Ann Lab Med 2017; 37(4): 313-319  
Detection of Serum Antibodies to Hepatitis E Virus Based on HEV Genotype 3 ORF2 Capsid Protein Expressed in Nicotiana benthamiana
Milena Mazalovska, M.S.1, Nikola Varadinov, M.S.1, Tsvetoslav Koynarski, Ph.D.2, Ivan Minkov, Ph.D.1, Pavel Teoharov, Ph.D.3,†, George P. Lomonossoff, Ph.D.4, and Gergana Zahmanova, Ph.D.1
Department of Plant Physiology and Molecular Biology1, University of Plovdiv “Paisii Hilendarski”, Plovdiv, Bulgaria; Department of Animal Genetics2, Faculty of Veterinary Medicine, Trakia University, Stara Zagora, Bulgaria; National Centre of Infectious and Parasitic Diseases3, Sofia, Bulgaria; Department of Biological Chemistry4, John Innes Centre, Norwich Research Park, Norwich, UK
Correspondence to: Gergana Zahmanova
Department of Plant Physiology and Molecular Biology, University of Plovdiv24 Tzar Assen str, Plovdiv 4000, Bulgaria
Tel: +359-32-261529
Fax: +359-32-261560
Received: November 5, 2016; Revised: December 8, 2016; Accepted: March 8, 2017; Published online: July 1, 2017.
© The Korean Society for Laboratory Medicine. All rights reserved.

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Background: Hepatitis E virus (HEV) causes epidemics in developing countries and is primarily transmitted through the fecal-oral route. There have been recent reports on the zoonotic spread of the virus, and several animal species, primarily pigs, have been recognized as reservoirs of HEV. Because of its possible spread, there is an urgent need of a method for the cost-effective production of HEV proteins that can be used as diagnostic antigens for the serological detection of anti-HEV antibodies.
Methods: The HEV open reading frame (ORF)2 protein was purified from plant tissue by using immobilized metal-anion chromatography (IMAC). The recombinant protein was used to develop an in-house ELISA for testing anti-HEV antibodies in both human and swine sera. Thirty-six serum samples collected from patients with serologically proven HEV infection with commercial kits were tested for anti-HEV IgG antibodies by using the plant-expressed protein. Forty-five serum samples collected from apparently healthy pigs in Bulgarian farms were also tested.
Results: We confirmed the transient expression and purification of a truncated version of the HEV genotype 3 capsid protein in Nicotiana benthamiana and its usefulness as a diagnostic antigen. ELISA showed the presence of anti-HEV IgG antibodies in 29 of the 36 human samples. The in-house ELISA showed anti-HEV IgG antibodies in 34 of the 45 pigs.
Conclusions: We describe a method for the production of HEV ORF2 protein in N. benthamiana and the usefulness of this protein for the serological detection of anti-HEV antibodies in both humans and swine.
Keywords: Hepatitis E virus, Transient expression, ELISA, Nicotiana benthamiana

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