Transcriptome analysis and verification of differentially expressed genes before and after enterovirus D68 infection

Xia TANG; Junzhuo SI; Nan LU; Yonglin HE; Lei XU; Jiajia BAO; Jialing TANG; Chun YANG

doi:10.16016/j.1000-5404.202009104

您当前的位置：

首页 >

文章列表页 >

Transcriptome analysis and verification of differentially expressed genes before and after enterovirus D68 infection

Basic Medicine | 更新时间：2026-01-09

- Transcriptome analysis and verification of differentially expressed genes before and after enterovirus D68 infection
- Journal of Third Military Medical University Vol. 43, Issue 4, Pages: 275-282(2021)
- 作者机构：
  
  400016 重庆，重庆医科大学基础医学院病原微生物学教研室
- 作者简介：
- 基金信息：
  
  the Natural Science Foundation of Chongqing(CSTC2016jcyjA0212)
- DOI：10.16016/j.1000-5404.202009104
  CLC： R373.2;R394-33;R394.3
- Received：13 September 2020，
  
  Revised：2020-11-25，
  
  Published：15 April 2021
- 稿件说明：
移动端阅览
Xia TANG, Junzhuo SI, Nan LU, et al. Transcriptome analysis and verification of differentially expressed genes before and after enterovirus D68 infection[J]. Journal of Third Military Medical University, 2021, 43(4): 275-282.
DOI：

Xia TANG, Junzhuo SI, Nan LU, et al. Transcriptome analysis and verification of differentially expressed genes before and after enterovirus D68 infection[J]. Journal of Third Military Medical University, 2021, 43(4): 275-282. DOI： 10.16016/j.1000-5404.202009104.

摘要

目的

探讨肠道病毒D68型（enterovirus D68

EV-D68)感染前后宿主细胞转录组的表达变化，寻找与EV-D68感染的相关基因及信号通路，为初步探索EV-D68的感染机制奠定基础。

方法

将病毒以感染复数（multiplicity of infection，MOI）0.01接种于人横纹肌瘤（rhabdomyosarcoma

RD）细胞，24 h后提取感染组和对照组细胞总RNA进行转录组测序，筛选差异表达基因(differentially expressed genes

DEGs)，对其进行GO和KEGG富集分析，并用RT-qPCR验证8个主要差异表达的基因（

EGR

1、

fos

、

ZNF

625、

ARRDC

3、

jun

、

ASNS

、

CHAC

1、

ADM

2）。

结果

从测序结果中共筛选出差异表达两倍以上的基因140个，其中上调基因71个，下调基因69个。GO功能富集分析结果主要涉及离子的调控与应答；KEGG富集分析结果主要与甲状旁腺激素的合成分泌和作用、GnRH信号通路、Toll样受体信号通路、IL-17信号通路和MAPK信号通路等有关。RT-qPCR验证结果表明选取的8个基因上调或下调的差异表达倍数趋势与测序结果基本一致，

fos

和

jun

的表达量随病毒MOI及感染时长的增加而增加。

结论

通过对差异表达基因的功能和信号通路富集分析及验证，EV-D68加重哮喘的机制可能与

fos

上调有关；而MAPK信号通路可能在EV-D68感染期间发挥促炎作用和抗病毒作用。

Abstract

Objective

To detect the alterations of the transcriptome in host cells infected by enterovirus D68 (EV-D68) and look for the genes involved in its infection so as to lay a foundation for the preliminary exploration of the infection mechanism.

Methods

The virus was inoculated into human rhabdomyosarcoma (RD) cells at a multiplicity of infection (MOI) of 0.01. After 24 h

RNA was extracted from the infected RD cells and control cells for transcriptome sequencing. Differentially expressed genes (DEGs) were screened and analyzed by using Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Eight DEGs

EGR

fos

ZNF

625

ARRDC

jun

ASNS

CHAC

1 and

ADM

2 were validated by RT-qPCR.

Results

A total of 140 genes with at least 2-fold changes at expression level were identified from the sequencing results

of which 71 were up-regulated and 69 were down-regulated. The results of GO functional enrichment analysis mainly involved the regulation and response to ions while the results of KEGG enrichment analysis mainly related to parathyroid hormone synthesis

secretion and action

IL-17 and GnRH signaling pathway

Toll-like receptor signaling pathway

and MAPK signaling pathway and so on. The RT-qPCR results showed that the trend of fold change of expression in the selected 8 genes was basically consistent with the sequencing results

and the expression levels of

fos

and

jun

were increased with the increase of virus MOI and infection duration.

Conclusion

By validating and using the functional and enrichment analysis

we infer that the mechanism of EV-D68 exacerbating asthma may be related to the up-regulation of

fos

and MAPK signaling pathway may play a pro-inflammatory and antiviral role during EV-D68 infection.

