Korean J Physiol Pharmacol.  2024 Sep;28(5):403-411. 10.4196/kjpp.2024.28.5.403.

Unraveling flavivirus pathogenesis: from bulk to single-cell RNA-sequencing strategies

• Kim D
• Jeong S
• Park SM

• KMID: 2559133
• DOI: http://doi.org/10.4196/kjpp.2024.28.5.403

Abstract

The global spread of flaviviruses has triggered major outbreaks worldwide, significantly impacting public health, society, and economies. This has intensified research efforts to understand how flaviviruses interact with their hosts and manipulate the immune system, underscoring the need for advanced research tools. RNA-sequencing (RNA-seq) technologies have revolutionized our understanding of flavivirus infections by offering transcriptome analysis to dissect the intricate dynamics of virus-host interactions. Bulk RNA-seq provides a macroscopic overview of gene expression changes in virus-infected cells, offering insights into infection mechanisms and host responses at the molecular level. Single-cell RNA sequencing (scRNAseq) provides unprecedented resolution by analyzing individual infected cells, revealing remarkable cellular heterogeneity within the host response. A particularly innovative advancement, virus-inclusive single-cell RNA sequencing (viscRNA-seq), addresses the challenges posed by non-polyadenylated flavivirus genomes, unveiling intricate details of virus-host interactions. In this review, we discuss the contributions of bulk RNA-seq, scRNA-seq, and viscRNA-seq to the field, exploring their implications in cell line experiments and studies on patients infected with various flavivirus species. Comprehensive transcriptome analyses from RNA-seq technologies are pivotal in accelerating the development of effective diagnostics and therapeutics, paving the way for innovative treatments and enhancing our preparedness for future outbreaks.

Keyword

Gene expression profiling; Infections; Single-cell analysis; Viruses

Figure

• 

  Fig. 1 The pathogenesis of flavivirus infections. Mosquitoes, the main transmitters of flavivirus, initiate the infection by injecting the virus into humans (left panel). Generally, infection with flavivirus causes mild symptoms such as fever and leads to various physiological changes within the body (middle panel). When symptoms become severe, the infection can affect multiple organs (right panel). ZIKV infection can cause ocular diseases and neurological inflammation, and it can also lead to infection of the placenta. DENV infection can cause damage to endothelial cells, leading to plasma leakage. YFV infection can cause damage to hepatocytes. DENV, YFV and WNV infections can cause acute kidney injury or renal failure.

• 

  Fig. 2 Comparative overview of RNA-sequencing (RNA-seq) technologies in flavivirus research. The distinct methodologies and outcomes associated with three different RNA-seq technologies applied in the study of flaviviruses. Bulk RNA-seq, a technique that analyzes the collective gene expression profiles from a population of cells, facilitating the discovery of virus-specific biomarkers. Single-cell RNA sequencing (scRNA-seq), which isolates and characterizes the gene expression of individual cells, revealing the cellular heterogeneity and the diversity of cellular responses to infection. Virus-inclusive single-cell RNA sequencing (viscRNA-seq), which allows for the detailed examination of the transcriptomes of both virus and host cells at the single-cell level, differing responses within a population of infected and uninfected cells.

Reference

1. Huang YJ, Higgs S, Horne KM, Vanlandingham DL. 2014; Flavivirus-mosquito interactions. Viruses. 6:4703–4730. DOI: 10.3390/v6114703. PMID: 25421894. PMCID: PMC4246245.

2. Coffey LL, Mertens E, Brehin AC, Fernandez-Garcia MD, Amara A, Després P, Sakuntabhai A. 2009; Human genetic determinants of Dengue virus susceptibility. Microbes Infect. 11:143–156. DOI: 10.1016/j.micinf.2008.12.006. PMID: 19121645.

3. Sharma A, Lal SK. 2017; Zika virus: transmission, detection, control, and prevention. Front Microbiol. 8:110. DOI: 10.3389/fmicb.2017.00110. PMID: 28217114. PMCID: PMC5290000.

4. Collins ND, Beck AS, Widen SG, Wood TG, Higgs S, Barrett ADT. 2018; Structural and nonstructural genes contribute to the genetic diversity of RNA viruses. mBio. 9:e01871–18. DOI: 10.1128/mBio.01871-18. PMID: 30377281. PMCID: PMC6212827.

5. Guarner J, Hale GL. 2019; Four human diseases with significant public health impact caused by mosquito-borne flaviviruses: West Nile, Zika, Dengue and Yellow Fever. Semin Diagn Pathol. 36:170–176. DOI: 10.1053/j.semdp.2019.04.009. PMID: 31006554.

