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)