1887

Abstract

Although RNA viruses have high mutation rates, host cells and organisms work as selective environments, maintaining the viability of virus populations by eliminating deleterious genotypes. In serial passages of RNA viruses in a single cell line, most of these selective bottlenecks are absent, with no virus circulation and replication in different tissues or host alternation. In this work, Aag-2 cells were accidentally infected with Chikungunya virus (CHIKV) and Mayaro virus (MAYV). After numerous passages to achieve infection persistency, the infectivity of these viruses was evaluated in C6/36 cells, African green monkey Vero cells and primary-cultured human fibroblasts. While these CHIKV and MAYV isolates were still infectious to mosquito cells, they lost their ability to infect mammalian cells. After genome sequencing, it was observed that CHIKV accumulated many nonsynonymous mutations and a significant deletion in the coding sequence of the hypervariable domain in the gene. Since MAYV showed very low titres, it was not sequenced successfully. Persistently infected Aag-2 cells also accumulated high loads of short and recombinant CHIKV RNAs, which seemed to have been originated from virus-derived DNAs. In conclusion, the genome of this CHIKV isolate could guide mutagenesis strategies for the production of attenuated or non-infectious (to mammals) CHIKV vaccine candidates. Our results also reinforce that a paradox is expected during passages of cells persistently infected by RNA viruses: more loosening for the development of more diverse virus genotypes and more pressure for virus specialization to this constant cellular environment.

Funding
This study was supported by the:
  • Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
    • Principle Award Recipient: BergmannM. Ribeiro
  • Conselho Nacional de Desenvolvimento Científico e Tecnológico
    • Principle Award Recipient: RenatoO. Resende
  • Fundação de Apoio à Pesquisa do Distrito Federal
    • Principle Award Recipient: RenatoO. Resende
Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.001700
2021-12-08
2022-01-28
Loading full text...

Full text loading...

