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Novel hepatitis E virus genotype in Norway rats, Germany.

Johne R, Heckel G, Plenge-Bönig A, Kindler E, Maresch C, Reetz J, Schielke A, Ulrich RG - Emerging Infect. Dis. (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Federal Institute for Risk Assessment, Berlin, 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.

Show MeSH

Related in: MedlinePlus

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.
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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.

Mentions: During July 8–16, 2009, a total of 6 Norway rats, 3 male and 3 female, 65–432 g, were trapped in manholes of the sewer system of Hamburg, northern Germany, at the same locations where ≈12 months before HEV RNA had been detected in rat feces (10). Standardized necropsy (9) found no morphologic abnormalities. Initial serologic screening with a commercial genotype 1–based ELISA (Axiom, Bürstadt, Germany) detected no reactive antibodies in transudates of any of the 6 rats. Liver RNA from 1 female (no. 68, 311 g) and 1 male (no. 63, 313 g) rat yielded an amplification product of the expected size (331–334 nt) and a sequence identity of 83.8%–94.6 % with the HEV sequences recently obtained from rat feces (data not shown). Using a strategy according to Schielke et al. (4), we determined the entire rat HEV genome sequences from each sample to be 6,945 nt and 6,948 nt; the sequences differed by an insertion–deletion polymorphism in the 3′ noncoding region. The sequence identity between each complete sequence was 95.3% and reached 55.1%–55.9% to HEV genotypes 1–4 and 49.3%–50.2% to avian HEV strains (Table). Using prediction software, we identified the major ORFs 1, 2, and 3 in the new genomes in an organization typical for HEV (Figure 1, panel A). In contrast to HEV genotypes 1–3, rat HEV ORFs 1 and 3 do not overlap. Three additional putative ORFs of 280–600 nt that overlap with ORFs 1 or 2 were predicted for each rat HEV genome (Figure 1, panel A). However, before the meaning of these findings can be verified, sequence information from additional rat HEV strains and experimental proof are needed. Phylogenetic analyses of a 1,576-nt segment available for all published rat HEV sequences demonstrated clear separation from mammalian genotypes 1–4 and avian strains (Figure 1, panel B). The same 3 phylogenetic clusters were obtained when the complete genomes were analyzed (Figure 1, panel C) and when the nucleotide and deduced amino acid sequences of ORF1, ORF2, and ORF3 were investigated separately (data not shown).


Novel hepatitis E virus genotype in Norway rats, Germany.

Johne R, Heckel G, Plenge-Bönig A, Kindler E, Maresch C, Reetz J, Schielke A, Ulrich RG - Emerging Infect. Dis. (2010)

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.
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Related In: Results  -  Collection

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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.
Mentions: During July 8–16, 2009, a total of 6 Norway rats, 3 male and 3 female, 65–432 g, were trapped in manholes of the sewer system of Hamburg, northern Germany, at the same locations where ≈12 months before HEV RNA had been detected in rat feces (10). Standardized necropsy (9) found no morphologic abnormalities. Initial serologic screening with a commercial genotype 1–based ELISA (Axiom, Bürstadt, Germany) detected no reactive antibodies in transudates of any of the 6 rats. Liver RNA from 1 female (no. 68, 311 g) and 1 male (no. 63, 313 g) rat yielded an amplification product of the expected size (331–334 nt) and a sequence identity of 83.8%–94.6 % with the HEV sequences recently obtained from rat feces (data not shown). Using a strategy according to Schielke et al. (4), we determined the entire rat HEV genome sequences from each sample to be 6,945 nt and 6,948 nt; the sequences differed by an insertion–deletion polymorphism in the 3′ noncoding region. The sequence identity between each complete sequence was 95.3% and reached 55.1%–55.9% to HEV genotypes 1–4 and 49.3%–50.2% to avian HEV strains (Table). Using prediction software, we identified the major ORFs 1, 2, and 3 in the new genomes in an organization typical for HEV (Figure 1, panel A). In contrast to HEV genotypes 1–3, rat HEV ORFs 1 and 3 do not overlap. Three additional putative ORFs of 280–600 nt that overlap with ORFs 1 or 2 were predicted for each rat HEV genome (Figure 1, panel A). However, before the meaning of these findings can be verified, sequence information from additional rat HEV strains and experimental proof are needed. Phylogenetic analyses of a 1,576-nt segment available for all published rat HEV sequences demonstrated clear separation from mammalian genotypes 1–4 and avian strains (Figure 1, panel B). The same 3 phylogenetic clusters were obtained when the complete genomes were analyzed (Figure 1, panel C) and when the nucleotide and deduced amino acid sequences of ORF1, ORF2, and ORF3 were investigated separately (data not shown).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Federal Institute for Risk Assessment, Berlin, 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.

Show MeSH
Related in: MedlinePlus