Skip to main content

Applying a pan-flavivirus RT-qPCR assay in Brazilian public health surveillance

Abstract

The aim of this study was to improve flavivirus field monitoring in Brazil using a reliable probe-based RT-qPCR assay. Standard flavivirus strains were employed to evaluate the performance of the assay, and its applicability was evaluated using 235 stored pools of Culicidae samples collected between 1993 and 1997 and in 2016. Flavivirus species were identified by sequencing. Sixteen (6.8%) samples tested positive: Ilheus virus, Iguape virus, and Saint Louis encephalitis virus were identified in historical specimens from 1993-1994, while insect-specific flaviviruses were detected in the samples from 2016. This approach was demonstrated to be accurate for flavivirus detection and characterization, and it can be successfully applied for vector surveillance and for monitoring and discovery of insect specific flaviviruses.

This is a preview of subscription content, access via your institution.

References

  1. 1.

    Gould EA, Solomon T (2008) Pathogenic flaviviruses. Lancet (London, England) 371:500–509. https://doi.org/10.1016/S0140-6736(08)60238-X

    CAS  Article  Google Scholar 

  2. 2.

    Heinz FX, Allison SL (2000) Structures and mechanisms in flavivirus fusion. Adv Virus Res 55:231–269

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Fauci AS, Morens DM (2016) Zika virus in the Americas-yet another arbovirus threat. N Engl J Med 374:601–604. https://doi.org/10.1056/NEJMp1600297

    Article  PubMed  Google Scholar 

  4. 4.

    Yun S-I, Lee Y-M (2017) Zika virus: an emerging flavivirus. J Microbiol 55:204–219. https://doi.org/10.1007/s12275-017-7063-6

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Guiana F (2019) Epidemiological Update Yellow Fever. 6–9

  6. 6.

    Junglen S, Kopp A, Kurth A et al (2009) A new flavivirus and a new vector: characterization of a novel flavivirus isolated from uranotaenia mosquitoes from a tropical rain forest. J Virol. https://doi.org/10.1128/JVI.00014-09

    Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Farfan-Ale JA, Lorono-Pino MA, Garcia-Rejon JE et al (2009) Detection of RNA from a novel West Nile-like virus and high prevalence of an insect-specific flavivirus in mosquitoes in the Yucatan Peninsula of Mexico. Am J Trop Med Hyg 80 (1):85–95

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Pauvolid-Corrêa A, Solberg O, Couto-Lima D et al (2015) Nhumirim virus, a novel flavivirus isolated from mosquitoes from the Pantanal, Brazil. Arch Virol 160:21–27. https://doi.org/10.1007/s00705-014-2219-8

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Rocco IM, Santos CLS, Bisordi I et al (2005) St. Louis encephalitis virus: first isolation from a human in Sao Paulo State, Brazil. Rev Inst Med Trop Sao Paulo 47:281–285. https://doi.org/10.1590/S0036-46652005000500008

    Article  PubMed  Google Scholar 

  10. 10.

    Gomes G, Causey OR (1959) Bussuquara, a new arthropod-borne virus. Proc Soc Exp Biol Med 101:275–279. https://doi.org/10.3181/00379727-101-24909

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Nassar ES, Coimbra TLM, Rocco IM et al (1997) Human disease caused by an arbovirus closely related to ilheus virus: report of five cases. Intervirology 40:247–252

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Batista WC, da Tavares GSB, Vieira DS et al (2011) Notification of the first isolation of Cacipacore virus in a human in the State of Rondonia, Brazil. Rev Soc Bras Med Trop 44:528–530

    Article  PubMed  Google Scholar 

  13. 13.

    Kuno G (2003) Serodiagnosis of flaviviral infections and vaccinations in humans. Adv Virus Res 61:3–65

    Article  PubMed  Google Scholar 

  14. 14.

    Mansfield KL, Horton DL, Johnson N et al (2011) Flavivirus-induced antibody cross-reactivity. J Gen Virol 92:2821–2829. https://doi.org/10.1099/vir.0.031641-0

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Kuno G (1998) Universal diagnostic RT-PCR protocol for arboviruses. J Virol Methods 72:27–41

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    de Bronzoni RVM, Baleotti FG, Ribeiro Nogueira RM et al (2005) Duplex reverse transcription-PCR followed by nested PCR assays for detection and identification of brazilian alphaviruses and flaviviruses. J Clin Microbiol 43:696–702. https://doi.org/10.1128/JCM.43.2.696-702.2005

    CAS  Article  PubMed Central  Google Scholar 

  17. 17.