References

  1. Huang YJS, Higgs S, Vanlandingham DL. Arbovirus-mosquito vector-host interactions and the impact on transmission and disease pathogenesis of arboviruses. Front Microbiol 2019; 10:22. [View Article] [PubMed]
    [Google Scholar]
  2. Kuno G, Chang GJJ. Biological transmission of arboviruses: Reexamination of and new insights into components, mechanisms, and unique traits as well as their evolutionary trends. Clin Microbiol Rev 2005; 18:608–637 [View Article] [PubMed]
    [Google Scholar]
  3. Lee WS, Webster JA, Madzokere ET, Stephenson EB, Herrero LJ. Mosquito antiviral defense mechanisms: A delicate balance between innate immunity and persistent viral infection. Parasit Vectors 2019; 12:165 [View Article] [PubMed]
    [Google Scholar]
  4. Sim S, Jupatanakul N, Dimopoulos G. Mosquito immunity against arboviruses. Viruses 2014; 6:4479–4504 [View Article] [PubMed]
    [Google Scholar]
  5. Ding SW, Voinnet O. Antiviral immunity directed by small RNAs. Cell 2007; 130:413–426 [View Article] [PubMed]
    [Google Scholar]
  6. Varjak M, Maringer K, Watson M, Sreenu VB, Fredericks AC et al. Aedes aegypti Piwi4 is a noncanonical PIWI protein involved in antiviral responses. mSphere 2017; 2: [View Article]
    [Google Scholar]
  7. Miesen P, Girardi E, van Rij RP. Distinct sets of PIWI proteins produce arbovirus and transposon-derived piRNAs in Aedes aegypti mosquito cells. Nucleic Acids Res 2015; 43:6545–6556 [View Article] [PubMed]
    [Google Scholar]
  8. Wagar ZL, Tree MO, Mpoy MC, Conway MJ. Low density lipopolyprotein inhibits flavivirus acquisition in Aedes aegypti. Insect Mol Biol 2017; 26:734–742 [View Article]
    [Google Scholar]
  9. Nguyet MN, Duong THK, Trung VT, Nguyen THQ, Tran CNB et al. Host and viral features of human dengue cases shape the population of infected and infectious Aedes aegypti mosquitoes. Proc Natl Acad Sci U S A 2013; 110:9072–9077 [View Article] [PubMed]
    [Google Scholar]
  10. Bottino-Rojas V, Talyuli OAC, Jupatanakul N, Sim S, Dimopoulos G et al. Heme signaling impacts global gene expression, immunity and dengue virus infectivity in Aedes aegypti. PLoS One 2015; 10:e0135985. [View Article] [PubMed]
    [Google Scholar]
  11. Liu J, Liu Y, Nie K, Du S, Qiu J et al. Flavivirus NS1 protein in infected host sera enhances viral acquisition by mosquitoes. Nat Microbiol 2016; 1:16087 [View Article] [PubMed]
    [Google Scholar]
  12. Apte-Deshpande A, Paingankar M, Gokhale MD, Deobagkar DN. Serratia odorifera a midgut inhabitant of Aedes aegypti mosquito enhances its susceptibility to dengue-2 virus. PLoS One 2012; 7:e40401 [View Article] [PubMed]
    [Google Scholar]
  13. Angleró-Rodríguez YI, Talyuli OA, Blumberg BJ, Kang S, Demby C et al. An Aedes aegypti-associated fungus increases susceptibility to dengue virus by modulating gut trypsin activity. eLife 2017; 6:e28844. [View Article] [PubMed]
    [Google Scholar]
  14. O’Neal ST, Samuel GH, Adelman ZN, Myles KM. Mosquito-borne viruses and suppressors of invertebrate antiviral RNA silencing. Viruses 2014; 6:4314–4331 [View Article] [PubMed]
    [Google Scholar]
  15. Goic B, Vodovar N, Mondotte JA, Monot C, Frangeul L et al. RNA-mediated interference and reverse transcription control the persistence of RNA viruses in the insect model Drosophila. Nat Immunol 2013; 14:396–403 [View Article]
    [Google Scholar]
  16. Poirier EZ, Goic B, Tomé-Poderti L, Frangeul L, Boussier J et al. Dicer-2-dependent generation of viral DNA from defective genomes of RNA viruses modulates antiviral immunity in insects. Cell Host Microbe 2018; 23:353–365 [View Article] [PubMed]
    [Google Scholar]
  17. Goic B, Stapleford KA, Frangeul L, Doucet AJ, Gausson V et al. Virus-derived DNA drives mosquito vector tolerance to arboviral infection. Nat Commun 2016; 7:12410. [View Article] [PubMed]
    [Google Scholar]
  18. Wesula Olivia L, Obanda V, Bucht G, Mosomtai G, Otieno V et al. Global emergence of Alphaviruses that cause arthritis in humans. Infect Ecol Epidemiology 2015; 5:29853 [View Article]
    [Google Scholar]
  19. Chen R, Mukhopadhyay S, Merits A, Bolling B, Nasar F et al. ICTV virus taxonomy profile: Togaviridae. J Gen Virol 2018; 99:761–762 [View Article]
    [Google Scholar]
  20. Dolan PT, Whitfield ZJ, Andino R. Mapping the evolutionary potential of RNA viruses. Cell Host Microbe 2018; 23:435–446 [View Article] [PubMed]
    [Google Scholar]
  21. Hughes AL, Hughes MAK. More effective purifying selection on RNA viruses than in DNA viruses. Gene 2007; 404:117–125 [View Article] [PubMed]
    [Google Scholar]
