Production and diagnostic application of recombinant domain III of West Nile envelope protein in Brazil

West Nile virus (WNV) is a flavivirus with a natural cycle involving mosquitoes and birds. Over the last 11 years, WNV has spread throughout the Americas with the imminent risk of its introduction in Brazil.

METHODS:

Envelope protein domain III of WNV (rDIII) was bacterially expressed and purified. An enzyme-linked immunosorbent assay with WNV rDIII antigen was standardized against mouse immune fluids (MIAFs) of different flavivirus.

RESULTS:

WNV rDIII reacted strongly with St. Louis encephalitis virus (SLEV) MIAF but not with other flaviviruses.

CONCLUSIONS:

This antigen may be a potentially useful tool for serologic diagnosis and may contribute in future epidemiological surveillance of WNV infections in Brazil.

Diagnosis; WNV antibodies; ELISA

Authorship

Juliana Helena Chávez

Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Centro de Pesquisa em Virologia, Ribeirão Preto, SPUniversidade de São PauloRibeirão Preto, SPUniversidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Centro de Pesquisa em Virologia, Ribeirão Preto, SP

Vinicius Pinho dos Reis

Jaqueline Raymondi Silva

Helen Julie Laure

Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Centro de Química de Proteínas, Ribeirão Preto, SPUniversidade de São PauloRibeirão Preto, SPUniversidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Centro de Química de Proteínas, Ribeirão Preto, SP

Universidade de São Paulo, Fundação Hemocentro de Ribeirão Preto, Ribeirão Preto, SPUniversidade de São PauloRibeirão Preto, SPUniversidade de São Paulo, Fundação Hemocentro de Ribeirão Preto, Ribeirão Preto, SP

José Cesar Rosa

Benedito Antônio Lopes da Fonseca

Luiz Tadeu Moraes Figueiredo

Address to Dr. Luiz Tadeu Moraes Figueiredo. Centro de Pesquisa em Virologia/FMRP/USP. Av. Bandeirantes 3900, 14049-900 Ribeirão Preto, SP, Brasil. Phone: 55 16 3602-3270 e-mail: ltmfigue@fmrp.usp.br; julianachavez@icbim.ufu.br

CONFLICT OF INTEREST The authors declare that there is no conflict of interest.

SCIMAGO INSTITUTIONS RANKINGS

Figures | Tables

Figures (2)
Tables (1)

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FIGURE 1
Expression and purification of rDIII-WNV from induced M15 Escherichia coli cells. Protein samples were subjected to 12% SDS-PAGE and visualized by Coomassie brilliant blue staining. Lane 1: Elution fraction obtained with elution buffer (NaH2PO4 100mM, Tris-HCL 10mM, and urea 8M, pH 4.5) by Ni-NTA purification in propylene columns, presence of a purified protein with 15kDa. Lanes 2-4: Crude extraction from induced M15 Escherichia coli cells with IPTG (1mM) after 4, 3, and 2h, respectively. Lane 5: Flow through fraction. Lane 6: Wash fraction. kDa, kilodaltons based on protein marker PageRulerTM Prestained Protein Ladder (Fermentas, USA). rDIII: Envelope protein domain III of WNV; WNV: West Nile virus; SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; NaH2PO4: sodium dihydrogen phosphate; IPTG: Isopropyl β-D-1-thiogalactopyranoside

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FIGURE 2
Immunoblotting of rDIII-WNV with IgM WNV monoclonal antibody. Protein samples were subjected to 12% SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad, USA) at 100V and 4oC, for 1h. Blocking was performed for 1h (3% BSA). Membranes were incubated with 1:100 dilution of IgM WNV monoclonal antibody (Pan-Bio, Australia) for 1h, and bound antibodies were detected using a goat immunoglobulin anti-human IgM conjugated with peroxidase (1:1.000) (Sigma, USA). The reaction was developed with 3,3'-diaminobenzidine (DAB) in a solution also including 0.1% of H2O2. Lane 1: rDIII WNV in crude extract from M15 Escherichia coli-induced cells. Lane 2: rDIII WNV in eluted fraction after Ni-NTA purification in propylene columns. rDIII: Envelope protein domain III of WNV; WNV: West Nile virus; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

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TABLE 1
Reactivity of murine polyclonal antibodies (MIAFs) with rDIIIWNV antigen in the standardized IgG-ELISA. rDIII-WNV antigen was used in 4μg/mL.

