Development of a method for detection of specific antibodies to E protein of yellow fever virus (Flaviviridae: Flavivirus) by enzyme immunoassay

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Abstract

Introduction. Yellow fever (YF) remains one of the most common natural focal infectious diseases in the world. In connection with the increasing tourist flow to countries endemic for YF, the discovery of stable populations of Aedes aegypti and Ae. albopictus which are the main vectors of the yellow fever virus (YFV), in the southern regions of Russia, and the fact that in medical institutions in our country it is possible to obtain a live attenuated vaccine against YF, but there is no way to evaluate the effectiveness of vaccination, the question arises of the development and implementation of diagnostic kits for detecting antibodies (AB) to the pathogen by enzyme immunoassay (ELISA).

The aim of this study was to develop a method for detecting specific IgG antibodies to the E protein of YFV by ELISA and assessing its diagnostic characteristics.

Materials and methods. A specific cDNA was synthesized by reverse transcription on an RNA template of YFV isolated on a cell culture of Aedes albopictus clone C6/36, and a fragment of the genome coding the YFV E protein was amplified and subsequently cloned into the plasmid pET160 (Thermo Fisher Scientific, USA). The resulting gene fragment was used as a DNA template to obtain a recombinant analog of the third domain of the YFV E protein in Escherichia coli cells (BL-21(DE3)). Next, the immunogenicity of the obtained antigen was evaluated and the analysis conditions were optimized.

Results. The optimal conditions for the production of the obtained recombinant E protein of YFV were determined, its specificity was confirmed by immunological methods (Western blot and ELISA), sorption buffers and blocking solutions were selected, and sensitivity and specificity of detection of antibodies to YFV using the recombinant antigen were assessed.

Conclusion. A method for the detection of specific IgG antibodies to the YFV E protein by ELISA was developed. This diagnostic kit can be used both to study the protective properties of the YF vaccine and to detect imported cases of infection in non-endemic areas.

About the authors

Ekaterina I. Krivosheina

FSSI State Scientific Center of Virology and Biotechnology “Vector”of the Federal Service for Surveillance of Consumer Rights Protection and Human Welfare (Rospotrebnadzor)

Email: mikkartash@yandex.ru
ORCID iD: 0000-0001-5181-0415
Russian Federation, Koltsovo, Novosibirsk region

Mikhail Y. Kartashov

FSSI State Scientific Center of Virology and Biotechnology “Vector”of the Federal Service for Surveillance of Consumer Rights Protection and Human Welfare (Rospotrebnadzor); Novosibirsk State University

Author for correspondence.
Email: mikkartash@yandex.ru
ORCID iD: 0000-0002-7857-6822

Ph.D. (Biol.), Senior Researcher, Department of Molecular Virology of Flaviviruses and Viral Hepatitis

Russian Federation, Koltsovo, Novosibirsk region; Novosibirsk

Ekaterina V. Naidenova

FSSI Russian Research Anti-Plague Institute «Microbe» of the Federal Service for Surveillance of Consumer Rights Protection and Human Welfare (Rospotrebnadzor)

Email: mikkartash@yandex.ru
ORCID iD: 0000-0001-6474-3696
Russian Federation, Saratov

Nikita D. Ushkalenko

FSSI State Scientific Center of Virology and Biotechnology “Vector”of the Federal Service for Surveillance of Consumer Rights Protection and Human Welfare (Rospotrebnadzor)

Email: mikkartash@yandex.ru
ORCID iD: 0000-0002-2171-7444
Russian Federation, Koltsovo, Novosibirsk region

Stepan A. Pyankov

FSSI State Scientific Center of Virology and Biotechnology “Vector”of the Federal Service for Surveillance of Consumer Rights Protection and Human Welfare (Rospotrebnadzor)

Email: mikkartash@yandex.ru
ORCID iD: 0000-0002-6593-6614
Russian Federation, Koltsovo, Novosibirsk region

Vladimir A. Ternovoi

FSSI State Scientific Center of Virology and Biotechnology “Vector”of the Federal Service for Surveillance of Consumer Rights Protection and Human Welfare (Rospotrebnadzor)

Email: mikkartash@yandex.ru
ORCID iD: 0000-0003-1275-171X
Russian Federation, Koltsovo, Novosibirsk region

Valery B. Loktev

FSSI State Scientific Center of Virology and Biotechnology “Vector”of the Federal Service for Surveillance of Consumer Rights Protection and Human Welfare (Rospotrebnadzor)

Email: mikkartash@yandex.ru
ORCID iD: 0000-0002-0229-321X
Russian Federation, Koltsovo, Novosibirsk region

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Supplementary files

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2. Fig. 1. Schematic representation of YFV viral RNA and polyprotein. Arrows indicate cleavage sites in the polyprotein that are processed by cellular (red arrows) or viral (green arrows) proteases.

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3. Fig. 2. Electrophoregram of E. coli BL-21 (DE3) cell lysates and purified recombinant protein in 15% PAGE-electrophoresis with SDS: 0 – before IPTG induction; 1–7 – incubation at 25°C under various conditions: 1–3 incubation for 5 hours after adding IPTG at concentrations of 0.1, 1 and 10 mM, respectively; 4–6 – incubation for 18 hours after adding IPTG at concentrations of 0.1, 1 and 10 mM, respectively; 7 – 18 hours after adding IPTG at concentration 1 mM/ml; 8 – purified recombinant protein. The arrow indicates the position of the recombinant YFV E protein . M – marker. (IPTG – isopropyl β-D-1-thiogalactopyranoside; YFV – yellow fever virus).

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4. Fig. 3. The results of the detection of the purified antigen by Western blotting: lane 1 – treated with serum containing antibodies to the dengue virus; lane 2 – serum that does not contain antibodies to YFV, TBEV, HCV, dengue virus; lane 3 – blood serum with antibodies to YFV. The arrow indicates the position of the recombinant YFV E protein. YFV – yellow fever virus; TBEV – tick-borne encephalitis virus; HCV – hepatitis C virus.

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5. Fig. 4. The results of ELISA performed to determine the optimal conditions for the sorption of the recombinant YFV antigen (dilution of blood serum 1 : 100).

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Copyright (c) 2022 Krivosheina E.I., Kartashov M.Y., Naidenova E.V., Ushkalenko N.D., Pyankov S.A., Ternovoi V.A., Loktev V.B.

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This work is licensed under a Creative Commons Attribution 4.0 International License.

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