Detection and Phylogenetic Characterization of Human Hepatitis E Virus Strains, Czech Republic – Quantity 17, Quantity 5—Could 2011 – Rising Infectious Ailments journal
Author connections: Author affiliations: Veterinary Research Institute, Brno, Czech Republic (P. Vasickova, M. Slany, I. Pavlik);; Charles University in Prague and University Hospital Bulovka, Prague, Czech Republic (P. Chalupa, M. Holub);; Faculty of Medicine of Masaryk University and Brno Faculty Hospital, Brno (R. Svoboda)
The hepatitis E virus (HEV) is a leading cause of epidemics and sporadic cases of enteric transmitted hepatitis worldwide. The zoonotic potential of HEV has recently been recognized, and pigs and other animal species have been viewed as natural reservoirs for the virus (1). Currently, mammalian HEV strains are divided into 4 major genotypes. The relative conservation of genotypes 1 and 2 corresponds to their primary circulation in humans. Genotype 1 consists of epidemic strains from developing countries in Asia and Africa, and representatives of genotype 2 have been described in Mexico and African countries. The diversity of genotypes 3 and 4 is related to their origin from a large number of animal species. Genotype 3 is widespread and has been isolated from patients with sporadic cases of acute hepatitis E worldwide. Genotype 4 contains strains of human and animal origin, especially in isolates from Asian countries (2,3).
In the Czech Republic, the incidence of hepatitis E has increased since the first case was described in 1996. From 1996 to 2005 a total of 159 cases of HEV infections were reported. 23% of these cases involved travel to industrialized countries (4). In 2005, 37 hepatitis E patients were reported in the Czech Republic, and in 2009 the number rose to 99 (5). Based on this data, the extensive genomic variability between HEV isolates and their known geographical distribution, we performed a phylogenetic analysis of HEV isolates from clinical samples from Czech patients with acute hepatitis E to determine the origin of the infection.
Stool samples from a total of 10 patients with serologically confirmed acute hepatitis E were tested (online attachment table, www.cdc.gov/EID/content/17/5/917-appT.htm). Informed consent was obtained from all patients involved in this study (ethics committee, University Hospital Bulovka; IRB00002721).
250 milligrams of stool sample were suspended in 2.25 ml of phosphate buffered saline, homogenized by vortexing, and clarified by centrifugation at 3,000 x g for 15 minutes. The RNA was obtained from 140 µl. l Supernatant extracted using the QIAamp Viral RNA Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. A positive template control was isolated from pig feces positive for HEV RNA from a previous study (6) and a water sample was included as a negative control.
Detection of HEV RNA was performed by nested reverse transcription PCR with 2 sets of degenerate primers targeting open reading frame 1 (ORF1) and the overlapping portion of ORF2 and ORF3 (ORF2 / 3) of the HEV genome, as before described (6). . Undiluted and 10-fold diluted samples of isolated RNA were analyzed. The specific PCR product (length 242 bp, ORF1 primer), which was obtained from 2 independent RNA isolations, was then produced for sequencing with the QIAquick PCR Purification Kit (QIAGEN). Both strands were sequenced at Eurofins MWG Operon (Ebersberg, Germany).
The sequencing and phylogenetic analysis were carried out with the software MEGA Version 3.1 (www.megasoftware.net). The 1000 replication neighborhood connection method in the bootstrap test was used for phylogenetic analysis (7), and bootstrap values> 50% were considered significant. The 5 most similar HEV sequences available in the GenBank database were compiled for each presented Czech human HEV isolate (isolate CZhHEV) according to the BLAST algorithm (http://blast.ncbi.nlm.nih.gov/Blast .cgi) selected. Selected sequences were supplemented with representatives of genotype 3e (strain G2, GenBank accession number AF110389), 3f (strain G1, accession number AF110388) and 3g (strain Osh 205, accession number AF455784) (2).
HEV RNA was detected in clinical specimens from 6 out of 10 patients whose specimens were tested. The results obtained using primers specific for ORF1 and ORF2 / 3 of the HEV genome were consistent with the exception of material from 1 patient. Specific PCR products from 5 CZhHEV isolates were sequenced (Table A1). The sequence analysis showed that sequences from CZhHEV isolates had a homology of 81.4% (CZhHEV107-09 and CZhHEV113-09) to 100.0% (CZhHEV113-09 and CZhHEV114-09).