6. Slon Campos JL, Mongkolsapaya J, Screaton GR. 2018; The immune response against flaviviruses. Nat Immunol. 19:1189–1198. DOI: 10.1038/s41590-018-0210-3. PMID: 30333606.

7. Cunha MDP, Duarte-Neto AN, Pour SZ, Ortiz-Baez AS, Černý J, Pereira BBS, Braconi CT, Ho YL, Perondi B, Sztajnbok J, Alves VAF, Dolhnikoff M, Holmes EC, Saldiva PHN, Zanotto PMA. 2019; Origin of the São Paulo Yellow Fever epidemic of 2017-2018 revealed through molecular epidemiological analysis of fatal cases. Sci Rep. 9:20418. DOI: 10.1038/s41598-019-56650-1. PMID: 31892699. PMCID: PMC6938505.

8. Dos Santos TH, Martin JLS, Castellanos LG, Espinal MA. 2019; Dengue in the Americas: Honduras' worst outbreak. Lancet. 394:2149. DOI: 10.1016/S0140-6736(19)32531-0. PMID: 31839185.

9. Gorshkov K, Shiryaev SA, Fertel S, Lin YW, Huang CT, Pinto A, Farhy C, Strongin AY, Zheng W, Terskikh AV. 2019; Zika virus: origins, pathological action, and treatment strategies. Front Microbiol. 9:3252. DOI: 10.3389/fmicb.2018.03252. PMID: 30666246. PMCID: PMC6330993.

10. Yun SI, Lee YM. 2014; Japanese encephalitis: the virus and vaccines. Hum Vaccin Immunother. 10:263–279. DOI: 10.4161/hv.26902. PMID: 24161909. PMCID: PMC4185882.

11. Dénes A, Ibrahim MA, Oluoch L, Tekeli M, Tekeli T. 2019; Impact of weather seasonality and sexual transmission on the spread of Zika fever. Sci Rep. 9:17055. DOI: 10.1038/s41598-019-53062-z. PMID: 31745123. PMCID: PMC6863851.

12. Pierson TC, Diamond MS. 2020; The continued threat of emerging flaviviruses. Nat Microbiol. 5:796–812. DOI: 10.1038/s41564-020-0714-0. PMID: 32367055. PMCID: PMC7696730.

13. Wang Z, Gerstein M, Snyder M. 2009; RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet. 10:57–63. DOI: 10.1038/nrg2484. PMID: 19015660. PMCID: PMC2949280.

14. Ozsolak F, Milos PM. 2011; RNA sequencing: advances, challenges and opportunities. Nat Rev Genet. 12:87–98. DOI: 10.1038/nrg2934. PMID: 21191423. PMCID: PMC3031867.

15. Tang F, Barbacioru C, Wang Y, Nordman E, Lee C, Xu N, Wang X, Bodeau J, Tuch BB, Siddiqui A, Lao K, Surani MA. 2009; mRNA-Seq whole-transcriptome analysis of a single cell. Nat Methods. 6:377–382. DOI: 10.1038/nmeth.1315. PMID: 19349980.

16. Park SM, Park M, Ban HJ, Baek SJ, Kim SY, Lee S, Jin HJ. 2022; Investigation of prodromal features in metabolic syndrome based on transcriptome analysis. Genes Dis. 10:708–711. DOI: 10.1016/j.gendis.2022.07.021. PMID: 37396528. PMCID: PMC10308104.

17. Park SM, Ban HJ, Lee M, Kim SY, Lee S, Jin HJ. 2023; Blood transcriptomic markers associated with immune abnormalities and sleep quality. Genes Dis. 101105. DOI: 10.1016/j.gendis.2023.101105.

18. Chun J, Park SM, Lee M, Ha IJ, Jeong MK. 2023; The sesquiterpene lactone-rich fraction of Inula helenium L. enhances the antitumor effect of anti-PD-1 antibody in colorectal cancer: integrative phytochemical, transcriptomic, and experimental analyses. Cancers (Basel). 15:653. DOI: 10.3390/cancers15030653. PMID: 36765611. PMCID: PMC9913754.

19. Kim A, Kim YR, Park SM, Lee H, Park M, Yi JM, Cha S, Kim NS. 2024; Jakyak-gamcho-tang, a decoction of Paeoniae Radix and Glycyrrhizae Radix et Rhizoma, ameliorates dexamethasone-induced muscle atrophy and muscle dysfunction. Phytomedicine. 123:155057. DOI: 10.1016/j.phymed.2023.155057. PMID: 37984121.

