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

Abstract

BackgroundHepatitis 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.MethodsWe 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.ResultsSamples 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).ConclusionBroad range methods for molecular characterization of HEV still need to be improved to detect all circulating strains.

Comprehensive Molecular Approach for Characterization of Hepatitis E Virus Genotype 3 Variants

“…Previous studies reported that quantitative HEV-specific real-time RT-PCR of a conserved region (ORF2/ORF3) in the HEV genome is the most sensitive method for HEV RNA detection (25). On the other hand, consensus nested RT-PCRs for the amplification of longer fragments (Ͼ300 nt) located in different HEV genome regions (ORF1 and ORF2) are useful for molecular characterization and genotyping/subtyping of HEV variants (38). However, full-length genome sequencing is definitely mandatory for proposing new HEV variants (genotype and subtype).…”

High Prevalence of Hepatitis E Virus in Swedish Moose – A Phylogenetic Characterization and Comparison of the Virus from Different Regions

“…However, failure to sequence HEV appear to be common, for example in a study, where only 9/16 (56%) and 3/9 (33%) of qPCR HEV-positive samples from wild boar and red deer could be sequenced after nested-PCR amplification [ 46 ]. In another study it was demonstrated that conventional PCR assays targeting different regions of the HEV genome could only detect a proportion of HEV positive (as demonstrated by serology and qPCR) reference samples [ 47 ]. This could be due to genetic variability of the strains, thereby explaining why highly similar moose RdRp sequences showed a similar ability to be sequenced in this study.…”

Molecular Epidemiology and Strain Comparison between Hepatitis E Viruses in Human Sera and Pig Livers during 2014 to 2016 in Hong Kong

“…Nested PCRs targeting HEV open reading frame 1 (ORF1; 133 nucleotides) and the ORF2/3 junction (97 nucleotides) were then performed separately using Phusion high-fidelity DNA polymerase (Thermo Fisher, USA)/Titanium Taq DNA polymerase (TaKaRa, Japan). Primers used are listed in Table S2 (47). Precautionary measures to minimize cross-contamination were implemented: (i) master mix preparation, DNA/RNA template addition, thermocycling, and post-PCR steps (e.g., gel electrophoresis) were performed in four separate rooms, and (ii) filtered pipette tips were used.…”