  22. Lequime S, Fontaine A, Ar Gouilh M, Moltini-Conclois I, Lambrechts L et al. Genetic drift, purifying selection and vector genotype shape dengue virus intra-host genetic diversity in mosquitoes. PLoS Genet 2016; 12:e1006111 [View Article]
    [Google Scholar]
  23. Riemersma KK, Coffey LL. Chikungunya virus populations experience diversity- dependent attenuation and purifying intra-vector selection in Californian Aedes aegypti mosquitoes. PLoS Negl Trop Dis 2019; 13:e0007853 [View Article] [PubMed]
    [Google Scholar]
  24. Lin J-J, Bhattacharjee MJ, Yu C-P, Tseng YY, Li W-H. Many human RNA viruses show extraordinarily stringent selective constraints on protein evolution. Proc Natl Acad Sci USA 2019; 116:19009–19018 [View Article]
    [Google Scholar]
  25. Silva ALG, Carvalho NV, Paterno LG, Moura LD, Filomeno CL et al. Methylene blue associated with maghemite nanoparticles has antitumor activity in breast and ovarian carcinoma cell lines. Cancer Nano 2021; 12:11 [View Article]
    [Google Scholar]
  26. Vasconcellos AF, Silva JMF, de Oliveira AS, Prado PS, Nagata T et al. Genome sequences of chikungunya virus isolates circulating in midwestern Brazil. Arch Virol 2019; 164:1205–1208 [View Article]
    [Google Scholar]
  27. Laposova K, Oveckova I, Tomaskova J. A simple method for isolation of cell-associated viral particles from cell culture. J Virol Methods 2017; 249:194–196 [View Article]
    [Google Scholar]
  28. Vasconcellos AF, Mandacaru SC, de Oliveira AS, Fontes W, Melo RM et al. Dynamic proteomic analysis of Aedes aegypti Aag-2 cells infected with Mayaro virus. Parasit Vectors 2020; 13:297 [View Article] [PubMed]
    [Google Scholar]
  29. Lundstrom K. Purification and concentration of alphavirus. Cold Spring Harb Protoc 2012 [View Article]
    [Google Scholar]
  30. SDS-PAGE Gel. Cold Spring Harb Protoc 2015 [View Article]
    [Google Scholar]
  31. Schrauf S, Tschismarov R, Tauber E, Ramsauer K. Current efforts in the development of vaccines for the prevention of zika and chikungunya virus infections. Front Immunol 2020; 11:592 [View Article] [PubMed]
    [Google Scholar]
  32. Gorchakov R, Wang E, Leal G, Forrester NL, Plante K et al. Attenuation of chikungunya virus vaccine strain 181/Clone 25 is determined by two amino acid substitutions in the E2 envelope glycoprotein. J Virol 2012; 86:6084–6096 [View Article] [PubMed]
    [Google Scholar]
  33. Mohamed Ali S, Amroun A, de Lamballerie X, Nougairède A. Evolution of Chikungunya virus in mosquito cells. Sci Rep 2018; 8:16175 [View Article] [PubMed]
    [Google Scholar]
  34. Brackney DE, Scott JC, Sagawa F, Woodward JE, Miller NA et al. C6/36 Aedes albopictus cells have a dysfunctional antiviral RNA interference response. PLoS Negl Trop Dis 2010; 4:e856. [View Article] [PubMed]
    [Google Scholar]
  35. Pohjala L, Utt A, Varjak M, Lulla A, Merits A et al. Inhibitors of alphavirus entry and replication identified with a stable Chikungunya replicon cell line and virus-based assays. PLoS ONE 2011; 6:e28923 [View Article] [PubMed]
    [Google Scholar]
  36. Hallengard D, Kakoulidou M, Lulla A, Kummerer BM, Johansson DX et al. Novel attenuated Chikungunya vaccine candidates elicit protective immunity in C57BL/6 mice. J Virol 2013; 88:2858–2866 [View Article]
    [Google Scholar]
  37. Götte B, Liu L, McInerney GM. The enigmatic alphavirus non-structural protein 3 (nsP3) revealing its secrets at last. Viruses 2018; 10:E105. [View Article] [PubMed]
    [Google Scholar]
  38. Galbraith SE, Sheahan BJ, Atkins GJ. Deletions in the hypervariable domain of the nsP3 gene attenuate Semliki Forest virus virulence. J Gen Virol 2006; 87:937–947 [View Article]
    [Google Scholar]
  39. Liljeström P, Lusa S, Huylebroeck D, Garoff H. In vitro mutagenesis of a full-length cDNA clone of Semliki Forest virus: the small 6,000-molecular-weight membrane protein modulates virus release. J Virol 1991; 65:4107–4113 [View Article]
    [Google Scholar]
  40. Nag DK, Brecher M, Kramer LD. DNA forms of arboviral RNA genomes are generated following infection in mosquito cell cultures. Virology 2016; 498:164–171 [View Article]
    [Google Scholar]
  41. Levi LI, Rezelj VV, Henrion-Lacritick A, Erazo D, Boussier J et al. Defective viral genomes from chikungunya virus are broad-spectrum antivirals and prevent virus dissemination in mosquitoes. PLoS Pathog 2021; 17:e1009110 [View Article]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/jgv.0.001700
Loading
/content/journal/jgv/10.1099/jgv.0.001700
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error