Improvement of a RT-PCR assay for Yellow Fever virus genome detection

Tatiana Carneiro da Rocha, Mario Antônio Navarro-Silva, Ana Caroline Dalla Bona, Marcio Roberto Teixeira Nunes, Walfrido Kühl Svoboda, Eliane Carneiro Gomes

Abstract


The aim of the present study was to describe an improved protocol of reverse transcription polymerase chain reaction (RT-PCR) for Yellow Fever virus genome detection. A strain of ribonucleic acid of Yellow Fever virus was submitted to the improved protocol of RT-PCR and the amplicons were visualized under ultraviolet transilluminator, purifed and sequenced. The nucleotide sequence obtained was compared with sequences available in GenBank using the tblastx tool. The amplicons produced by the strain of ribonucleic acid of Yellow Fever virus exhibited fragments of 400 and 800 base pairs and the consensus sequence exhibited a similarity of 100% with Yellow Fever virus sequences recorded in GenBank. The improved protocol described in this study allowed Yellow Fever virus genome detection and enabled the elimination of the nested-PCR step, which has been frequently associated with contamination. In addition, it reduced the time of reaction, the cost of reagents and the possibility of sample contamination. New methods of investigating these infections must be elaborated and a continuous vigilance of these viruses in their different vectors and hosts is required to avoid negative impacts on human health, tourism and trade

Keywords


Yellow Fever virus; Flavivirus; Reverse Transcriptase Polymerase Chain Reaction.

Full Text:

PDF

References


Araújo FAA, Ramos DG, Santos AL, Passos PHO, Elkhoury ANSM, Costa ZGA, Leal SG, Romano APMR. Epizootias em primatas não humanos durante reemergência do vírus da febre amarela no Brasil, 2007 a 2009. Epidemiol Serv Saúde. 2011;20(4):527-36.

Araújo JMG, Gomes GM, Faria NRC, Araújo ESM, Filippis AMB, Santos FB, Schatzmayr HG, Nogueira RM. Evaluation of a generic RT-nested-PCR for detection of flaviviruses in suspected fatal cases of dengue infection, Rio de Janeiro, Brazil. J Virol Methods. 2012;186(1-2):167-70.

Ayers M, Adachi D, Johnson G, Andonova M, Drebot M, Tellier R. A single tube RT-PCR assay for the detection of mosquito-borne flaviviruses. J Virol Methods. 2006; 135(2):235-9.

Bona ACD, Twerdochlib AL, Navarro-Silva MA. Genetic diversity of dengue virus sorotypes 1 and 2 in state of Paraná, Brazil, based on a fragment of the capisid/ premembrane junction region. Rev Soc Bras Med Trop. 2012;45(3):297-300.

Brasil. Ministério da Saúde, Departamento de Vigilância em Saúde. Boletim Epidemiológico 2015. Available from: portalsaude.saude.gov.br/images/pdf/2015/ agosto/07/2015-008---FA.pdf.

Calisher CH, Karabatsos N, Dalrymple JM, Shope RE, Porterfield JS, Westaway EG, Brandt WE. Antigenic relationships between flaviviruses as determined by crossneutralization tests with polyclonal antisera. J Gen Virol. 1989;70(Pt 1):37-43.

Eldadah ZA, Asher DM, Godec MS, Pomeroy KL, Goldfarb LG, Feinstone SM, Levitan H, Gibbs CJ Jr, Gajdusek DC. Detection of Flaviviruses by reverse-transcriptase polymerase chain reaction. J Med Virol. 1991;33(4):260-7.

Gubler DJ. The changing epidemiology of yellow fever and dengue, 1900 to 2003: full circle? Comp Immunol Microbiol Infect Dis. 2004;27(5):319-30.

Heinz FX, Stiasny K. Flaviviruses and their antigenic structure. J Clin Virol. 2012;55(4):289-95.

ICTV. International Committee on Taxonomy of Viruses 2014. Available from: http://www.ictv online.org/virusTaxonomy.asp.

Kuno G. Universal diagnostic RT-PCR protocol for arboviruses. J Virol Methods. 1998;72(1):27-41.

Lanciotti RS. Molecular amplification assays for the detection of flaviviruses. Adv Virus Res. 2003;61:67-99.

Maher-Sturgess SL, Forrester NL, Wayper PJ, Gould EA, Hall RA, Barnard RT, Gibbs MJ. Universal primers that amplify RNA from all three Flavivirus subgroups. Virol J. 2008;5(16):1-10.

Meiyu F, Huosheng C, Cuihua C, Xiaodong T, Lianhua J, Yifei P, Weijun C, Huiyu G. Detection of flaviviruses by reverse transcriptase polymerase chain reaction with the universal primer set. Microbiol Immunol. 1997;41(3):209-13.

Monath TP, Heinz FX. Flaviviruses. In Fields BN, Knipe DM, Howley PM. Virology. Philadelphia: Lippincott-Raven Publishers; 1996. p. 691-1034.

Moraes-Bronzoni RV, Baleotti FG, Nogueira RMR, Nunes M, Figueiredo LTM. Duplex reverse transcription-PCR followed by nested PCR assays for detection and identification of Brazilian alphaviruses and flaviviruses. J Clin Microbiol. 2005;43(2): 696-702.

Moreli ML, Aquino VH, Cruz AC, Figueiredo LTM. Diagnosis of Oropouche virus infection by RT-nested-PCR. J Med Virol. 2002;66(1):139-42.

Nunes MRT, Palacios G, Nunes KNB, Casseb SMM, Martins LC, Quaresma JAS, Savji N, Lipkin WI, Vasconcelos PF. Evaluation of two molecular methods for the detection of Yellow fever virus genome. J Virol Methods. 2011;174(1-2):29-34.

Pabbaraju K, Ho KCY, Wong S, Fox JD, Kaplen B, Tyler S, Debrot M, Tilley PA. Surveillance of mosquito-borne viruses in Alberta using reverse transcription polymerase chain reaction with generic primers. J Med Entomol. 2009;46(3):640-8.

Pierre V, Drouet M-T, Deubel V. Identification of mosquito-borne flavivirus sequences using universal primers and reverse transcription/polymerase chain reaction. Res Virol. 1994;145(2):93-104.

Rice CM, Lenches EM, Eddy SR, Shin SJ, Sheets RL, Strauss JH. Nucleotide sequence of yellow fever virus: implications for flavivirus gene expression and evolution. Science. 1985;229(4715):726-33.

Scaramozzino N, Crance JM, Jouan A, Debriel DA, Stoll F, Garin D. Comparison of flavivirus universal primer pair and development of a rapid, highly sensitive heminested reverse transcription-PCR assay for detection of flaviviruses target to a conserved region of the NS5 gene sequences. J Clin Microbiol. 2001;39(5):1922-7.

Seah CLK, Chow VTK, Chan YC. Semi-nested PCR using NS3 primers for the detection and typing of dengue viruses in clinical serum specimens. Clin Diagn Virol. 1995;4(2):113-20.

Tanaka M 1993. Rapid identification of flavivirus using the polymerase chain reaction. J Virol Methods. 1993;41(3):311-22.


Refbacks

  • There are currently no refbacks.



Licença Creative Commons
Este obra está licenciado com uma Licença Creative Commons Atribuição-NãoComercial 4.0 Internacional.