Mem Inst Oswaldo Cruz, Rio de Janeiro, VOLUME 115 | DECEMBER 2020
Original Article

Dengue, Yellow Fever, Zika and Chikungunya epidemic arboviruses in Brazil: ultrastructural aspects

Debora Ferreira Barreto-Vieira1,+, Dinair Couto-Lima2, Fernanda Cunha Jácome1, Gabriela Cardoso Caldas1, Ortrud Monika Barth1

1Fundação Oswaldo Cruz-Fiocruz, Instituto Oswaldo Cruz, Laboratório de Morfologia e Morfogênese Viral, Rio de Janeiro, RJ, Brasil
2Fundação Oswaldo Cruz-Fiocruz, Instituto Oswaldo Cruz, Laboratório de Mosquitos Transmissores de Hematozoários, Rio de Janeiro, RJ, Brasil

DOI: 10.1590/0074-02760200278
451 views 4 downloads

BACKGROUND The impact of arbovirus cocirculation in Brazil is unknown. Dengue virus (DENV) reinfection may result in more intense viraemia or immunopathology, leading to more severe disease. The Zika virus (ZIKV) epidemic in the Americas provided pathogenicity evidence that had not been previously observed in flavivirus infections. In contrast to other flaviviruses, electron microscopy studies have shown that ZIKV may replicate in viroplasm-like structures. Flaviviruses produce an ensemble of structurally different virions, collectively contributing to tissue tropism and virus dissemination.
OBJECTIVES AND METHODS In this work, the Aedes albopictus mosquito cell lineage (C6/36 cells) and kidney epithelial cells from African green monkeys (Vero cells) were infected with samples of the main circulating arboviruses in Brazil [DENV-1, DENV-2, DENV-3, DENV-4, ZIKV, Yellow Fever virus (YFV) and Chikungunya virus (CHIKV)], and ultrastructural studies by transmission electron microscopy were performed.
FINDINGS We observed that ZIKV, the DENV serotypes, YFV and CHIKV particles are spherical. ZIKV, DENV-1, -2, -3 and -4 presented diameters of 40-50 nm, and CHIKV presented approximate diameters of 50-60 nm. Viroplasm-like structures was observed in ZIKV replication cycle.
MAIN CONCLUSIONS The morphogenesis of these arboviruses is similar to what has been presented in previous studies. However, we understand that further studies are needed to investigate the relationship between viroplasm-like structures and ZIKV replication dynamics.

