Molecular Neurobiology

, Volume 54, Issue 2, pp 1541–1551 | Cite as

Differential Expression and Significance of Circulating microRNAs in Cerebrospinal Fluid of Acute Encephalitis Patients Infected with Japanese Encephalitis Virus

  • Saptamita Goswami
  • Atoshi Banerjee
  • Bharti Kumari
  • Bhaswati Bandopadhyay
  • Nemai Bhattacharya
  • Nandita Basu
  • Sudhanshu Vrati
  • Arup BanerjeeEmail author


Changes in circulating microRNAs (miRNAs) in the cerebrospinal fluid (CSF) have been associated with different neurological diseases. Here, we presented results of a pilot study aimed at determining the feasibility of detecting miRNAs in the CSF of Japanese Encephalitis virus (JEV) infected individuals with acute encephalitis syndrome (AES). We demonstrated the circulating miRNA profile in CSF of acute encephalitis patients infected with JEV. Using a quantitative real-time PCR-based miRNA array, we examined the level of 87 miRNAs expressed in human exosomes isolated from CSF. Subsequently, correlation between cytokine level and miRNAs expression in CSF samples was examined. In this study, we identified and validated the upregulated expression of three miRNAs, miR-21-5p, miR-150-5p, and miR-342-3p that were specifically circulated in CSF of acute encephalitis patients infected with JEV. CSF miR-21-5p, miR-150-5p, and miR-342-3p expressions were also elevated in infected mice brain. However, the expression pattern of these miRNAs differed in neuronal cells, microglial cells, and the exosome derived from JEV-infected cell culture supernatant. Interestingly, neuronal cells infected with vaccine strain (SA-14-14) did not lead to any upregulation of these three miRNAs. Further, miR-150-5p expression was found to be negatively correlated(r = −0.5279, p = 0.016) with TNFα level. Pathway analysis of putative target genes of these miRNAs indicated involvement of TGF-β, NGF, axon guidance, and MAPK signaling pathways in JEV/AES patients. This study for the first time represents the circulating miRNA in CSF of AES patients and identified the upregulated miRNAs in JEV-infected patients and offers the basis for future investigation.


Exosome Circulating miRNAs Acute encephalitis 



This work is supported by the funding from DBT, India (BT/PR6714/MED/29/617/2012) to A. B (Arup Banerjee). We acknowledge Dr. Manpreet Kaur, Vaccine Technologist at VIDRC, THSTI, for assisting in flow cytometric data analysis. We also acknowledge Suman Ghosal and Shaoli Das, Senior Research Fellow at Computational Biology Group, Indian Association for the Cultivation of Science, Kolkata, India for helping in pathway and network analysis. We are also thankful to all the technical staff of Department of Virology, School of Tropical Medicine for collecting and processing the CSF samples.

Compliance with Ethical Standards

CSF samples were collected by diagnostic lumbar puncture after written informed consent and after exclusion of large intracranial mass lesions or increased intracranial pressure or both. The STM ethical committee had approved CSF sample collections. All CSF samples  were collected in accordance with the approved guidelines and regulations.


