Novel hepatitis E virus genotype in Norway rats, Germany

Abstract

Human hepatitis E virus infections may be caused by zoonotic transmission of virus genotypes 3 and 4. To determine whether rodents are a reservoir, we analyzed the complete nucleotide sequence of a hepatitis E-like virus from 2 Norway rats in Germany. The sequence suggests a separate genotype for this hepatotropic virus.

Figure 1

Genome structure and localization of putative open reading frames (ORFs) and functional domains in ORF1 of hepatitis E virus (HEV) sequences from Norway rats nos. 63 and 68, collected in Germany, July 2009 (A); phylogenetic trees based on a partial nucleotide sequence of 1,576 nt (B); and the complete genomes (C). RNA was isolated from liver samples by using the RNeasy Mini Kit and a QIAshredder (QIAGEN, Hilden, Germany). The entire rat HEV genome sequences of each rat were determined by a primer walking strategy and rapid amplification of cDNA ends protocols (GenBank accession nos. GU345042 and GU345043). ORFs were predicted by using the SeqBuilder Module of the DNASTAR software package (Lasergene, Madison, WI, USA). Putative functional domains in ORF1 were compared with those predicted in the corresponding regions of ORF1 from HEV genotypes 1–4 (11). The methyltransferase (MeT), helicase (Hel), and RNA-dependent RNA polymerase (RdRp, GDD motif indicated) domains are conserved and in the same order in the rat HEV genomes. In contrast, the papain-like protease domain (PLP) and the proline-rich domain (Prol) were more variable. Three additional ORFs (4, 5, 6) were predicted for both rat HEV genomes, for which no similar amino acid sequence could be found by BLASTp (http://blast.ncbi.nlm.nih.gov/Blast.cgi) search, sequence profile search in Pfam, and no functional pattern by Prosite (www.expasy.ch/prosite/) search; however, several similar sequences were retrieved from the Uniprot collection by comparison of translated nucleotide sequences with BLASTx (http://blast.ncbi.nlm.nih.gov/Blast.cgi). In addition, these ORFs showed less difference to the host codon usage than ORF3 as determined by Graphical Codon Usage Analyzer (http://gcua.schoedl.de/) and STRAP (http://3d-alignment.eu/). Phylogenetic relationships were reconstructed by using neighbor-joining and Bayesian algorithms after substitution model estimation (12). Robustness of nodes in phylogenetic trees is given above branches for Bayesian algorithms (sampling every 10 of 1 million generations; first 25,000 samples discarded as burn-in) and below branches for neighbor joining (10,000 bootstrap replicates). Only support values for main nodes that connect genotypes or major evolutionary lineages are displayed. *Indicates that neighbor-joining algorithms suggest instead a closer phylogenetic relationship between genotypes 3 and 4 with genotype 1 basal to these 2. Scale bar indicates phylogenetic distances in nucleotide substitutions per site.

Figure 2

Immunohistochemical staining (peroxidase-antiperoxidase (PAP) technique) of liver samples from 2 rat-hepatitis E virus (HEV)–positive Norway rats from Germany, July 2009. Arrows indicate immunohistochemical positive reactions in the cytoplasm of single hepatocytes (A) and in a few foci in hepatocytes and stellate cells (B). For PAP staining, deparaffinized slides of liver samples were incubated with anti-HEV–positive human serum, which had been previously used to detect rat HEV by using solid phase immunoelectron microscopy (10), for 1 h at 37°C with protein A (Sigma-Aldrich, Steinheim, Germany) at a dilution of 1:100 for 45 min at 37°C and finally with PAP complexes from rabbits (Sigma, St. Louis, MO, USA) at a dilution 1:200 for 45 min at 37°C. AEC (3-amino-9-ethylcarbazol; Sigma Chemie GmbH, Deisenhofen, Germany) was used as the substrate chromogene. The slides were counterstained with hematoxylin and subsequently analyzed by light microscopy. Scale bars = 20 µm.

Figure A1

Results of real-time reverse transcription–PCR showing cycle threshold (Ct) values of viral load in different tissues of Norway rats nos. 63 (white bars) and 68 (gray bars). For RNA isolation, 10 mg of tissue or 10 µL of blood were homogenized and used. The PCR was selective for a region in the open reading frame 2 of rat hepatitis E virus (rHEV) and was based on primers rHEV-forward (5′-TACCCGATGCCGGGCAGT-3′) and rHEV-reverse (5′-ATCCACATCTGGGACAGG-3′) and probe (5′-6FAM-AATGACAGCACAGGCACC-BBQ-3′). Error bars indicate SD. *Brain sample from rat no. 63 contained blood.