The phylogenetic analysis showed that the isolates CZhHEV107-09 and CZhHEV197-09 were genetically most similar to subtype 3e (strain G2) and, together with sequences from a pig from Hungary (HUN-072, accession number EU718650), a person from Germany (V0713286, Accession number EU7879117), pigs from Japan (swJ8-5, accession number AB248521; swJ12-4, accession number AB248522; swJB-E10, accession number AB481226) and a patient from Japan (JNH-Ehi04L, accession number AB291958). The isolate CZhHEV107-09 had the highest sequence homology (88.8%) with the strain HUN-072, while the isolate CZhHEV197-09 and the Greek strain G2 had a homology of 93.0%.
The isolate CZhHEV108-09 belonged together with other Czech strains of wild boar to subtype 3g (CZwb51-09, accession number GU299814; CZwbHEV71-09, accession number GU299816), Czech domestic pigs (CZswHEV21, accession number EU1174;, accession number EU1174B10 .229 V0714, German strain Accession number EU879118) and strain Osh205. CZhHEV108-09 shared the highest homology (91.3%) with strain CZwbHEV51-09 and strain V0714229.
Number. Phylogenetic tree, created with MEGA Version 3.1 software (www.megasoftware.net) using the neighbor connection method with 1,000 replications in the bootstrap test based on 242 bp long sequences within the open reading frame 1 (OFR1) …
Identical sequences from CZhHEV113-09 and CZhHEV114-09 isolates were clustered into subtype 3f (strain G1). A human strain from Spain (VH2, accession number AF195065), pig strains from the Netherlands (NLSW28, accession number AF336003; NLSW82, accession number AF336009) and from Spain (SWP6; accession number EU723514) also belonged to subtype 3f. The highest sequence homology (95.0%) was found for the isolates CZhHEV113-09, CZhHEV114-09 and strain VH2 (figure). The sequences of the isolates CZhHEV107-09, CZhHEV108-09, CZhHEV113-09 and CZhHEV197-09 have been deposited in GenBank under the accession numbers. GU299817, GU299812, GU299813 or GU299815.
We tested stool samples from 10 patients with serologically confirmed acute hepatitis E and, despite this confirmation, only detected HEV-RNA in 6 of these patients (Table A1; Figure). Most serological tests used to diagnose hepatitis E use recombinant genotypes 1 and 2 proteins, and these tests may be less sensitive and specific for detecting genotypes 3 and 4 of the genotype Hepevirus (8,9). These results were confirmed by a study conducted in the Czech Republic, which showed that 28 (47.5%) out of 59 IgM anti-HEV positive cases were actually false positive (unpublished data).
According to Lu et al. (2) Subtypes 3c, 3e, 3f, 3h and 3i were identified mainly in Europe and subtype 3g in Asia. In our study, isolates CZhHEV107-09 and CZhHEV197-09 were genetically related to subtype 3e, while identical CZhHEV113-09 and CZhHEV114-09 belonged to subtype 3f. Isolate CZhHEV108-09, clustered with subtype 3g and a strain of a wild boar from the Czech Republic (CZwbHEV51-09) and a strain of a human in Germany that has been associated with the consumption of wild boar meat, offal and locally produced meat products (10) . These results supported the idea of zoonotic transmission of HEV. In addition, other Czech lines of domestic pigs also belonged to subtypes 3g and 3f (Figure).
The patient, from whose stool isolate CZhHEV108-09 was identified, reported that he had eaten homemade slaughter products 1 month before the first symptoms of hepatitis E and had also visited sushi bars in Germany that served grilled pork. As Lupulovic et al. (11) reported that the prevalence of anti-HEV immunoglobulin (Ig) G in pigs raised in family backyards is similar to that in pigs raised on commercial farms. Therefore, infection obtained during home slaughter is likely. On the other hand, Germany is one of the largest exporters of domestic pork and pork to the Czech Republic. Due to the geographical proximity, subtype 3g can circulate between the two countries. In addition, this HEV infection cannot be excluded from the consumption of inadequately heat-treated meat in sushi bars.
The identity of the sequences of isolates CZhHEV113-09 and CZhHEV114-09 strongly suggested an identical HEV source. Both patients lived in the same city and had overlapping hospital stays. In addition, the patients reported having consumed pork and using the same knife and cutting board for raw and cooked meat (Table A1). Therefore, cross contamination is likely during meat processing.
Ms. Vasickova is a researcher in the Food and Feed Safety Department at the Veterinary Research Institute in Brno, Czech Republic. Her primary research interests include the qualitative and quantitative detection of HEV in biological samples and the genetic variability of HEV.
The conclusions, findings, and opinions of the authors who contributed to this journal do not necessarily reflect the official position of the U.S. Department of Health, the public health service, the Centers for Disease Control and Prevention, or any of the institutions affiliated with the authors. The use of trade names is for identification only and does not imply endorsement by any of the groups mentioned above.