In house ELISA based on recombinant ORF2 protein underline high prevalence of IgG anti-hepatitis E virus amongst blood donors in south Brazil

PLOS ONE, Dec 2019

Hepatitis E Virus (HEV) is a zoonotic pathogen responsible for causing acute hepatitis in human, especially in developing countries. Diagnosis of HEV usually relies on the detection of antibodies mostly by enzyme-linked immunosorbent assay (ELISA). In the present study, we designed a new indirect ELISA (iELISA) based on a short recombinant peptide derived from the capsid protein (ORF2p) and demonstrated its potential for detecting human IgG against HEV genotype 3. The best polystyrene plate (Maxisorp®), optimal ORF2p coating antigen concentration (0,67μg/well) and primary antibody dilution (1:100) were determined. This iELISA showed a sensitivity of 91.4% and specificity of 95.9%. The comparison of our in house iELISA with a commercial assay (RecomWell, Mikrogen®) showed 94.25% of agreement and a kappa index of 0.88. The ORF2 recombinant ELISA was used to screen 780 blood donors for anti-HEV IgG and we found that 314 (40,25%) of these donors were IgG positive. This high prevalence of antibodies suggests, for the first time, that the Southern Brazil region might be endemic to Hepatitis E Virus genotype 3.

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In house ELISA based on recombinant ORF2 protein underline high prevalence of IgG anti-hepatitis E virus amongst blood donors in south Brazil

May In house ELISA based on recombinant ORF2 protein underline high prevalence of IgG anti- hepatitis E virus amongst blood donors in south Brazil Rafael Pandolfi 0 1 Denise Ramos de Almeida 0 1 Marcelo Alves Pinto 1 Luiz Carlos Kreutz 0 1 Rafael Frandoloso 0 1 0 LaboratoÂrio de Microbiologia e Imunologia AvancËada, Faculdade de Agronomia e Medicina VeterinaÂria, Universidade de Passo Fundo , Passo Fundo, Rio Grande do Sul , Brazil , 2 LaboratoÂrio de Desenvolvimento TecnoloÂgico em Virologia/IOC - FundacËão Instituto Oswaldo Cruz , Rio de Janeiro, Rio de Janeiro , Brazil 1 Editor: Yury E Khudyakov, Centers for Disease Control and Prevention , UNITED STATES Hepatitis E Virus (HEV) is a zoonotic pathogen responsible for causing acute hepatitis in human, especially in developing countries. Diagnosis of HEV usually relies on the detection of antibodies mostly by enzyme-linked immunosorbent assay (ELISA). In the present study, we designed a new indirect ELISA (iELISA) based on a short recombinant peptide derived from the capsid protein (ORF2p) and demonstrated its potential for detecting human IgG against HEV genotype 3. The best polystyrene plate (Maxisorp®), optimal ORF2p coating antigen concentration (0,67μg/well) and primary antibody dilution (1:100) were determined. This iELISA showed a sensitivity of 91.4% and specificity of 95.9%. The comparison of our in house iELISA with a commercial assay (RecomWell, Mikrogen®) showed 94.25% of agreement and a kappa index of 0.88. The ORF2 recombinant ELISA was used to screen 780 blood donors for anti-HEV IgG and we found that 314 (40,25%) of these donors were IgG positive. This high prevalence of antibodies suggests, for the first time, that the Southern Brazil region might be endemic to Hepatitis E Virus genotype 3. Introduction Viral hepatitis stands up as a major public health issue worldwide and is caused by several different types of enterically and parenterally transmitted viruses. Hepatitis E (HE), for instance, caused by hepatitis E virus (HEV) is endemic in several developing African, Asian and South American countries [ 1 ] and autochthones cases are found at increasing and steadily frequency in developed countries [ 2 ]. The infection by HEV is usually unnoticed except in pregnant women and patients with liver-related problems [ 3 ]. However, a recent report indicated that the infection might become chronic mainly in immunocompromised individual such as kidney and liver-transplanted [ 4 ] and HIV-positive subjects [ 5 ]. In this scenario, one of the major question relates to the role HEV might take in causing chronic hepatitis in individual under immunosuppressive medication treatments [ 6 ] and recipients of blood derived product [ 7 ]. Thus, screening for anti-HEV antibodies or HEV RNA amongst blood donor should become mandatory. HEV is a small icosahedral non-enveloped RNA virus with a single-stranded positive-sense genome with approximately 7.3 kilobases classified in the genus Hepevirus, Hepeviridae family [ 8 ]. The viral genome contains three open reading frames (ORFs) that encode the structural and nonstructural proteins. There are 4 well-known genotypes with distinct epidemiological distributions: genotypes 1 and 2 are epidemically found in Asia, Central and South America and in some African countries [ 9 ] and infect exclusively humans [ 10 ] whereas genotypes 3 and 4 are found mostly in Asia and developing countries and might cause infection in humans and animals, mainly domestic pigs [ 11 ] and wild boars [ 12 ]. Although there is a strong evidence of cross species transmission infection between human and pigs [ 13 ] the role of other animal species in the epidemiology of HEV infection remains to be evaluated. In endemic countries the transmission of HEV occurs mostly by the oral-fecal route [ 14 ]; conversely, in developed countries, foodborne transmission [ 15 ], blood transfusion [ 7 ] and transplants of solid organs such as heart, lung, liver and kidney [ 16 ] are becoming major source of viral dissemination. The ingestion of undercooked contaminated pork meat and pork-related product might also constitute a potential source of infection [ 17 ]. However, the scarcity of data on these routes of infection hampers further evaluation on HEV epidemiology and the impact on public health. Nonetheless, the detection of anti-HEV antibodies or HEV nuclei acid amongst blood donors [ 18, 19 ] indicates that viral spread might be long occurring and the prevalence and incidence of HEV might be even higher than previously thought. In Brazil, HEV genotype 3 is commonly found in pig farms [20] and autochthonous human cases of HEV have already been described here [ 21 ], in Germany [ 22 ], France [ 23 ], Cambodia [ 24 ] and Israel [ 25 ] strengthening the zoonotic potential of HEV. HE diagnosis is based mostly on the detection of anti-HEV antibodies (IgM, IgG and IgA) towards ORF-2 and ORF-3 encod (...truncated)


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Rafael Pandolfi, Denise Ramos de Almeida, Marcelo Alves Pinto, Luiz Carlos Kreutz, Rafael Frandoloso. In house ELISA based on recombinant ORF2 protein underline high prevalence of IgG anti-hepatitis E virus amongst blood donors in south Brazil, PLOS ONE, 2017, Volume 12, Issue 5, DOI: 10.1371/journal.pone.0176409