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Genetic characterization of the complete coding regions of genotype 3 hepatitis E virus isolated from Spanish swine herds
Virus Research 139 (2009) 111–116
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Short communication
Genetic characterization of the complete coding regions of genotype 3 hepatitis E virus isolated from Spanish swine herds夽 Bibiana Peralta a,∗ , Enric Mateu a,b , Maribel Casas a , Nilsa de Deus a , Marga Martín a,b , Sonia Pina a,c a
Centre de Recerca en Sanitat Animal (CRESA), UAB-IRTA, Campus de la Universitat Autònoma de Barcelona, 08193 Barcelona, Spain Departament de Sanitat i d’Anatomia Animal, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain c Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Barcelona, Spain b
a r t i c l e
i n f o
Article history: Received 30 June 2008 Received in revised form 17 September 2008 Accepted 19 September 2008 Available online 14 November 2008 Keywords: Complete coding regions Swine HEV Spain Genotype 3
a b s t r a c t The complete coding regions of five hepatitis E virus isolates of swine origin from two different pig farms and the complete genome sequence of two of these strains were obtained and compared to other full length or partial HEV sequences. Based on the nucleotide sequence, the examined Spanish isolates were 87.1–99.7% similar among them being the closest known strain a Mongolian porcine strain (swMN06C1056) which shares 84.5–86.1% of the nucleotide sequence, and are also close to other HEV porcine strains from Japan. Two isolates from the same farm presented an 87 nucleotide insertion in the polyproline hinge unique among all HEV isolates known so far. Comparison with partial HEV sequenced strains indicates that the isolates described in this study form a cluster containing human and porcine HEV strains from Europe, being the only representatives of the subtype 3f that were completely sequenced. Evolutive pressure analysis indicates that microevolution of HEV seems to be driven by negative selection. Further studies should be carried out in order to clarify the HEV origin and evolution. © 2008 Elsevier B.V. All rights reserved.
Hepatitis E virus (HEV) is a small non-enveloped virus belonging to the Hepeviridae family (Emerson et al., 2004). The viral genome consists of a 7.2 kb single-strand positive-sense RNA containing three partially overlapping open reading frames (ORF), a 5 and 3 non-translated regions and a poly-A tract. ORF1 encodes for non-structural proteins such as methyltransferase, helicase and RNA-dependent RNA polymerase; ORF2 encodes the capsid protein, and ORF3 encodes the cytoskeleton-associated phosphoprotein (Reyes et al., 1990; Huang et al., 1992). Four HEV genotypes and several sub-genotypes have been described so far (Lu et al., 2006), which seem to be geographically distributed. Genotype 1 is common in epidemics in Asia, particularly in India (Arankalle et al., 1999). Genotype 2 was originally detected in Mexico but later on has been reported in Africa (Huang et al., 1992; Buisson et al., 2000). Genotype 3 strains are responsible of sporadic infections in humans and also are widespread in swine. This genotype accounts for the largest number of HEV isolates in humans, pig and wild-boars in Europe (Van der Poel et al., 2001; De Deus et al., 2007; Kaci et al., 2008) and have recently
夽 The GenBank accession numbers for the sequences of SW626, SW627, SWP6, SWP7 and SWP8 are EU723512, EU723513, EU723514, EU723515 and EU723516, respectively. ∗ Corresponding author. Tel.: +34 93 5814527; fax: +34 93 5814490. E-mail address: [email protected] (B. Peralta). 0168-1702/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2008.09.008
been described in other animal species in Asia as well (Takahashi et al., 2004; Nakamura et al., 2006). Genotype 4 strains seem to be autochthonous of Asia and have been detected in humans and pigs (Nishizawa et al., 2003). HEV genotypes 3 and 4 are thought to be zoonotic or, at least, have the potential to be transmitted from animals to humans. Thus, porcine, wild-boar and deer strains can be very close to human isolates (Takahashi et al., 2004; Nakamura et al., 2006) and recent reports documented cases of human hepatitis E in which all evidences pointed to the consumption of uncooked or undercooked meat or viscera as the source of infection (Takahashi et al., 2004; Tei et al., 2004). Europe is considered a non-endemic region for human hepatitis E, with few clinical