FIGURE 1 Expression and purification of rDIII-WNV from induced M15 Escherichia coli cells. Protein samples were subjected to 12% SDS-PAGE and visualized by Coomassie brilliant blue staining. Lane 1: Elution fraction obtained with elution buffer (NaH2PO4 100mM, Tris-HCL 10mM, and urea 8M, pH 4.5) by Ni-NTA purification in propylene columns, presence of a purified protein with 15kDa. Lanes 2-4: Crude extraction from induced M15 Escherichia coli cells with IPTG (1mM) after 4, 3, and 2h, respectively. Lane 5: Flow through fraction. Lane 6: Wash fraction. kDa, kilodaltons based on protein marker PageRulerTM Prestained Protein Ladder (Fermentas, USA). rDIII: Envelope protein domain III of WNV; WNV: West Nile virus; SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; NaH2PO4: sodium dihydrogen phosphate; IPTG: Isopropyl β-D-1-thiogalactopyranoside

FIGURE 2 Immunoblotting of rDIII-WNV with IgM WNV monoclonal antibody. Protein samples were subjected to 12% SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad, USA) at 100V and 4oC, for 1h. Blocking was performed for 1h (3% BSA). Membranes were incubated with 1:100 dilution of IgM WNV monoclonal antibody (Pan-Bio, Australia) for 1h, and bound antibodies were detected using a goat immunoglobulin anti-human IgM conjugated with peroxidase (1:1.000) (Sigma, USA). The reaction was developed with 3,3'-diaminobenzidine (DAB) in a solution also including 0.1% of H2O2. Lane 1: rDIII WNV in crude extract from M15 Escherichia coli-induced cells. Lane 2: rDIII WNV in eluted fraction after Ni-NTA purification in propylene columns. rDIII: Envelope protein domain III of WNV; WNV: West Nile virus; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

TABLE 1 Reactivity of murine polyclonal antibodies (MIAFs) with rDIIIWNV antigen in the standardized IgG-ELISA. rDIII-WNV antigen was used in 4μg/mL.

^*
Absorbance values represent the mean of quadruplicate wells. Equivalent values were reached in three independent experiments. SLEV MIAF (1:100) and other MIAFS were used as primary antibody. Secondary antibody (1:2,000) was an anti-mouse IgG conjugated with peroxidase (Sigma, USA). Expression vector PQE31 alone was induced and purified to be used as a control for nonspecific binding of antibodies. MIAFs of Oropouche virus and Mayaro virus were used as primary antibody negative controls. MIAFs: mouse immune ascitic fluids; rDIII: envelope protein domain III of WNV; WNV: West Nile virus; IgG-ELISA: immunoglobulin G-enzymelinked immunosorbent assay; PQE31: Expression vector; SLEV: Saint Louis encephalitis virus; ROCV: Rocio virus; CPCV: Cacipacoré virus; OROV: Oropouche virus; MAYV: Mayaro virus.

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Sociedade Brasileira de Medicina Tropical - SBMT Caixa Postal 118, 38001-970 Uberaba MG Brazil, Tel.: +55 34 3318-5255 / +55 34 3318-5636/ +55 34 3318-5287, http://rsbmt.org.br/ - Uberaba - MG - Brazil
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Production and diagnostic application of recombinant domain III of West Nile envelope protein in Brazil

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[1] Address to Dr. Luiz Tadeu Moraes Figueiredo. Centro de Pesquisa em Virologia/FMRP/USP. Av. Bandeirantes 3900, 14049-900 Ribeirão Preto, SP, Brasil. Phone: 55 16 3602-3270 e-mail: ltmfigue@fmrp.usp.br; julianachavez@icbim.ufu.br