    Patel P, Landt O, Kaiser M et al (2013) Development of one-step quantitative reverse transcription PCR for the rapid detection of flaviviruses. Virol J 10:58. https://doi.org/10.1186/1743-422X-10-58

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Biacchesi S, Skiadopoulos MH, Yang L et al (2005) Rapid human metapneumovirus microneutralization assay based on green fluorescent protein expression. J Virol Methods 128:192–197. https://doi.org/10.1016/j.jviromet.2005.05.005

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Gubler DJ, Kuno G, Sather GE et al (1984) Mosquito cell cultures and specific monoclonal antibodies in surveillance for dengue viruses. Am J Trop Med Hyg 33:158–165

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Domingo C, Patel P, Yillah J et al (2012) Advanced Yellow fever virus genome detection in point-of-care facilities and reference laboratories. J Clin Microbiol 50:4054–4060. https://doi.org/10.1128/JCM.01799-12

    Article  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Dyer J, Chisenhall DM, Mores CN (2007) A multiplexed TaqMan assay for the detection of arthropod-borne flaviviruses. J Virol Methods 145:9–13. https://doi.org/10.1016/j.jviromet.2007.05.001

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Lanciotti RS, Kosoy OL, Laven JJ et al (2008) Genetic and serologic properties of Zika Virus associated with an epidemic, Yap State, Micronesia, 2007. Emerg Infect Dis 14:1232–1239. https://doi.org/10.3201/eid1408.080287

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Cunha MS, da Costa AC, de Azevedo Fernandes NCC et al (2019) Epizootics due to Yellow Fever Virus in Sao Paulo State, Brazil: viral dissemination to new areas (2016–2017). Sci Rep 9:5474. https://doi.org/10.1038/s41598-019-41950-3

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Choi KH, Groarke JM, Young DC et al (2004) The structure of the RNA-dependent RNA polymerase from bovine viral diarrhea virus establishes the role of GTP in de novo initiation. Proc Natl Acad Sci USA 101:4425–4430. https://doi.org/10.1073/pnas.0400660101

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Scaramozzino N, Crance JM, Jouan A et al (2001) Comparison of flavivirus universal primer pairs and development of a rapid, highly sensitive heminested reverse transcription-PCR assay for detection of flaviviruses targeted to a conserved region of the NS5 gene sequences. J Clin Microbiol 39:1922–1927. https://doi.org/10.1128/JCM.39.5.1922-1927.2001

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Tanaka M (1993) Rapid identification of flavivirus using the polymerase chain reaction. J Virol Methods 41:311–322

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Kuno G, Chang G, Tsuchiya K et al (1998) Phylogeny of the genus Flavivirus. J Virol 72:73–83

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Romeiro MF, de Souza WM, Tolardo AL et al (2016) Evaluation and optimization of SYBR Green real-time reverse transcription polymerase chain reaction as a tool for diagnosis of the Flavivirus genus in Brazil. Rev Soc Bras Med Trop 49:279–285. https://doi.org/10.1590/0037-8682-0444-2015

    Article  PubMed  Google Scholar 

  29. 29.

    Moureau G, Temmam S, Gonzalez JP et al (2007) A real-time RT-PCR method for the universal detection and identification of flaviviruses. Vector Borne Zoonotic Dis 7:467–477. https://doi.org/10.1089/vbz.2007.0206

    CAS  Article  PubMed  Google Scholar 

  30. 30.

    Coimbra TL, Nassar ES, Nagamori AH et al (1993) Iguape: a newly recognized flavivirus from Sao Paulo State, Brazil. Intervirology 36:144–152

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Bhatt S, Gething PW, Brady OJ et al (2013) The global distribution and burden of dengue. Nature 496:504–507. https://doi.org/10.1038/nature12060

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Moureau G, Cook S, Lemey P et al (2015) New insights into flavivirus evolution, taxonomy and biogeographic history, extended by analysis of canonical and alternative coding sequences. PLoS One. https://doi.org/10.1371/journal.pone.0117849

    Article  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Simmonds P, Becher P, Bukh J et al (2017) ICTV virus taxonomy profile: flaviviridae. J Gen Virol 98:2–3. https://doi.org/10.1099/jgv.0.000672

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Pauvolid-Correa A, Kenney JL, Couto-Lima D et al (2013) Ilheus virus isolation in the Pantanal, west-central Brazil. PLoS Negl Trop Dis 7:e2318. https://doi.org/10.1371/journal.pntd.0002318

    Article  PubMed  PubMed Central  Google Scholar 

  35. 35.

    da Vieira CJSP, Andrade de CD, Kubiszeski JR et al (2019) Detection of Ilheus virus in mosquitoes from southeast Amazon, Brazil. Trans R Soc Trop Med Hyg 113:424–427. https://doi.org/10.1093/trstmh/trz031

    Article  PubMed  CAS  Google Scholar 

  36. 36.

    Pereira LE, Suzuki A, Coimbra TL et al (2001) Ilheus arbovirus in wild birds (Sporophila caerulescens and Molothrus bonariensis). Rev Saude Publica 35:119–123

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Reisen WK (2003) Epidemiology of St. Louis encephalitis virus. Adv Virus Res 61:139–183

    Article  PubMed  Google Scholar 

  38. 38.