01. Nunes PCG, Daumas RD, Sánchez-Arcila JC, Nogueira RMN, Horta MAP, dos Santos FB. 30 years of fatal dengue cases in Brazil: a review. BMC Public Health. 2019; 19(1): 329-40.
02. Lowe R, Barcellos C, Brasil P, Cruz OG, Honório NA, Kuper H, et al. The Zika virus epidemic in Brazil: from discovery to future implications. Int J Environ Res Public Health. 2018; 5(1): 96-14.
03. Souza TML, Vieira YR, Delatorre E, Barbosa-Lima G, Luiz RLF, Vizzoni A, et al. Emergence of the East-Central-South-African genotype of Chikungunya virus in Brazil and the city of Rio de Janeiro may have occurred years before surveillance detection. Sci Rep. 2019; 9(1): 2760-7.
04. Possas C, Lourenço-de-Oliveira R, Tauil PL, Pinheiro FP, Pissinatti A, da Cunha RV, et al. Yellow fever outbreak in Brazil: the puzzle of rapid viral spread and challenges for immunisation. Mem Inst Oswaldo Cruz. 2018; 113(10): 1-12.
05. Lima-Camara TN. Arboviroses emergentes e novos desafios para a saúde pública no Brasil. Rev Saude Publica. 2016; 50: 36-43.
06. Medeiros AS, Costa DMP, Branco MSD, Souza DM, Monteiro JD, Galvão SPM, et al. Dengue virus in Aedes aegypti and Aedes albopictus in urban areas in the state of Rio Grande do Norte, Brazil: importance of virological and entomological surveillance. PLoS One. 2018; 13(3): 1-11.
07. Donalisio MRI, Freitas ARR, Zuben APBV. Arboviroses emergentes no Brasil: desafios para a clínica e implicações para a saúde pública. Rev Saude Publica. 2017; 51: 30-6.
08. Barreto-Vieira DF, Jácome FC, Silva MAN, Caldas GC, Filippis AMB, Sequeira PC, et al. Structural investigation of C6/36 and Vero cell cultures infected with a Brazilian Zika virus. PLoS One. 2017; 12(9): 1-18.
09. Simizu B, Yamamoto K, Hashimoto K, Ogata T. Structural proteins of Chikungunya virus. J Virol. 1984; 51(1): 254-8.
10. Cao S, Zhang W. Characterization of an early-stage fusion intermediate of sindbis virus using Cryoelectron microscopy. Proc Natl Acad Sci USA. 2013; 110(33): 13362-7.
11. Jose J, Snyder JE, Kuhn RJ. A structural and functional perspective of alphavirus replication and assembly. Future Microbiol. 2009; 4: 837-56.
12. Kalvodova L, Sampaio JL, Cordo S, Ejsing CS, Shevchenko A, Simons K. The lipidomes of vesicular Stomatitis virus, Semliki forest virus, and the host plasma membrane analyzed by quantitative shotgun mass spectrometry. J Virol. 2009; 83: 7996-8003.
13. Gubler DJ, Kuno G, Sather GE, Velez M, Oliver A. Mosquito cell cultures and specific monoclonal antibodies in surveillance for dengue viruses. Am J Trop Med Hyg. 1984; 33: 158-65.
14. Drosten C, Göttig S, Schilling S, Asper M, Panning M, Schmit H, et al. Rapid detection and quantification of RNA of Ebola and Marburg viruses, Lassa virus, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, dengue virus and yellow fever virus by real-time reverse transcription-PCR. J Clin Microbiol. 2002; 40: 2323-30.
15. Lanciotti RS, Calisher CH, Gubler DJ, Chang GJ, Vorndam AV. Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. J Clin Microbiol. 1992; 30(3): 545-51.
16. Mukhopadhyay S, Kuhn RJ, Rossmann MG. A structural perspective of the flavivirus life cycle. Nat Rev Microbiol. 2005; 3(1): 13-22.
17. Cruz-Oliveira C, Freire JM, Conceição TM, Higa LM, Castanho ARB, Da Poian AT. Receptors and routes of dengue virus entry into the host cells. FEMS Microbiol Rev. 2015; 39(2): 155-70.
18. Barth OM. Atlas of dengue viruses morphology and morphogenesis. Rio de Janeiro: Imprinta; 2000. 209 pp.
19. Kostyuchenko VA, Zhang Q, Tan JL, Ng T-S, Lok S-M. Immature and mature dengue serotype 1 virus structures provide insight into the maturation process. J Virol. 2013; 87: 7700-7.
20. Prasad M, Miller AS, Klose T, Sirohi D, Buda G, Jiang W, et al. Structure of the immature Zika virus at 9A resolution. Nat Struct Mol Biol. 2017; 24(2): 184-6.
21. Yu M, Zhang W, Holdaway HA, Li L, Kostyuchenko VA, Chipman PR, et al. Structure of the immature dengue virus at low pH primes proteolytic maturation. Science. 2008; 319(5871): 1834-7.
22. Zhang Y, Corver J, Chipman PR, Zhand W, Pletnev SV, Sedlak D, et al. Rossmann structures of immature flavivirus particles. Embo J. 2003; 22: 2604-13.
23. Barth OM. Replication of dengue viruses in mosquito cell cultures - a model from ultrastructural observations. Mem Inst Oswaldo Cruz. 1992; 87(4): 565-74.
24. Mackenzie JM, Jones MK, Young PR. Immunolocalization of the dengue virus nonstructural glycoprotein NS1 suggests a role in viral RNA replication. Virology. 1996; 220: 232-40.
25. Ng ML, Yeong FM, Tan SH. Cryosubstitution technique reveals new morphology of flavivirus-induced structures. J Virol Methods. 1994; 49: 305-14.
26. Cortese M, Goellner S, Acosta EG, Neufeldt CJ, Oleksiuk O, Lampe M, et al. Ultrastructural characterization of Zika virus replication factories. Cell Rep. 2017; 18(9): 2113-23.
27. Morita K, Nabeshima T, Buerano CC. Japanese encephalitis. Rev Sci Tech. 2015; 34(2): 441-52.