cases, although seroprevalences among healthy population can vary from 1% to 16% (Pavia et al., 1998; Buti et al., 2006; Mansuy et al., 2008). In contrast, recent studies showed that seropositive animals are present in up to 97% of the pig herds (Rutjes et al., 2007; Seminati et al., 2008). In both cases, humans and pigs, European autochthonous isolates belong to genotype 3 (Pina et al., 2000; Clemente-Casares et al., 2003; Buti et al., 2004; De Deus et al., 2007). Up to now, the molecular epidemiology of HEV genotype 3 in Europe, and particularly the relationship existing between human and swine strains, has been studied by comparing very short viral sequences and not a single complete European genotype 3 sequence is available yet. In the present study, five HEV isolates from two pig farms were sequenced, analysed and compared to
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other HEV sequences in order to gain insight in the understanding of the epidemiology and evolution of HEV. Bile samples of five 14-week-old pigs from two different herds (A and B) were selected. Those animals had been submitted to the Veterinary Pathology Diagnostic Service of the Veterinary Faculty of Barcelona for necropsy and suffered subclinical hepatitis as revealed by the histopathological analysis. Total viral RNA was extracted using Nucleospin® RNA virus kit (Macherey-Nagel) and the presence of genomic HEV RNA was determined by means of a seminested RT-PCR as described previously (De Deus et al., 2007). Viral RNA was quantified by real-time PCR as described by Jothikumar et al. (2006) in 2–8 × 106 genomes/ml of bile. The nearly full-length genomes were obtained using the SuperScriptTM III One Step RT-PCR System with Platinum® Taq High Fidelity (Invitrogen) for the cDNA synthesis and first round PCR amplification, and the PfuUltra® II Fusion HS DNA Polymerase system (Stratagene) for the seminested amplification, according to the manufacturer’s instructions. For the analysis of the fragment comprising the entire ORF1 and the 5 -end of the ORF2, primers F25 (5 -GGTCGAYGCCATGGAGGCCC-3 ) and R5500 (5 -GVGGGGCGCTGGGACTGGTC-3 ) were used for the first round, and primers F25 and R5470 (5 -TGGGACTGGTCRCGCCAAG3 ) for the second round of the PCR. For the amplification of an overlapping fragment containing the 3 -end portion of the ORF1 and the complete ORF2 and ORF3, including the 3 -NTR, primers F5000 (5 -AATGTKGCKCAGGTYTGTG-3 ) and R7260 (5 -TTTTTTTTTTTTTCCKGGGRGCGCG-3 ) were used for the first round of amplification, and the primers F5160 (5 MGGSTRGAATGAATAACATG-3 ) and R7260 were used for the seminested amplification. The products were analysed by electrophoresis and stained with SYBR Gold® (Molecular Probes). The amplified products of about 5400 and 2100 bp respectively, were gel purified using the NucleoSpin® Extract II (MachereyNagel) following manufacturer’s instructions and cloned into pCR® II-Blunt-TOPO® (Invitrogen). Recombinant plasmids were used to transform competent E. coli DH10B (Invitrogen). Positive clones were cultured and recombinant plasmids were extracted with the NucleoSpin® Plasmid kit (Macherey–Nagel). Sequencing of both strands was performed from five colonies of each fragment using the ABI PRISM 3700 DNA analyzer (Applied Biosystems, Foster City, USA), using specific primers designed along the genomes. The 5 -NTR of one strain of each farm was determined using the 5 RACE System for Rapid amplification of cDNA Ends, Version 2.0 (Invitrogen) following manufacturer’s instructions. Sequence assembly was accomplished using the programs Phred (Ewing and Green, 1998; Ewing et al., 1998), Phrap and Consed (Gordon et al., 1998), and Bioedit (Hall, 1999). Homology and identity search with respect to HEV strains available at the GenBank was performed by using the BLAST utilities (http://www.ncbi.nlm.nih.gov/BLAST). Alignments were carried out with the ClustalX 1.8 program (ftp://ftpigbmc.ustras/pub/clustalX) (Thompson et al., 1997) and the phylogenetic trees for the entire genome or partial ORFs were constructed with the neighbour-joining method using MEGA4.0 (Tamura et al., 2007). Bootstrap test of 1000 replicates was done to evaluate the reliability of the different groups (Felsenstein, 1985). Selection pressures along the genome were assessed by calculating the difference between non-synonymous (dN) and synonymous (dS) rates (dN − dS) for each ORF, using the SNAP (http://www.hiv.lanl.gov/content/sequence/SNAP/SNAP.html) and DataMonkey (http://www.datamonkey.org) web servers. Sequence assembly yielded a consensus sequence of 7216 nucleotides for the completed sequenced strain from farm A (SW626) and 7304 nucleotides for the sample completely