关键词

Keywords

references

SCHIEBLE J H, FOX V L, LENNETTE E H. A probable new human picornavirus associated with respiratory disease[J]. Am J Epidemiol, 1967, 85(2): 297-310. DOI: 10.1093/oxfordjournals.aje.a120693.

MIDGLEY C M, WATSON J T, NIX W A, et al . Severe respiratory illness associated with a nationwide outbreak of enterovirus D68 in the USA (2014): a descriptive epidemiological investigation[J ] . Lancet Respir Med, 2015, 3(11): 879-887. DOI: 10.1016/s2213-2600(15)00335-5.

POELMAN R, SCHUFFENECKER I, VAN LEER-BUTER C, et al . European surveillance for enterovirus D68 during the emerging North-American outbreak in 2014[J ] . J Clin Virol, 2015, 71: 1-9. DOI: 10.1016/j.jcv.2015.07.296.

OBERSTE M S, MAHER K, SCHNURR D, et al .Enterovirus 68 is associated with respiratory illness and shares biological features with both the enteroviruses and the rhinoviruses[J ] . J Gen Virol, 2004, 85(9): 2577-2584. DOI: 10.1099/vir.0.79925-0.

RAJPUT C, HAN M Y, BENTLEY J K, et al .Enterovirus D68 infection induces IL-17-dependent neutrophilic airway inflammation and hyperresponsiveness[J ] . JCI Insight, 2018, 3(16): 1-13. DOI: 10.1172/jci.insight.121882.

BOWERS J R, VALENTINE M, HARRISON V, et al . Genomic analyses of acute flaccid myelitis cases among a cluster in Arizona provide further evidence of enterovirus D68 role[J ] . mBio, 2019, 10(1). DOI: 10.1128/mbio.02262-18.

MEIJER A, BENSCHOP K S, DONKER G A, et al . Continued seasonal circulation of enterovirus D68 in the Netherlands, 2011-2014[J ] . Euro Surveill, 2014, 19(42). DOI: 10.2807/1560-7917.es2014.19.42.20935.

KRAMER R, SABATIER M, WIRTH T, et al . Molecular diversity and biennial circulation of enterovirus D68: a systematic screening study in Lyon, France, 2010 to 2016[J ] . Euro Surveill, 2018, 23(37). DOI: 10.2807/1560-7917.es.2018.23.37.1700711.

UPRETY P, CURTIS D, ELKAN M, et al . Association of enterovirus D68 with acute flaccid myelitis, Philadelphia, Pennsylvania, USA,2009-2018[J ] . Emerg Infect Dis, 2019, 25(9): 1676-1682. DOI: 10.3201/eid2509.190468.

[American Centers for Disease Control and Prevention: AFM investigation[EB/OL ] .[2020-09-13 ] https://www.cdc.gov/acute-flaccid-myelitis/afm-surveillance.html https://www.cdc.gov/acute-flaccid-myelitis/afm-surveillance.html .

MEYERS L, BARD J D, GALVIN B, et al . Enterovirus D68 outbreak detection through a syndromic disease epidemiology network[J ] . J Clin Virol, 2020, 124:104262.doi:10.1016/j.jcv.2020.104262.

XIANG Z, GONZALEZ R, WANG Z, et al . Coxsackievirus A21, enterovirus 68, and acute respiratory tract infection, China[J ] . Emerg Infect Dis, 2012, 18(5): 821-824. DOI: 10.3201/eid1805.111376.

XIAO Q, REN L, ZHENG S, et al . Prevalence and molecular characterizations of enterovirus D68 among children with acute respiratory infection in China between 2012 and 2014[J ] . Sci Rep, 2015, 5: 16639. DOI: 10.1038/srep16639.

LEE J T, SHIH W L, YEN T Y, et al .Enterovirus D68 seroepidemiology in Taiwan, a cross sectional study from 2017[J ] . PLoS ONE, 2020, 15(3): e0230180. DOI: 10.1371/journal.pone.0230180.

WANG H Y, DIAZ A, MOYER K, et al . Molecular and clinical comparison of enterovirus D68 outbreaks among hospitalized children, Ohio, USA, 2014 and 2018[J ] . Emerg Infect Dis, 2019, 25(11): 2055-2063. DOI: 10.3201/eid2511.190973.

DREWS S J, SIMMONDS K, USMAN H R, et al . Characterization of enterovirus activity, including that of enterovirus D68, in pediatric patients in Alberta, Canada, in 2014[J ] . J Clin Microbiol, 2015, 53(3): 1042-1045. DOI: 10.1128/jcm.02982-14.

MERTZ D, ALAWFI A, PERNICA J M, et al . Clinical severity of pediatric respiratory illness with enterovirus D68 compared with rhinovirus or other enterovirus genotypes[J ] . CMAJ, 2015, 187(17): 1279-1284. DOI: 10.1503/cmaj.150619.

