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Identification of rabbit hepatitis E virus (HEV) and novel HEV clade in Irish blood donors

Published:April 25, 2022DOI:https://doi.org/10.1016/j.jhep.2022.04.015

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      • Risk of transfusion-transmitted hepatitis E virus infection from pool-tested platelets and plasma
        Journal of HepatologyVol. 76Issue 1
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          Immunocompromised patients are at risk of chronic hepatitis E which can be acquired by blood transfusions. Currently, screening of blood donors (BDs) for HEV RNA with a limit of detection (LOD) of 2,000 IU/ml is required in Germany. However, this may result in up to 440,000 IU of HEV RNA in blood products depending on their plasma volume. We studied the residual risk of transfusion-transmitted (tt) HEV infection when an LOD of 2,000 IU/ml is applied.
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      To the Editor:
      We read with interest that Cordes et al.
      • Cordes A.K.
      • Goudeva L.
      • Lütgehetmann M.
      • Wenzel J.J.
      • Behrendt P.
      • Wedemeyer H.
      • et al.
      Risk of transfusion-transmitted hepatitis E virus infection from pool-tested platelets and plasma.
      undertook a study wherein they performed individual donor nucleic acid testing (ID-NAT) for hepatitis E virus (HEV) in Germany using the Procleix HEV assay (Grifols, Barcelona, Spain). Using a similar approach, between January 4th, 2016 and March 17th, 2017, 172,277 blood donations were screened for HEV by the Irish Blood Transfusion Service. Screening was performed by ID-NAT using the Procleix HEV assay on the automated Procleix Panther System (Grifols). Irish blood donors consented for their samples to be used for the purposes of anonymous research. Forty-three confirmed HEV RNA-positive blood donations were identified, resulting in an overall incidence of 1:4,006. The HEV viral loads ranged from ∼0.5-4.3 log10 IU/ml. Twenty-seven donations were window period donations; one was anti-HEV IgM positive; 4 anti-IgG positive; the remaining donations were positive for both IgM and IgG (Fortress Diagnostics, Antrim, Northern Ireland).
      Cordes et al.,
      • Cordes A.K.
      • Goudeva L.
      • Lütgehetmann M.
      • Wenzel J.J.
      • Behrendt P.
      • Wedemeyer H.
      • et al.
      Risk of transfusion-transmitted hepatitis E virus infection from pool-tested platelets and plasma.
      performed molecular typing of the HEV-positive donations with subtype 3c predominating, with a further virus identified as subtype 3f consistent with the prevalence of these subtypes in Germany. Sequence analysis was performed on the HEV RNA-positive Irish donor samples using a combination of methods targeting the methytransferase (Met) and RNA-dependent (directed) RNA polymerase (RdRp) in open reading frame 1 (ORF1).
      • van der Poel W.H.
      • Verschoor F.
      • van der Heide R.
      • Herrera M.I.
      • Vivo A.
      • Kooreman M.
      • et al.
      Hepatitis E virus sequences in swine related to sequences in humans, The Netherlands.
      ,
      • Drexler J.F.
      • Seelen A.
      • Corman V.M.
      • Fumie Tateno A.
      • Cottontail V.
      • Melim Zerbinati R.
      • et al.
      Bats worldwide carry hepatitis E virus-related viruses that form a putative novel genus within the family Hepeviridae.
      Sequences were generated for thirty (70%) of the HEV RNA-positive samples; 13 were HEV subtype 3c, 12 were 3e, 2 were 3f and 3 were unclassified. Of the unclassified viruses, unlike Cordes et al.,
      • Cordes A.K.
      • Goudeva L.
      • Lütgehetmann M.
      • Wenzel J.J.
      • Behrendt P.
      • Wedemeyer H.
      • et al.
      Risk of transfusion-transmitted hepatitis E virus infection from pool-tested platelets and plasma.
      who reported that they failed to identify rabbit HEV (HEV-3ra) in their cohort, one Irish donor virus (IE-568) was clearly related to HEV-3ra isolates. The Met sequence for IE-568 shares 87.6% nucleotide identity with a HEV-3ra from Japan (LC535077) whilst the RpRp sequence for IE-568 shares 92.58% identity with HEV 3ra sequences obtained from rabbits in China and Mongolia (KX227751 and AB740222, respectively).
