Ann Lab Med.  2017 Jul;37(4):313-319. 10.3343/alm.2017.37.4.313.

Detection of Serum Antibodies to Hepatitis E Virus Based on HEV Genotype 3 ORF2 Capsid Protein Expressed in Nicotiana benthamiana

• Mazalovska M
• Varadinov
• Koynarski
• Minkov I
• Teoharov P
• Lomonossoff GP
• Zahmanova

• KMID: 2376787
• DOI: http://doi.org/10.3343/alm.2017.37.4.313

Abstract

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.

Keyword

Hepatitis E virus; Transient expression; ELISA; Nicotiana benthamiana

MeSH Terms

Agriculture
Animals
Antibodies*
Capsid Proteins*
Capsid*
Chromatography
Developing Countries
Enzyme-Linked Immunosorbent Assay
Genotype*
Hepatitis E virus*
Hepatitis E*
Hepatitis*
Humans
Immunoglobulin G
Methods
Open Reading Frames
Plants
Swine
Tobacco*
Antibodies
Capsid Proteins
Immunoglobulin G

Figure

• 

  Fig. 1 (A) SDS-PAGE of expressed and purified hepatitis E virus (HEV) 110-610 C-end His-tag protein; (B) Western blot of the gel with anti-HEV ORF2 antibody; (C) SDS-PAGE of expressed and purified HEV 110-610 N-end His-tag protein; (D) Western blot of the gel with anti-HEV ORF2 antibody. M, protein marker; 1, crude extract; 2, supernatant; 3, pellet; 4, flow-through; 5, washing step; 6–9, elutes of the 54.6 kDa protein.Abbreviations: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; ORF2, open reading frame 2.

• 

  Fig. 2 Western blot of purified hepatitis E virus (HEV) 110-610 His-tag 54.6 kDa protein. Membranes were incubated with swine serum (A), human serum (B), and monoclonal antibody (anti-hepatitis ORF2 antibody) (C). M, protein marker; 1, HEV 110-610 N-end His-tag protein; 2, HEV 110-610 C-end His-tag protein (arrows).

• 

  Fig. 3 In–house ELISA of serum samples based on the hepatitis E virus (HEV) 110-610 C-end His-tag protein. The cut-off value (CO) is indicated by a horizontal line. Samples were considered positive (in red), if the absorbance was above the cut-off value (P/N) (see Methods).

• 

  Fig. 4 Western blot probed with hepatitis E virus (HEV) 110-610-based ELISA negative human sera from this study (lanes 1-7). Arrow indicates the 54.6 kDa protein of interest. There are some aggregates and degraded products. Five HEV negative human sera previously characterized by commercial kit were used as control for western blot specificity (data not shown). M, protein marker.

• 

  Fig. 5 Western blot of the hepatitis E virus (HEV) 110-610 C-end His-tag protein, probed with ELISA negative swine sera from this study (lanes 1–4), and with positive serum (lane 5). M, protein marker. Swine sera recognized a protein band of 54.6 kDa (arrow). Four HEV negative swine sera previously characterized by commercial kit were used as control for western blot specificity (data not shown).

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