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Molecular characterisation of human hepatitis E virus from Italy: comparative analysis of five reverse transcription-PCR assays

Giuseppina La Rosa1*, Marta Fratini1, Michele Muscillo1, Marcello Iaconelli1, Stefania Taffon2, Michele Equestre3, Paola Chionne2, Elisabetta Madonna2, Giulio Pisani4, Roberto Bruni2 and Anna Rita Ciccaglione2*

Author Affiliations

1 Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy

2 Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy

3 Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome, Italy

4 Center for Immunobiologicals Research and Evaluation, Istituto Superiore di Sanità, Rome, Italy

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Virology Journal 2014, 11:72  doi:10.1186/1743-422X-11-72

Published: 22 April 2014

Abstract

Background

Hepatitis E (HEV) is an important public-health concern as a major cause of enterically transmitted hepatitis worldwide. In industrialised countries it is considered rare, and largely confined to travellers returning from endemic areas. However, autochthonous (locally acquired) HEV infection is also emerging in these regions. The infection is caused by different genotypes, depending on whether it is travel-related or autochthonous. Conventional RT-PCR followed by sequencing of PCR products can identify HEV genotype and, depending on the region, the subtype, thus helping in defining the origin of infection and tracing the source of contamination.

Methods

We re-analysed a collection of serum samples previously confirmed as hepatitis E positive by anti-HEV IgM and IgG assays as well as by Real-Time PCR, with the aim to compare the performances of five different broad range RT-PCR assays that could be provided for molecular characterisation of HEV. This approach is certainly valuable to investigate the molecular epidemiology of acute hepatitis E in countries where co-circulation of different genotypes occurs, like Italy.

Results

Samples were analyzed by five assays targeting the ORF1, ORF2, and ORF2/3 regions. The sensitivity of these assays varied significantly, depending on the target region. Only 46% of samples tested positive by nested PCR; moreover, no single method was able to detect all positive samples. Most sequences originated from patients who had travelled to endemic areas (genotype 1), while the minority originated from Italian patients with no travel history (genotype 3).

Conclusion

Broad range methods for molecular characterization of HEV still need to be improved to detect all circulating strains.

Keywords:
Hepatitis E virus; RT-PCR assays; Molecular characterization; Sequencing; Genotyping