HASEGAWA S, HIRANO R, OKAMOTO-NAKAGAWA R, et al .Enterovirus 68 infection in children with asthma attacks: virus-induced asthma in Japanese children[J ] . Allergy, 2011, 66(12): 1618-1620. DOI: 10.1111/j.1398-9995.2011.02725.x.

PAN H H, TSAI C R, TING P J, et al . Respiratory presentation of patients infected with enterovirus D68 in Taiwan[J ] . Pediatr Neonatol, 2020, 61(2): 168-173. DOI: 10.1016/j.pedneo.2019.09.006

WEI W, GUO H R, CHANG J L, et al . ICAM-5/telencephalin is a functional entry receptor for enterovirus D68[J ] . Cell Host Microbe, 2016, 20(5): 631-641. DOI: 10.1016/j.chom.2016.09.013.

LIU Y, SHENG J, BAGGEN J, et al .Sialic acid-dependent cell entry of human enterovirus D68[J ] . Nat Commun, 2015, 6: 8865. DOI: 10.1038/ncomms9865.

DEMOLY P, BASSET-SEGUIN N, CHANEZ N, et al . C-fos proto-oncogene expression in bronchial biopsies of asthmatics[J ] . Am J Respir Cell Mol Biol, 1992, 7(2): 128-133. DOI: 10.1165/ajrcmb/7.2.128.

LIU H, YANG X, HOU W. Correlation of c-fos protein expression with neuropeptide content in the lung of bronchial asthmatic rat[J]. Int J Clin Exp Pathol, 2014, 7(12): 8657-8665.

GUO R B, SUN P L, ZHAO A P, et al . Chronic asthma results in cognitive dysfunction in immaturemice[J ] . Exp Neurol, 2013, 247: 209-217. DOI: 10.1016/j.expneurol.2013.04.008.

CHEN S D, WEN Z H, CHANG W K, et al . Acute effect of methylprednisolone on the b rain in a rat model of allergic asthma[J ] . Neurosci Lett, 2008, 440(2): 87-91. DOI: 10.1016/j.neulet.2008.03.092.

CHEN Z, LIU N N, XIAO J, et al . The amygdala via the paraventricular nucleus regulates asthma attack in rats[J ] . CNS Neurosci Ther, 2020, 26(7): 730-740. DOI: 10.1111/cns.13293.

ZHU L, LI W H, QI G J, et al . The immune mechanism of intestinal tract Toll-like receptor in mediating EV71 virus type severe hand-foot-and-mouth disease and the MAPK pathway[J ] . Exp Ther Med, 2017, 13(5): 2263-2266. DOI: 10.3892/etm.2017.4245.

ZHANG H, FENG H, LUO L, et al . Distinct effects of knocking down MEK1 and MEK2 on replication of Herpes simplex virus type 2[J ] . Virus Res, 2010, 150(1/2): 22-27. DOI: 10.1016/j.virusres.2010.02.007.

SI X N, LUO H L, MORGAN A, et al . Stress-activated protein kinases are involved in coxsackievirus B3 viral progeny release[J ] . J Virol, 2005, 79(22): 13875-13881. DOI: 10.1128/jvi.79.22.13875-13881.2005.

TUNG W H, HSIEH H L, LEE I T, et al .Enterovirus 71 modulates a COX-2/PGE2/cAMP-dependent viral replication in human neuroblastoma ce lls: Role of the c-Src/EGFR/p42/p44 MAPK/CREB signaling pathway[J ] . J Cell Biochem, 2011, 112(2): 559-570. DOI: 10.1002/jcb.22946.

CEBALLOS-OLVERA I, CHÁVEZ-SALINAS S, MEDINA F, et al . JNK phosphorylation, induced during dengue virus infection, is important for viral infection and requires the presence of cholesterol[J ] . Virology (Lond), 2010, 396(1): 30-36. DOI: 10.1016/j.virol.2009.10.019.

FEJER G, FREUDENBERG M, GREBER U F, et al . Adenovirus-triggered innate signalling pathways[J ] . Eur J Microbiol Immunol, 2011, 1(4): 279-288. DOI: 10.1556/eujmi.1.2011.4.3.

Views

下载量

CSCD

Alert me when the article has been cited

提交

Tools

Publicity Resources

Screening and identification of key genes involved in lymphatic metastasis of oral squamous cell carcinoma

Downregulating RRM2B expression to increase radiation sensitivity of breast cancer cells

Screening and identification of synaptic regulatory genes in the inner ear of mice with presbycusis

Screening of differentially expressed genes in ankylosing spondylitis by cDNA microarray

Related Author

Ying CHEN

Li LUO

Yajing ZHONG

Qin ZENG

李安琪

孙海燕

祁孟瑶

林培颖

Related Institution

Department of Stomatology, Chongqing People's Hospital

Department of Stomatology, Second Affiliated Hospital, Army Medical University (Third Military Medical University)

⁰