2017). Nosocomial transmission and genetic diversity of rhinovirus in a neonatal intensive care unit. J. Pediatr. 193 252.e1–255.e1. 10.1016/j.jpeds.2017.09.013
    1. Maza E., Frasse P., Senin P., Bouzayen M., Zouine M. (2013). Comparison of normalization methods for differential gene expression analysis in RNA-Seq experiments: a matter of relative size of studied transcriptomes. Commun. Integr. Biol. 6:e25849. 10.4161/cib.25849
    1. Menachery V. D., Eisfeld A. J., Schafer A., Josset L., Sims A. C., Proll S., et al. (2014). Pathogenic influenza viruses and coronaviruses utilize similar and contrasting approaches to control interferon-stimulated gene responses. mBio 5:e1174-14.
    1. Morrison J., Josset L., Tchitchek N., Chang J., Belser J. A., Swayne D. E., et al. (2014). H7N9 and other pathogenic avian influenza viruses elicit a three-pronged transcriptomic signature that is reminiscent of 1918 influenza virus and is associated with lethal outcome in mice. J. Virol. 88 10556–10568. 10.1128/jvi.00570-14
    1. Papi A., Contoli M., Adcock I. M., Bellettato C., Padovani A., Casolari P., et al. (2013). Rhinovirus infection causes steroid resistance in airway epithelium through nuclear factor kappaB and c-Jun N-terminal kinase activation. J. Allergy Clin. 132 1075.e6–1085.e6.
    1. Papi A., Johnston S. L. (1999). Rhinovirus infection induces expression of its own receptor intercellular adhesion molecule 1 (ICAM-1) via increased NF-kappaB-mediated transcription. J. Biol. Chem. 274 9707–9720. 10.1074/jbc.274.14.9707
    1. Prill M. M., Dahl R. M., Midgley C. M., Chern S. W., Lu X., Feikin D. R., et al. (2018). Severe respiratory illness associated with rhinovirus during the enterovirus D68 outbreak in the United States, August 2014-November 2014. Clin. Infect. Dis. 66 1528–1534. 10.1093/cid/cix1034
    1. Rajan D., Gaston K. A., McCracken C. E., Erdman D. D., Anderson L. J. (2013). Response to rhinovirus infection by human airway epithelial cells and peripheral blood mononuclear cells in an in vitro two-chamber tissue culture system. PLoS One 8:e66600. 10.1371/journal.pone.0066600
    1. Rajan D., McCracken C. E., Kopleman H. B., Kyu S. Y., Lee F. E., Lu X., et al. (2014). Human rhinovirus induced cytokine/chemokine responses in human airway epithelial and immune cells. PLoS One 9:e114322. 10.1371/journal.pone.0114322
    1. Raudvere U., Kolberg L., Kuzmin I., Arak T., Adler P., Peterson H., et al. (2019). g:Profiler: a web server for functional enrichment analysis and conversions of gene lists (2019 update). Nucleic Acids Res. 47 W191–W198.
    1. Ritchie M. E., Phipson B., Wu D., Hu Y., Law C. W., Shi W., et al. (2015). limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43:e47. 10.1093/nar/gkv007
    1. Robinson M. D., McCarthy D. J., Smyth G. K. (2010). edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26 139–140. 10.1093/bioinformatics/btp616
    1. Roe J., Venturini C., Gupta R. K., Gurry C., Chain B. M., Sun Y., et al. (2019). Blood transcriptomic stratification of short-term risk in contacts of tuberculosis. Clin. Infect. Dis. 70 731–737. 10.1093/cid/ciz252
    1. Seelbinder B., Wolf T., Priebe S., McNamara S., Gerber S., Guthke R., et al. (2019). GEO2RNAseq: an easy-to-use R pipeline for complete pre-processing of RNA-seq data. bioRxiv [preprint]. 10.1101/771063
    1. Seng L. G., Daly J., Chang K. C., Kuchipudi S. V. (2014). High basal expression of interferon-stimulated genes in human bronchial epithelial (BEAS-2B) cells contributes to influenza A virus resistance. PLoS One 9:e109023. 10.1371/journal.pone.0109023
    1. Shinya K., Gao Y., Cilloniz C., Suzuki Y., Fujie M., Deng G., et al. (2012). Integrated clinical, pathologic, virologic, and transcriptomic analysis of H5N1 influenza virus-induced viral pneumonia in the rhesus macaque. J. Virol. 86 6055–6066. 10.1128/jvi.00365-12