  1. 1.
    Kakkar M, Rogawski ET, Abbas SS, Chaturvedi S, Dhole TN, Hossain SS, Krishnan SK (2013) Acute encephalitis syndrome surveillance, Kushinagar district, Uttar Pradesh, India, 2011–2012. Emerg Infect Dis 19(9):1361–1367. doi: 10.3201/eid1909.121855 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Vlassov AV, Magdaleno S, Setterquist R, Conrad R (2012) Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials. Biochim Biophys Acta 1820(7):940–948. doi: 10.1016/j.bbagen.2012.03.017 CrossRefPubMedGoogle Scholar
  3. 3.
    ELA S, Mager I, Breakefield XO, Wood MJ (2013) Extracellular vesicles: biology and emerging therapeutic opportunities. Nat Rev Drug Discov 12(5):347–357. doi: 10.1038/nrd3978 CrossRefGoogle Scholar
  4. 4.
    Lo Cicero A, Stahl PD, Raposo G (2015) Extracellular vesicles shuffling intercellular messages: for good or for bad. Curr Opin Cell Biol 35:69–77. doi: 10.1016/ CrossRefPubMedGoogle Scholar
  5. 5.
    Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO (2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 9(6):654–659. doi: 10.1038/ncb1596 CrossRefPubMedGoogle Scholar
  6. 6.
    Fruhbeis C, Frohlich D, Kramer-Albers EM (2012) Emerging roles of exosomes in neuron-glia communication. Front Physiol 3:119. doi: 10.3389/fphys.2012.00119 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Kalani A, Tyagi A, Tyagi N (2014) Exosomes: mediators of neurodegeneration, neuroprotection and therapeutics. Mol Neurobiol 49(1):590–600. doi: 10.1007/s12035-013-8544-1 CrossRefPubMedGoogle Scholar
  8. 8.
    Alevizos I, Illei GG (2010) MicroRNAs as biomarkers in rheumatic diseases. Nat Rev Rheumatol 6(7):391–398. doi: 10.1038/nrrheum.2010.81 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Cho WC (2010) MicroRNAs: potential biomarkers for cancer diagnosis, prognosis and targets for therapy. Int J Biochem Cell Biol 42(8):1273–1281. doi: 10.1016/j.biocel.2009.12.014 CrossRefPubMedGoogle Scholar
  10. 10.
    De Smaele E, Ferretti E, Gulino A (2010) MicroRNAs as biomarkers for CNS cancer and other disorders. Brain Res 1338:100–111. doi: 10.1016/j.brainres.2010.03.103 CrossRefPubMedGoogle Scholar
  11. 11.
    Clifford DB, Fagan AM, Holtzman DM, Morris JC, Teshome M, Shah AR, Kauwe JS (2009) CSF biomarkers of Alzheimer disease in HIV-associated neurologic disease. Neurology 73(23):1982–1987. doi: 10.1212/WNL.0b013e3181c5b445 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Muller M, Jakel L, Bruinsma IB, Claassen JA, Kuiperij HB, Verbeek MM (2015) MicroRNA-29a is a candidate biomarker for Alzheimer’s disease in cell-free cerebrospinal fluid. Mol Neurobiol. doi: 10.1007/s12035-015-9156-8 PubMedCentralGoogle Scholar
  13. 13.
    van Harten AC, Mulders J, Scheltens P, van der Flier WM, Oudejans CB (2015) Differential expression of microRNA in cerebrospinal fluid as a potential novel biomarker for Alzheimer’s disease. J Alzheimers Dis 47(1):243–252. doi: 10.3233/JAD-140075 CrossRefPubMedGoogle Scholar
  14. 14.
    Zhu B, Ye J, Nie Y, Ashraf U, Zohaib A, Duan X, Fu ZF, Song Y et al (2015) MicroRNA-15b modulates Japanese encephalitis virus-mediated inflammation via targeting RNF125. J Immunol 195(5):2251–2262. doi: 10.4049/jimmunol.1500370 CrossRefPubMedGoogle Scholar
  15. 15.
    Pareek S, Roy S, Kumari B, Jain P, Banerjee A, Vrati S (2014) MiR-155 induction in microglial cells suppresses Japanese encephalitis virus replication and negatively modulates innate immune responses. J Neuroinflammation 11:97. doi: 10.1186/1742-2094-11-97 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Thounaojam MC, Kaushik DK, Kundu K, Basu A (2014) MicroRNA-29b modulates Japanese encephalitis virus-induced microglia activation by targeting tumor necrosis factor alpha-induced protein 3. J Neurochem 129(1):143–154. doi: 10.1111/jnc.12609 CrossRefPubMedGoogle Scholar
  17. 17.