28. Gillespie LK, Hoenen A, Morgan G, Mackenzie JM. The endoplasmic reticulum provides the membrane platform for biogenesis of the flavivirus replication complex. J Virol. 2010; 84: 10438-47.
29. Junjhon JG, Pennington JG, Edwards TJ, Perera R, Lanman J, Kuhn RJ. Ultrastructural characterization and three-dimensional architecture of replication sites in dengue virus-infected mosquito cells. J Virol. 2014; 88: 4687-97.
30. Miorin L, Romero-Brey I, Maiuri P, Hoppe S, Krijnse-Locker J, Bartenschlager R, et al. Three-dimensional architecture of tickborne encephalitis virus replication sites and trafficking of the replicated RNA. J Virol. 2013; 87: 6469-81.
31. Welsch S, Miller S, Romero-Brey I, Merz A, Bleck CKE, Walther P, et al. Composition and three-dimensional architecture of the dengue virus replication and assembly sites. Cell Host Microbe. 2009; 5: 365-75.
32. Romero-Brey I, Bartenschlager R. Membranous replication factories induced by plus-strand RNA viruses. Viruses. 2014; 6: 2826-57.
33. Romero-Brey I, Bartenschlager R. Endoplasmic reticulum: the favorite intracellular ciche for viral replication and assembly. Viruses. 2016; 8: 1-26.
34. Christopher N, Moffat K, Brooks E, Wileman T. A guide to viral inclusions, membrane rearrangements, factories, and viroplasm produced during virus replication. Adv Virus Res. 2007; 70: 101-82.
35. Moshe A, Gorovits R. Virus-induced aggregates in infected cells. Viruses. 2012; 4(10): 2218-32.
36. Eichwald C, Arnoldi F, Laimbacher AS, Schraner EM, Fraefel C, Wild P, et al. Rotavirus viroplasm fusion and perinuclear localization are dynamic processes requiring stabilized microtubules. PLoS One. 2012; 7(10): 1-16.
37. Majerowicz S, Kubelka CF, Stephens P, Barth OM. Ultrastructural study of experimental infection of rotavirus in a murine heterologous model. Mem Inst Oswaldo Cruz. 1994; 89(3): 395-402.
38. Szajner P, Weisberg AS, Wolffe EJ, Moss B. Vaccinia virus A30L protein is required for association of viral membranes with dense viroplasm to form immature virions. J Virol. 2001; 75(13): 5752-61.
39. Marie S-M, Scola BL, Barrassi L, Espinosa L, Raoult D. Ultrastructural Characterization of the giant volcano-like virus factory of Acanthamoeba polyphaga Mimivirus. PLoS One. 2007; 2(3): e328.
40. Sourisseau M, Schilte C, Casartelli N, Trouillet C, Guivel- Benhassine, Rudnicka D, et al. Characterization of reemerging chikungunya virus. PLoS Pathog. 2007; 3(6): 1-14.
41. van Duijl-Richter MK, Hoornweg TE, Rodenhuis-Zybert IA, Smit JM. Early events in Chikungunya virus infection-from virus cell binding to membrane fusion. Viruses. 2015; 7(7): 3647-74.
42. Bernard E, Solignant M, Gay B, Chazal N, Higgs S, Devaux C, et al. Endocytosis of Chikungunya virus into mammalian cells: role of clathrin and early endosomal compartments. PLoS One. 2010; 5(7): 1-11.
43. Wu D, Zhang Y, Zhouhui Q, Kou J, Liang W, Zhang H, et al. Chikungunya virus with E1-A226V mutation causing two outbreaks in 2010, Guangdong, China. Virol J. 2013; 10: 1-9.
44. Frolova EI, Gorchakov R, Pereboeva L, Atasheva S, Frolov I. Functional Sindbis virus replicative complexes are formed at the plasma membrane. J Virol. 2010; 84(22): 11679-95.
45. Spuul P, Balistreti G, Kääriäinen L, Ahola T. Phosphatidylinositol 3-kinase-, actin-, and microtubule-dependent transport of Semliki Forest virus replication complexes from the plasma membrane to modified lysosomes. J Virol. 2010; 84(15): 7543-57.
46. Kujala P, Ikäheimonen A, Ehsani N, Vihinen H, Auvinen P, Kääriäinen L. Biogenesis of the Semliki Forest virus RNA replication complex. J Virol. 2001; 75(8): 3873-84.
47. Thaa B, Biasiotto R, Eng K, Neuvonen M, Gotte B, Rheinemann L, et al. Differential PI3K-Akt-mTOR activation by Semliki Forest and Chikungunya virus, dependent on nsP3 and connected to replication complex internalization. J Virol. 2015; 89(22): 11420-37.
48. Utt A, Quirin T, Saul S, Hellstrom K, Ahola T, Merits A. Versatile trans-replication systems for Chikungunya virus allow functional analysis and tagging of every replicase protein. PLoS One. 2016; 11(3): 1-27.
49. Silva LA, Dermody TS. Chikungunya virus: epidemiology, replication, disease mechanisms, and prospective intervention strategies. J Clin Invest. 2017; 127(3): 737-49.
50. Jin J, Galaz-Montoya G, Sherman MB, Sun SY, Goldsmith CS, O’Toole ET, et al. Neutralizing antibodies inhibit Chikungunya virus budding at the plasma membrane. Cell Host Microbe. 2018; 24(3): 417-28.

+ Corresponding author:
Received 01 June 2020
Accepted 21 December 2020

Our Location

Memórias do Instituto Oswaldo Cruz

Av. Brasil 4365, Castelo Mourisco 
sala 201, Manguinhos, 21040-900 
Rio de Janeiro, RJ, Brazil

Tel.: +55-21-2562-1222

This email address is being protected from spambots. You need JavaScript enabled to view it.

Support Program


fiocruz governo
faperj cnpq capes