      Two further unclassified HEV isolates (IE-112 and IE-224) were found to share 88% and 89% nucleotide identity with each other for the Met and RdRp sequences, respectively; however, they were more distantly related to other HEV isolates in the GenBank database. Phylogenetic analysis of concatenated Met and RdRp sequence fragments (525 bp in total) demonstrated that IE-568 lies firmly within the HEV 3ra clade,
      • Tamura K.
      • Stecher G.
      • Kumar S.
      MEGA 11: molecular evolutionary genetics analysis version 11.
      and IE-112 and IE-224 form a distinct clade clustering outside of HEV-3ra and basal to most HEV-3 sequences (Fig. 1A). In order to improve resolution of this new phylogenetic clade, it was possible to perform next generation sequencing for IE-112 using an HEV-sequence enriched library approach.
      • Schilling-Loeffler K.
      • Viera-Segura O.
      • Corman V.M.
      • Schneider J.
      • Gadicherla A.K.
      • Schotte U.
      • et al.
      Cell culture isolation and whole genome characterization of hepatitis E virus strains from wild boars in Germany.
      The full-length sequence shared, at best, only ∼77-80% nucleotide identity with HEV-3ra and genotype 3 sequences in GenBank. Phylogenetic analysis of the full-length IE-112 sequence confirmed a clustering outside the HEV-3ra clade and basal to other HEV-3 clades, but distinct from the non-HEV-3 Orthohepevirus A genotypes (Fig. 1B); separate analysis of ORF1, ORF2 and ORF3 of IE-112 showed overall similar phylogeny for the novel sequence (not shown). Using several different approaches, there was no evidence of recombination in the IE-112 sequence, including the hypervariable region in ORF1 (Fig. S1-S3). Although IE-112 does not contain the 90/93 bp insert, within the ORF1 X region, characteristic of HEV-3ra viruses, neither does a recently identified virus (rab81) isolated from a wild rabbit in Germany
      • Cierniak F.
      • von Arnim F.
      • Heckel G.
      • Ulrich R.G.
      • Groschup M.H.
      • Eiden M.
      A Putative novel hepatitis E virus genotype 3 subtype identified in rabbit, Germany 2016.
      (Fig. S4). Both IE-II2 and IE-224 form a distinct and novel HEV clade distantly related to HEV-3ra.
      Figure thumbnail gr1
      Fig. 1Phylogenetic analysis of HEV-positive Irish donations IE-568, IE-112 and IE-224.
      (A) Cladogram of maximum likelihood phylogenetic tree. Phylogenies were generated using Geneious Prime® 2022.0.1 and nucleotide alignments of the Met (242 bp), the RdRp (283 bp) fragments of IE-586 and IE-224, and complete genome data for IE-112 and HEV reference sequences.
      • Smith D.B.
      • Izopet J.
      • Nicot F.
      • Simmonds P.
      • Jameel S.
      • Meng X.J.
      • et al.
      Update: proposed reference sequences for subtypes of hepatitis E virus (species Orthohepevirus A).
      Numbers at nodes, represent bootstrap values of repetitive analyses for confidence testing. (B) Bayesian phylogenetic analysis of the full-length sequence of IE-112 and reference sequences, here all alignment position containing any ambiguous data or gaps have been removed from the dataset. A GTR model with a γ distribution (G) across sites and a proportion of invariant sites (I) (GTR + G + I) was used as the substitution model; HEV-1 was used as the out-group. Trees were run for 1 million generations and sampled every 500 steps. Circles, at nodes, represent Bayesian posterior probability support of 1 and the scale bar indicates genetic distance. In addition to proposed reference sequences, full-length HEV 3ra isolates (HTL-10 and JP-59), published since the latest HEV reference sequence update,
      • Smith D.B.
      • Izopet J.
      • Nicot F.
      • Simmonds P.
      • Jameel S.
      • Meng X.J.
      • et al.
      Update: proposed reference sequences for subtypes of hepatitis E virus (species Orthohepevirus A).
      have been included in the analysis. The HEV isolate identified in a wild rabbit in Germany (rab81
      • Cierniak F.
      • von Arnim F.
      • Heckel G.
      • Ulrich R.G.
      • Groschup M.H.
      • Eiden M.
      A Putative novel hepatitis E virus genotype 3 subtype identified in rabbit, Germany 2016.