sequenced from farm B (SWP6). For the rest of the samples the 5 NTR was not determined, thus sequence assembly yield a consensus sequence of 7192 nt for the strain from farm A (SW627), and 7279 nt for the strains from farm B (SWP7 and SWP8). In farm B isolates, ORF1 had an in-frame insertion of 87 nucleotides in the poly-proline hinge, unique among all of the HEV isolates known so far. To confirm this feature an RT-PCR analysis was performed in bile from both herds. The sequencing of the amplicons was in perfect agreement with those of the sequences showing a correct assembly. Over the entire genome, nucleotide similarity for samples obtained within the same herd ranged from 96.6% to 99.7%; in contrast, nucleotide sequences from herd A and B were only 87.1–89.5% similar. The closest HEV full-genome sequence available in GenBank was swMN06-C1056, a genotype 3 porcine isolate from Mongolia that shared 84.5% and 86.1% of the nucleotide sequence when compared to the Spanish farm A or B isolates, respectively. Identities with other genotype 3 strains ranged between 79.3% and 85.1%. Similarity to strains of other genotypes was below 75%. When considering individual ORFs, the examined Spanish isolates have a nucleotide identity among them of 85.8–99.7% for ORF1; 90.1–99.9% for ORF2 and 95.0–100% for ORF3. Comparison of the predicted translated frame displayed aminoacid sequence identities were 95.2–99.9% for ORF1; 97.7–100% for ORF2 and 96.4–100% for ORF3, indicating that most of the changes corresponded to synonymous mutations. The comparative analysis of the individual ORFs among the Spanish strains and other 27 genotype 3 strains available at Genbank, showed nucleotide identities of about 77.9–84.3% for ORF1, 83.5–88.5% for ORF2 and 91.1–97% for ORF3, respectively. As before, comparison in terms of the predicted aminoacid sequence produced higher identity scores: 91.1–96% for ORF1 polyprotein, 95.3–97.7% for ORF2 capsid protein, and 91.1–99.1% for ORF3 protein. In ORF3, however, nucleotide identity was often higher than aminoacid identity suggesting that non-synonymous mutations may occur more frequently in this fragment (Supplementary Table 1). The 5 NTR was 25 nt long and the homology was 96–100% with other genotype 3 strains and between 92% and 96% with sequences of other genotypes. In the 3 NTR, an identity of 73.3–98.3% was observed between the Spanish isolates, and 58–75% with respect to other genotype 3 strains, and 49.1–62.9% when compared to genotypes 1, 2 and 4. In order to determine the genetic relationship of the examined Spanish strains with other known European sequences, phylogenetic trees using the neighbour-joining method based on short fragments were constructed. A phylogenetic tree based on a 168 nucleotide fragment located between positions 6083 and 6250 of the capsid gene was done (Fig. 1A). In that tree, the Spanish isolates reported in this study were grouped in the subtype 3f according to the classification proposed by Lu et al. (2006). Closest sequences were other European pig or human genotype 3 HEV strains reported previously in Spain and Netherlands (Pina et al., 2000; Van der Poel et al., 2001; Fernandez-Barredo et al., 2006; De Deus et al., 2007; Martín et al., 2007) although this grouping was not supported by significant bootstrap values. Selection of a region of 232 nucleotides in the 5 -end of the ORF1 permitted the inclusion of more genotype 3 strains (Fig. 1B). The resulting phylogenetic tree grouped the isolates of the present study again within subtype 3f with the Spanish human isolates VH1, VH2 and VH3 (Pina et al., 2000; Buti et al., 2004), and also with sequences from Greece (Schlauder et al., 1999), the Netherlands (Van der Poel et al., 2001) and Japan. A third phylogenetic tree was constructed based on the alignment of all coding regions of 79 HEV strains from human or animal origin available at GenBank and the Spanish strains SW626, SW627, SWP6, SWP7 and SWP8 (Fig. 2). The analysis showed
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Fig. 1. Phylogenetic trees constructed by the neighbor joining method using: (A) 168 bp sequence located in the capsid gene of HEV and (B) 232 bp sequence located in the 5- end of the ORF1. Bootstrap values of >70% are indicated for the major nodes. Sequences reported in this study are marked as (䊉).