20. Kim A, Park SM, Kim NS, Lee H. 2023; Ginsenoside Rc, an active component of Panax ginseng, alleviates oxidative stress-induced muscle atrophy via improvement of mitochondrial biogenesis. Antioxidants (Basel). 12:1576. DOI: 10.3390/antiox12081576. PMID: 37627571. PMCID: PMC10451796.

21. Park SM, Kim A, Lee H, Baek SJ, Kim NS, Park M, Yi JM, Cha S. 2022; Systematic transcriptome analysis reveals molecular mechanisms and indications of bupleuri radix. Front Pharmacol. 13:1010520. DOI: 10.3389/fphar.2022.1010520. PMID: 36304143. PMCID: PMC9592978.

22. Bojkova D, Klann K, Koch B, Widera M, Krause D, Ciesek S, Cinatl J, Münch C. 2020; Proteomics of SARS-CoV-2-infected host cells reveals therapy targets. Nature. 583:469–472. DOI: 10.1038/s41586-020-2332-7. PMID: 32408336.

23. Winkler ES, Bailey AL, Kafai NM, Nair S, McCune BT, Yu J, Fox JM, Chen RE, Earnest JT, Keeler SP, Ritter JH, Kang LI, Dort S, Robichaud A, Head R, Holtzman MJ, Diamond MS. 2020; SARS-CoV-2 infection of human ACE2-transgenic mice causes severe lung inflammation and impaired function. Nat Immunol. 21:1327–1335. DOI: 10.1038/s41590-020-0778-2. PMID: 32839612. PMCID: PMC7578095.

24. Lowe R, Shirley N, Bleackley M, Dolan S, Shafee T. 2017; Transcriptomics technologies. PLoS Comput Biol. 13:e1005457. DOI: 10.1371/journal.pcbi.1005457. PMID: 28545146. PMCID: PMC5436640.

25. Rashmi SH, Disha KS, Sudheesh N, Karunakaran J, Joseph A, Jagadesh A, Mudgal PP. 2023; Repurposing of approved antivirals against Dengue virus serotypes: an in silico and in vitro mechanistic study. Mol Divers. doi: 10.1007/s11030-023-10716-5. [Epub ahead of print.] Erratum in: Mol Divers. 2023. doi: 10.1007/s11030-023-10748-x. DOI: 10.1007/s11030-023-10748-x.

26. Pérez-Pérez MJ, Saiz JC, Priego EM, Martín-Acebes MA. 2021; Antivirals against (re)emerging flaviviruses: should we target the virus or the host? ACS Med Chem Lett. 13:5–10. DOI: 10.1021/acsmedchemlett.1c00617. PMID: 35059112. PMCID: PMC8762743.

27. O'Neal JT, Upadhyay AA, Wolabaugh A, Patel NB, Bosinger SE, Suthar MS. 2019; West Nile virus-inclusive single-cell RNA sequencing reveals heterogeneity in the type I interferon response within single cells. J Virol. 93:e01778–18. DOI: 10.1128/JVI.01778-18. PMID: 30626670. PMCID: PMC6401468.

28. Hanley JP, Tu HA, Dragon JA, Dickson DM, Rio-Guerra RD, Tighe SW, Eckstrom KM, Selig N, Scarpino SV, Whitehead SS, Durbin AP, Pierce KK, Kirkpatrick BD, Rizzo DM, Frietze S, Diehl SA. 2021; Immunotranscriptomic profiling the acute and clearance phases of a human challenge Dengue virus serotype 2 infection model. Nat Commun. 12:3054. DOI: 10.1038/s41467-021-22930-6. PMID: 34031380. PMCID: PMC8144425.

29. Katze MG, He Y, Gale M Jr. 2002; Viruses and interferon: a fight for supremacy. Nat Rev Immunol. 2:675–687. DOI: 10.1038/nri888. PMID: 12209136.

30. Yadav A, Shamim U, Ravi V, Devi P, Kumari P, Maurya R, Das P, Somani M, Budhiraja S, Tarai B, Pandey R. 2023; Early transcriptomic host response signatures in the serum of Dengue patients provides insights into clinical pathogenesis and disease severity. Sci Rep. 13:14170. DOI: 10.1038/s41598-023-41205-2. PMID: 37644081. PMCID: PMC10465479.

31. Sarkar MMH, Rahman MS, Islam MR, Rahman A, Islam MS, Banu TA, Akter S, Goswami B, Jahan I, Habib MA, Uddin MM, Mia MZ, Miah MI, Shaikh AA, Khan MS. 2023; Comparative phylogenetic analysis and transcriptomic profiling of Dengue (DENV-3 genotype I) outbreak in 2021 in Bangladesh. Virol J. 20:127. Erratum in: Virol J. 2023;20:184. DOI: 10.1186/s12985-023-02030-1. PMID: 37337232. PMCID: PMC10278332.