    1. Subramanian A., Tamayo P., Mootha V. K., Mukherjee S., Ebert B. L., Gillette M. A., et al. (2005). Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. U.S.A. 102 15545–15550. 10.1073/pnas.0506580102
    1. Syedbasha M., Egli A. (2017). Interferon lambda: modulating immunity in infectious diseases. Front. Immunol. 8:119. 10.3389/fimmu.2017.00119
    1. To K. K., Hung I. F., Li I. W., Lee K. L., Koo C. K., Yan W. W., et al. (2010). Delayed clearance of viral load and marked cytokine activation in severe cases of pandemic H1N1 2009 influenza virus infection. Clin. Infect. Dis. 50 850–859. 10.1086/650581
    1. To K. K., Lau C. C., Woo P. C., Lau S. K., Chan J. F., Chan K. H., et al. (2009). Human H7N9 virus induces a more pronounced pro-inflammatory cytokine but an attenuated interferon response in human bronchial epithelial cells when compared with an epidemiologically-linked chicken H7N9 virus. Virol. J. 13:42.
    1. To K. K., Lau S. K., Chan K. H., Mok K. Y., Luk H. K., Yip C. C., et al. (2016a). Pulmonary and extrapulmonary complications of human rhinovirus infection in critically ill patients. J. Clin. Virol. 77 85–91. 10.1016/j.jcv.2016.02.014
    1. To K. K., Lau C. C., Woo P. C., Lau S. K., Chan J. F., Chan K. H., et al. (2016b). Human H7N9 virus induces a more pronounced pro-inflammatory cytokine but an attenuated interferon response in human bronchial epithelial cells when compared with an epidemiologically-linked chicken H7N9 virus. Virol. J. 13:42.
    1. To K. K., Lu L., Fong C. H., Wu A. K., Mok K. Y., Yip C. C., et al. (2018). Rhinovirus respiratory tract infection in hospitalized adult patients is associated with TH2 response irrespective of asthma. J. Infect. 76 465–474. 10.1016/j.jinf.2018.02.005
    1. To K. K., Lu L., Yip C. C., Poon R. W., Fung A. M., Cheng A., et al. (2017). Additional molecular testing of saliva specimens improves the detection of respiratory viruses. Emerg. Microbes Infect. 6:e49.
    1. To K. K., Mok K. Y., Chan A. S., Cheung N. N., Wang P., Lui Y. M., et al. (2016). Mycophenolic acid, an immunomodulator, has potent and broad-spectrum in vitro antiviral activity against pandemic, seasonal and avian influenza viruses affecting humans. J. Gen. Virol. 97 1807–1817. 10.1099/jgv.0.000512
    1. To K. K. W., Chan K. H., Ho J., Pang P. K. P., Ho D. T. Y., Chang A. C. H., et al. (2019). Respiratory virus infection among hospitalized adult patients with or without clinically apparent respiratory infection: a prospective cohort study. Clin. Microbiol. Infect. 25 1539–1545. 10.1016/j.cmi.2019.04.012
    1. Wei W., Guo H., Chang J., Yu Y., Liu G., Zhang N., et al. (2016). ICAM-5/telencephalin is a functional entry receptor for enterovirus D68. Cell Host Microbe 20 631–641. 10.1016/j.chom.2016.09.013
    1. Zaas A. K., Chen M., Varkey J., Veldman T., Hero A. O., Lucas J., et al. (2009). Gene expression signatures diagnose influenza and other symptomatic respiratory viral infections in humans. Cell Host Microbe 6 207–217. 10.1016/j.chom.2009.07.006
    1. Zhai Y., Franco L. M., Atmar R. L., Quarles J. M., Arden N., Bucasas K. L., et al. (2015). Host transcriptional response to influenza and other acute respiratory viral infections–a prospective cohort study. PLoS Pathog. 11:e1004869. 10.1371/journal.ppat.1004869
    1. Zhao H., To K. K. W., Chu H., Ding Q., Zhao X., Li C., et al. (2018). Dual-functional peptide with defective interfering genes effectively protects mice against avian and seasonal influenza. Nat. Commun. 9:2358.
    1. Zheng B., Chan K. H., Zhang A. J., Zhou J., Chan C. C., Poon V. K., et al. (2010). D225G mutation in hemagglutinin of pandemic influenza H1N1 virus enhances virulence in mice. Exp. Biol. Med. 235 981–988. 10.1258/ebm.2010.010071

Source: PubMed

Sponsorer og samarbejdspartnere

Medicinske tilstande

Narkotikainterventioner

3
Abonner