    Bocato-Chamelet EL et al (2001) Isolamento do flavivírus Iguape a partir de mosquitos Anopheles (Kerteszia) cruzii em Juquitiba – Estado de São Paulo – Brasil. Rev Inst Adolfo Lutz 60(1):65–69

    Google Scholar 

  39. 39.

    Guzman H, Contreras-Gutierrez MA, Travassos da Rosa APA et al (2018) Characterization of three new insect-specific flaviviruses: their relationship to the mosquito-borne flavivirus pathogens. Am J Trop Med Hyg 98:410–419. https://doi.org/10.4269/ajtmh.17-0350

    CAS  Article  PubMed  Google Scholar 

  40. 40.

    Gravina HD, Suzukawa AA, Zanluca C et al (2019) Identification of insect-specific flaviviruses in areas of Brazil and Paraguay experiencing endemic arbovirus transmission and the description of a novel flavivirus infecting Sabethes belisarioi. Virology 527:98–106. https://doi.org/10.1016/j.virol.2018.11.008

    CAS  Article  PubMed  Google Scholar 

  41. 41.

    Carrera J-P, Guzman H, Beltran D et al (2015) Mercadeo virus: a novel mosquito-specific flavivirus from Panama. Am J Trop Med Hyg 93:1014–1019. https://doi.org/10.4269/ajtmh.15-0117

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Roiz D, Vazquez A, Seco MP et al (2009) Detection of novel insect flavivirus sequences integrated in Aedes albopictus (Diptera: Culicidae) in Northern Italy. Virol J. https://doi.org/10.1186/1743-422X-6-93

    Article  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Bolling BG, Eisen L, Moore CG, Blair CD (2011) Insect-specific flaviviruses from Culex Mosquitoes in Colorado, with evidence of vertical transmission. Am J Trop Med Hyg 85:169–177. https://doi.org/10.4269/ajtmh.2011.10-0474

    Article  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Hoshino K, Takahashi-Nakaguchi A, Isawa H et al (2012) Entomological surveillance for flaviviruses at migratory bird stopover sites in Hokkaido, Japan, and a new insect flavivirus detected in Aedes galloisi (Diptera: Culicidae). J Med Entomol 49:175–182

    Article  PubMed  Google Scholar 

  45. 45.

    Kenney JL, Solberg OD, Langevin SA, Brault AC (2014) Characterization of a novel insect-specific flavivirus from Brazil: potential for inhibition of infection of arthropod cells with medically important flaviviruses. J Gen Virol 95:2796–2808. https://doi.org/10.1099/vir.0.068031-0

    CAS  Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank the staff of the Vector-Borne Diseases Laboratory of Adolfo Lutz Institute, Akemi Suzuki, Luis Eloy Pereira, Iray Maria Rocco, Terezinha Lisieux Moraes Coimbra and Renato Pereira de Sousa, for laboratory assistance. We thank the staff of the Cellular Cultures Laboratory of Adolfo Lutz Institute, Aurea Silveira Cruz Garçon and Ana Cristina Scarparo de Miranda, for supplying cell lines. We would also like to thank Antonio Erculiani Junior for technical assistance, and the Superintendência de Controle de Endemias-SUCEN for assistance in mosquito collection and identification.

Funding

This work was also supported by FAPESP #2012/23645-4 (Dr. Paulo Cesar Mayorca) and FAPESP #2013/21719–3 (Dr. Maurício Lacerda Nogueira).

Author information

Affiliations

Authors

Contributions

M.S.C. and P.C.M. conceived the study and designed the study protocol; M.S.C., A.L., F.C.P.S., J.S.N., A.Y.M, M.L.N., and P.C.M. participated in conducting the study; M.S.C., J.N.S. and A.Y.M. acquired the data. M.S.C. performed the plaque, TCID50 and IFA assays; M.S.C., G.S.C. and F.C.P.S. performed the RT-qPCR assays; M.S.C. and A.L. conducted the sequencing assays; M.S.C., A.L., F.C.P.S., M.L.N. and P.C.M. analyzed and interpreted the data; M.S.C. and A.L. drafted the manuscript; F.C.P.S, J.S.N., G.S.C., A.Y.M., M.L.N. and P.C.M. critically revised the manuscript for intellectual content. All authors read and approved the final version. M.S.C. is the guarantor of the paper.

Corresponding author

Correspondence to Mariana Sequetin Cunha.

Ethics declarations

Ethical approval

Prior approval was granted by the Ethics Committee for the Use of Animals, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil (CEUA no. 5400050214).

Conflict of interest

None to declare.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Handling Editor: Tim Skern.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 931 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Cunha, M.S., Luchs, A., dos Santos, F.C.P. et al. Applying a pan-flavivirus RT-qPCR assay in Brazilian public health surveillance. Arch Virol 165, 1863–1868 (2020). https://doi.org/10.1007/s00705-020-04680-w

Download citation