Transfusion-associated Hepatitis E, France
Figure. Outline of phylogenetic tree (complete figure available online, http://www.cdc.gov/EID/13/4/zzz-T.htm) constructed by the neighbor-joining method on the basis of partial nucleotide sequences of the open reading frame (ORF) 2 region of hepatitis E virus (HEV) genome obtained from the blood donor and the blood recipient, with sequences of other local HEV strains previously identified, and published HEV sequences. Local HEV sequences were obtained after reverse transcription–PCR amplification that used SuperScript One-Step RT-PCR System (Invitrogen Life Technologies with in-house protocols that used outer primers HevMrsFwd1: 5’-AATTATGCYCAGTAYCGRGTT-3’, 5663 nt (primer location is defined in reference to GenBank sequence accession no. NC_001434); HevMrsRev1: 5′-CCTTTRTCYTGCTGRGCATTCTC-3’, 6393, then inner primers HEVMrsFwd2 5’-GTWATGCTYTGCATACATGGCT-3’, 5,948 and HevMrs2Rev2: 5’-AGCCGACGAAATCAATTCTGT-3’, 6,295, to obtain a fragment of 347 bp. PCR fragments were purified then directly sequenced by using the inner primers and the Big Dye Terminator cycle sequencing kit version 1.1 on ABI Prism 3130 genetic analyzer (Applied Biosystems, Branchburg, NJ, USA). HEV genotype was determined by using phylogenetic analysis with a set of published HEV sequences (genotype and subtype are indicated with the GenBank accession no.) (8). Bootstrap values are indicated when >50% as a percentage obtained from 100 resamplings of the data. HEV sequences from the blood donor and recipient (indicated by black squares) have been submitted to GenBank (accession nos. EF028801 and EF028802); white squares indicate HEV sequences from other individuals with hepatitis E living in Marseille and its geographic area. Black inverted triangles indicate sequences involved in transfusion-transmitted hepatitis E cases in Japan (Japan A ); white inverted triangles indicate other Japanese HEV sequences from the same geographic area (Japan A ). White triangles indicate HEV sequences from the United Kingdom (3) that have an 80%–100% homology at the nucleotide level with those obtained by Boxall et al. (7). White circles indicate sequences from patients living in Hokkaido, Japan (Japan B; ); ORF 1 sequences from these strains were very similar to those characterized in a transfusion-transmitted hepatitis E case that occurred in Hokkaido.