      ) has been tentatively proposed as a novel subtype of HEV genotype 3. The red lines indicate the donor isolates described in the study. The sequences of IE-112, IE-224 and IE-568 are listed under GenBank accession numbers OM777188, OM777189 and OM777190, respectively. GTR, general time-reversible.
      Re-testing of IE-568, IE-112 and IE-224 using the altona Diagnostics realStar® HEV RT-PCR kit (altona Diagnostics, Hamburg, Germany) yielded viral loads of 1.1, 4.3 and 2.8 log10 IU/ml, respectively. Using the Cobas® HEV/Cobas® 6800 System (Roche Diagnostics, Mannheim, Germany) IE-112 and IE-224 had viral loads of 4.4 and 2.5 log10 IU/ml compared to the World Health Organization International Standard (6329/10), similar to the Altona Diagnostics assay values; IE-568 was not detected since the viral load was below the 95% cut-off of the Cobas® HEV assay (18.6 IU/ml). Nevertheless, we have previously observed reduced sensitivity (∼2 log10 IU/ml) for one HEV-3ra isolate (MG211750)
      • Baylis S.A.
      • Hanschmann K.O.
      • Matsubayashi K.
      • Sakata H.
      • Roque-Afonso A.M.
      • Kaiser M.
      • et al.
      Development of a World Health Organization International Reference Panel for different genotypes of hepatitis E virus for nucleic acid amplification testing.
      with the Cobas® HEV assay, but not the Procleix assay, although it is not known if this is the case for other HEV-3ra viruses.
      All 3 donors ate pork with donor IE-112 reported to have consumed “black pudding” (pork/beef blood sausage) as well as cured meats. Whilst donor IE-224 reported eating medium/rare venison, the other donors denied eating game meat including rabbit. One donor (IE-112) owned a dog, the other donors had no pets. No donors reported contact with farm animals; donor IE-568 reported contact with a koala in an Australian zoo ∼4 months prior to donation. Although donors IE-112 and IE-224 donated within 1 week of each other, one lived in Dublin whilst the other lived in a rural area on the West coast. None of the donors travelled abroad in the 9 weeks prior to donation suggesting autochthonous transmission of HEV. No post-donation illnesses were reported. HEV-3ra sequences have mainly been identified in immunosuppressed individuals
      • Abravanel F.
      • Lhomme S.
      • El Costa H.
      • Schvartz B.
      • Peron J.M.
      • Kamar N.
      • et al.
      Rabbit hepatitis E virus infections in humans, France.
      ,
      • Sahli R.
      • Fraga M.
      • Semela D.
      • Moradpour D.
      • Gouttenoire J.
      Rabbit HEV in immunosuppressed patients with hepatitis E acquired in Switzerland.
      and the detection of IE-568 in a healthy blood donor is noteworthy. With the identification of the novel isolates IE-112 and IE-224, forming a new clade and potentially a tentative new genotype, further investigations are required to understand potential animal origins associated with these Irish isolates and potential routes of transmission which, in the case of HEV-3ra, where it has been identified in humans, remain elusive.

      Financial support

      The Irish Department of Health provided financial support for HEV blood donor screening by the Irish Blood Transfusion Service. In addition, this work was funded in part by core funding by the German Federal Ministry of Health (Bundesministerium für Gesundheit) (S.A.B.) and a grant ZMVI1-2518FSB705 (V.M.C).

      Authors’ contributions

      S.A.B - analysis of HEV-positive donor samples, writing and review of manuscript; N.O’F - HEV screening, review of manuscript; L.B. - HEV screening, review of manuscript; B.H. - analysis of HEV-positive samples, review of manuscript; VMC - analysis of HEV-positive donor samples, writing and review of manuscript.

      Conflicts of interest

      The authors declare no conflicts of interest.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Acknowledgements

      We thank Michel Molier and Roswitha Kleiber for excellent technical assistance and Julia Schneider, Jörn Beheim-Schwarzbach and Tobias Bleicker for help with sequencing.

      Supplementary data

      The following are the supplementary data to this article:

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