32. Tang H, Hammack C, Ogden SC, Wen Z, Qian X, Li Y, Yao B, Shin J, Zhang F, Lee EM, Christian KM, Didier RA, Jin P, Song H, Ming GL. 2016; Zika virus infects human cortical neural progenitors and attenuates their growth. Cell Stem Cell. 18:587–590. DOI: 10.1016/j.stem.2016.02.016. PMID: 26952870. PMCID: PMC5299540.

33. Chen J, Yang YF, Chen J, Zhou X, Dong Z, Chen T, Yang Y, Zou P, Jiang B, Hu Y, Lu L, Zhang X, Liu J, Xu J, Zhu T. 2017; Zika virus infects renal proximal tubular epithelial cells with prolonged persistency and cytopathic effects. Emerg Microbes Infect. 6:e77. DOI: 10.1038/emi.2017.67. PMID: 28831192. PMCID: PMC5583673.

34. Chen J, Yang YF, Yang Y, Zou P, Chen J, He Y, Shui SL, Cui YR, Bai R, Liang YJ, Hu Y, Jiang B, Lu L, Zhang X, Liu J, Xu J. 2018; AXL promotes Zika virus infection in astrocytes by antagonizing type I interferon signalling. Nat Microbiol. 3:302–309. DOI: 10.1038/s41564-017-0092-4. PMID: 29379210.

35. Dang J, Tiwari SK, Lichinchi G, Qin Y, Patil VS, Eroshkin AM, Rana TM. 2016; Zika virus depletes neural progenitors in human cerebral organoids through activation of the innate immune receptor TLR3. Cell Stem Cell. 19:258–265. DOI: 10.1016/j.stem.2016.04.014. PMID: 27162029. PMCID: PMC5116380.

36. Chen ZL, Yin ZJ, Qiu TY, Chen J, Liu J, Zhang XY, Xu JQ. 2022; Revealing the characteristics of ZIKV infection through tissue-specific transcriptome sequencing analysis. BMC Genomics. 23:697. DOI: 10.1186/s12864-022-08919-5. PMID: 36209057. PMCID: PMC9546753.

37. Muñoz LS, Barreras P, Pardo CA. 2016; Zika virus-associated neurological disease in the adult: Guillain-Barré syndrome, encephalitis, and myelitis. Semin Reprod Med. 34:273–279. DOI: 10.1055/s-0036-1592066. PMID: 27612158.

38. de Paula Freitas B, de Oliveira Dias JR, Prazeres J, Sacramento GA, Ko AI, Maia M, Belfort R Jr. 2016; Ocular findings in infants with microcephaly associated with presumed Zika virus congenital infection in Salvador, Brazil. JAMA Ophthalmol. 134:529–535. DOI: 10.1001/jamaophthalmol.2016.0267. PMID: 26865554. PMCID: PMC5444996.

39. Ryan FJ, Carr JM, Furtado JM, Ma Y, Ashander LM, Simões M, Oliver GF, Granado GB, Dawson AC, Michael MZ, Appukuttan B, Lynn DJ, Smith JR. 2021; Zika virus infection of human iris pigment epithelial cells. Front Immunol. 12:644153. DOI: 10.3389/fimmu.2021.644153. PMID: 33968035. PMCID: PMC8100333.

40. Brand C, Deschamps-Francoeur G, Bullard-Feibelman KM, Scott MS, Geiss BJ, Bisaillon M. 2023; Kunjin virus, Zika virus, and Yellow Fever virus infections have distinct effects on the coding transcriptome and proteome of brain-derived U87 cells. Viruses. 15:1419. DOI: 10.3390/v15071419. PMID: 37515107. PMCID: PMC10385720.

41. Svensson V, Vento-Tormo R, Teichmann SA. 2018; Exponential scaling of single-cell RNA-seq in the past decade. Nat Protoc. 13:599–604. DOI: 10.1038/nprot.2017.149. PMID: 29494575.

42. Ratnasiri K, Wilk AJ, Lee MJ, Khatri P, Blish CA. 2023; Single-cell RNA-seq methods to interrogate virus-host interactions. Semin Immunopathol. 45:71–89. DOI: 10.1007/s00281-022-00972-2. PMID: 36414692. PMCID: PMC9684776.

43. Hein MY, Weissman JS. 2022; Functional single-cell genomics of human cytomegalovirus infection. Nat Biotechnol. 40:391–401. DOI: 10.1038/s41587-021-01059-3. PMID: 34697476.

