Full coding hepatitis E virus genotype 3 genome amplification method

Abstract

Hepatitis E virus (HEV) genotype 3 produces zoonotic infection associated with the consumption of infected animals. HEV infections can become chronic in immunocompromised (IC) patients. The viral genome has three well defined open reading frames (ORF1, ORF2 and ORF3) within which various domains and functions have been described. This paper (i) describes a new method of complete sequencing of the HEV coding region through overlapping PCR systems, (ii) establishes a consensus sequence and polymorphic positions (PP) for each domain, and (iii) analyzes the complete coding sequence of an IC patient. With regard to the consensus, a high percentage of PP was observed in protease (PP=19%) and the X domain (PP=22%) within ORF1, the N-terminal region of the S domain (PP=22%) in ORF2, and the P1 (PP=35%) and P2 (PP=25%) domains in ORF3. In contrast, the ORF1 Y, ORF2 S, ORF2 M and ORF3 D1 domains were conserved in the reference sequences (0.40, 1, 0.70 and 0% of PP, respectively). The sequence from the IC patient had more mutations in the RpRp (D1235G, Q1242R, S1454T, V1480I, I1502 V, K1511R, G1373 V, E1442D, V1693 M), the terminal ORF2 S- domain (F10L, S26T, G36S, S70P, A105 V, I113 V), the X domain (T938 M, T856 V, S898A) and the helicase (S1014N, S975T, Q1133 K).

Update: proposed reference sequences for subtypes of hepatitis E virus (species Orthohepevirus A)

“…Four sequences of the proposed subtype 3chi-new (MF444089, KU176130, KU513561 and MF444030) [18–20] fall into two groups, with the first three closely related and MF444030 more distant. We propose that this new subtype be called 3m, following previous naming conventions, with KU513561 as the reference sequence, being the first to be reported.…”

Emergent subtype of hepatitis E virus genotype 3 in wild boar in Spain

“…Phylogenetic analysis of the ORF2 region was performed using nested RT‐PCR, following the protocol described elsewhere (Muñoz‐Chimeno et al, ), with modified primers in the second round (5´‐AATTATGCYCAGTAYCGRGTTG‐3´). The second amplification product of 649 bp was sequenced using the BigDye Terminator Cycle Sequencing Ready Reaction Kit on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems).…”

Hepatitis E virus in Spanish donors and the necessity for screening

“…Individual positive samples were genotyped. For phylogenetic analysis, nested RT‐PCR was performed, targeting the ORF2 region (viral capsid protein), following the protocol described by Muñoz‐Chimeno et al and applying primer modification (5′‐AATTATGCYCAGTAYCGRGTTG‐3′) in the second round. The second amplification product of 649 bp was sequenced using the BigDye Terminator Cycle Sequencing Ready Reaction Kit on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).…”