    Thounaojam MC, Kundu K, Kaushik DK, Swaroop S, Mahadevan A, Shankar SK, Basu A (2014) MicroRNA 155 regulates Japanese encephalitis virus-induced inflammatory response by targeting Src homology 2-containing inositol phosphatase 1. J Virol 88(9):4798–4810. doi: 10.1128/JVI.02979-13 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Sengupta N, Ghosh S, Vasaikar SV, Gomes J, Basu A (2014) Modulation of neuronal proteome profile in response to Japanese encephalitis virus infection. PloS One 9(3):e90211. doi: 10.1371/journal.pone.0090211 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Shelke GV, Lasser C, Gho YS, Lotvall J (2014) Importance of exosome depletion protocols to eliminate functional and RNA-containing extracellular vesicles from fetal bovine serum. J Extracell Vesicles 3:24783. doi: 10.3402/jev.v3.24783
  20. 20.
    Drusco A, Bottoni A, Lagana A, Acunzo M, Fassan M, Cascione L, Antenucci A, Kumchala P et al (2015) A differentially expressed set of microRNAs in cerebro-spinal fluid (CSF) can diagnose CNS malignancies. Oncotarget 6(25):20829–20839CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Pacifici M, Delbue S, Ferrante P, Jeansonne D, Kadri F, Nelson S, Velasco-Gonzalez C, Zabaleta J et al (2013) Cerebrospinal fluid miRNA profile in HIV-encephalitis. J Cell Physiol 228(5):1070–1075. doi: 10.1002/jcp.24254 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Yelamanchili SV, Lamberty BG, Rennard DA, Morsey BM, Hochfelder CG, Meays BM, Levy E, Fox HS (2015) MiR-21 in extracellular vesicles leads to neurotoxicity via TLR7 signaling in SIV neurological disease. PLoS Pathog 11(7):e1005032. doi: 10.1371/journal.ppat.1005032 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Lugli G, Cohen AM, Bennett DA, Shah RC, Fields CJ, Hernandez AG, Smalheiser NR (2015) Plasma exosomal miRNAs in persons with and without Alzheimer disease: altered expression and prospects for biomarkers. PLoS One 10(10):e0139233. doi: 10.1371/journal.pone.0139233 CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Moran J, Ramirez-Martinez G, Jimenez-Alvarez L, Cruz A, Perez-Patrigeon S, Hidalgo A, Orozco L, Martinez A et al (2015) Circulating levels of miR-150 are associated with poorer outcomes of A/H1N1 infection. Exp Mol Pathol 99(2):253–261. doi: 10.1016/j.yexmp.2015.07.001 CrossRefPubMedGoogle Scholar
  25. 25.
    Sang W, Wang Y, Zhang C, Zhang D, Sun C, Niu M, Zhang Z, Wei X et al (2015) MiR-150 impairs inflammatory cytokine production by targeting ARRB-2 after blocking CD28/B7 costimulatory pathway. Immunol Lett. doi: 10.1016/j.imlet.2015.11.001 PubMedGoogle Scholar
  26. 26.
    Swarup V, Ghosh J, Duseja R, Ghosh S, Basu A (2007) Japanese encephalitis virus infection decrease endogenous IL-10 production: correlation with microglial activation and neuronal death. Neurosci Lett 420(2):144–149. doi: 10.1016/j.neulet.2007.04.071 CrossRefPubMedGoogle Scholar
  27. 27.
    Zhou L, Pupo GM, Gupta P, Liu B, Tran SL, Rahme R, Wang B, Rua R et al (2012) A parallel genome-wide mRNA and microRNA profiling of the frontal cortex of HIV patients with and without HIV-associated dementia shows the role of axon guidance and downstream pathways in HIV-mediated neurodegeneration. BMC Genomics 13:677. doi: 10.1186/1471-2164-13-677 CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Tesseur I, Zou K, Esposito L, Bard F, Berber E, Can JV, Lin AH, Crews L et al (2006) Deficiency in neuronal TGF-beta signaling promotes neurodegeneration and Alzheimer’s pathology. J Clin Invest 116(11):3060–3069. doi: 10.1172/JCI27341 CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Peng F, Dhillon NK, Yao H, Zhu X, Williams R, Buch S (2008) Mechanisms of platelet-derived growth factor-mediated neuroprotection—implications in HIV dementia. Eur J Neurosci 28(7):1255–1264. doi: 10.1111/j.1460-9568.2008.06444.x CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Kolkata School of Tropical MedicineKolkataIndia
  2. 2.Vaccine and Infectious Disease Research Center (VIDRC)Translational Health Science and Technology Institute (THSTI)FaridabadIndia

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