44. Zanini F, Robinson ML, Croote D, Sahoo MK, Sanz AM, Ortiz-Lasso E, Albornoz LL, Rosso F, Montoya JG, Goo L, Pinsky BA, Quake SR, Einav S. 2018; Virus-inclusive single-cell RNA sequencing reveals the molecular signature of progression to severe Dengue. Proc Natl Acad Sci U S A. 115:E12363–E12369. DOI: 10.1073/pnas.1813819115. PMID: 30530648. PMCID: PMC6310786.

45. Arora JK, Opasawatchai A, Poonpanichakul T, Jiravejchakul N, Sungnak W, Matangkasombut O, Teichmann SA, Matangkasombut P, Charoensawan V. DENFREE Thailand. 2022; Single-cell temporal analysis of natural Dengue infection reveals skin-homing lymphocyte expansion one day before defervescence. iScience. 25:104034. DOI: 10.1016/j.isci.2022.104034. PMID: 35345453. PMCID: PMC8957021.

46. Robinson M, Sweeney TE, Barouch-Bentov R, Sahoo MK, Kalesinskas L, Vallania F, Sanz AM, Ortiz-Lasso E, Albornoz LL, Rosso F, Montoya JG, Pinsky BA, Khatri P, Einav S. 2019; A 20-gene set predictive of progression to severe Dengue. Cell Rep. 26:1104–1111.e4. DOI: 10.1016/j.celrep.2019.01.033. PMID: 30699342. PMCID: PMC6352713.

47. Waickman AT, Friberg H, Gromowski GD, Rutvisuttinunt W, Li T, Siegfried H, Victor K, McCracken MK, Fernandez S, ikiatkhachorn A Sr, Ellison D, Jarman RG, Thomas SJ, Rothman AL, Endy T, Currier JR. 2021; Temporally integrated single cell RNA sequencing analysis of PBMC from experimental and natural primary human DENV-1 infections. PLoS Pathog. 17:e1009240. DOI: 10.1371/journal.ppat.1009240. PMID: 33513191. PMCID: PMC7875406.

48. Giovannoni F, Bosch I, Polonio CM, Torti MF, Wheeler MA, Li Z, Romorini L, Rodriguez Varela MS, Rothhammer V, Barroso A, Tjon EC, Sanmarco LM, Takenaka MC, Modaresi SMS, Gutiérrez-Vázquez C, Zanluqui NG, Dos Santos NB, Munhoz CD, Wang Z, Damonte EB, et al. 2020; AHR is a Zika virus host factor and a candidate target for antiviral therapy. Nat Neurosci. 23:939–951. Erratum. DOI: 10.1038/s41593-020-0664-0. PMID: 32690969. PMCID: PMC7897397.

49. Espada CE, da Rocha EL, Ricciardi-Jorge T, Dos Santos AA, Soares ZG, Malaquias G, Patrício DO, Gonzalez Kozlova E, Dos Santos PF, Bordignon J, Sanford TJ, Fajardo T, Sweeney TR, Báfica A, Mansur DS. 2024; ISG15/USP18/STAT2 is a molecular hub regulating IFN I-mediated control of Dengue and Zika virus replication. Front Immunol. 15:1331731. DOI: 10.3389/fimmu.2024.1331731. PMID: 38384473. PMCID: PMC10879325.

50. Zanini F, Pu SY, Bekerman E, Einav S, Quake SR. 2018; Single-cell transcriptional dynamics of flavivirus infection. Elife. 7:e32942. DOI: 10.7554/eLife.32942. PMID: 29451494. PMCID: PMC5826272.

51. Brown MG, Hermann LL, Issekutz AC, Marshall JS, Rowter D, Al-Afif A, Anderson R. 2011; Dengue virus infection of mast cells triggers endothelial cell activation. J Virol. 85:1145–1150. DOI: 10.1128/JVI.01630-10. PMID: 21068256. PMCID: PMC3019992.

52. Ab-Rahman HA, Rahim H, AbuBakar S, Wong PF. 2016; Macrophage activation syndrome-associated markers in severe Dengue. Int J Med Sci. 13:179–186. DOI: 10.7150/ijms.13680. PMID: 26941578. PMCID: PMC4773282.

53. Leis AA, Grill MF, Goodman BP, Sadiq SB, Sinclair DJ, Vig PJS, Bai F. 2020; Tumor necrosis factor-alpha signaling may contribute to chronic West Nile virus post-infectious proinflammatory state. Front Med (Lausanne). 7:164. DOI: 10.3389/fmed.2020.00164. PMID: 32426358. PMCID: PMC7203783.