that swine strains of this study clustered in genotype 3; being the only representatives of the subtype 3f that were completely sequenced, and closely related to the yet unclassified Mongolian strain swMN06-C1056 (Lorenzo et al., 2007) and to the Japanese strains HE-JA04-1911, SWJ12-4 and SWJ8-5 (Inoue et al., 2006) of subtype 3e and OSH205 from Kyrgyzstan (Lu et al., 2004) representing subtype 3 g (Lu et al., 2006). The results obtained in this work showed that each examined herd had its own HEV variant of which one had a unique insertion in the poly-proline hinge of ORF1. The significance of that insertion in terms of virulence or survival is unknown and should be further investigated. The fact that the insertion was present in the virus infecting different animals suggest that this could be a target for an epidemiological marker, and could be useful for monitoring the spreading of the HEV infection in the same farm and between farms. Also this study shows that most changes in ORF1 and ORF2 correspond to synonymous mutations, while changes in ORF3 were mainly non-synonymous ones. Although in ORF1 mean dN − dS was −0.31, positive selection was observed in the poly-proline hinge (dN − dS = 0.171). Also, ORF2, where mean dN − dS was −0.35 positive selection was predominant in the N-terminal region. Then, in the ORF3, a mean value dN − dS = 0.017 indicated that ORF3 is subjected to positive selection. Therefore, these results showed that microevolution of HEV seems to be driven by negative selection (dN < dS) except for ORF3 (Fig. 3 summarizes these results). This would be consistent with the expected behaviour of a small genome
virus in which most parts would probably be essential for viral viability. On the other hand, as occurs with all the sequences described, the aminoacid sequence of the capsid gene of the Spanish isolates was very similar (91–97%) to that of other isolates whatever its origin or genotype. Since most of the available ELISAs are based on entire or N-terminal truncated recombinant ORF2 proteins of genotypes 1 and 2 cross-reactivity has been demonstrated (Engle et al., 2002; Arankalle et al., 2007). Thus, the selective pressure map presented here pinpoints some of the regions of interest for further epidemiological studies and virus evolution. The phylogenetic tree based on short nucleotide fragments showed that the Spanish isolates grouped with European genotype 3f strains according to the classification by Lu et al. (2006), while Japanese and American isolates formed different clusters within the same genotype. A similar analysis was obtained using the RNAdependent RNA polymerase region which have demonstrated to be the one who better correlates with the complete genome (Zhai et al., 2006) (not shown), but any other European sequences was available to be included in that analysis. Nevertheless, partial regions used for the analysis usually showed inconsistency in the grouping although Spanish isolates always grouped in genotype 3f (Fig. 1). Otherwise, when comparing the nearly full-length genome, the position of the Spanish strains was confirmed with a significant bootstrap value. The closest isolate was a Mongolian strain of porcine origin, but this finding must be considered a bias, since no other European
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Fig. 2. Phylogenetic tree constructed by the neighbour joining method using the HEV complete coding region. Bootstrap values >70% are indicated for the major nodes.
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Fig. 3. Differences between non-synonymous and synonymous (dN − dS) rates plot for HEV ORF1, ORF2 and ORF3. ORF1 domains are numbered as follows: (1) methyltransferase domain, (2) Y domain, (3) papain-like protease, (4) poly-proline hinge, (5) X domaine, (6) RNA helicase, (7) RNA-dependent RNA polymerase. Arrow indicates the insertion in the poly-proline hinge. * indicates positions with significant positive selection (p < 0.1).
sequences are available. Previous reports suggested that despite the diversity of HEV genotype 3, a geographical pattern can be observed, each continent having autochthonous HEV strains different but related to the ones from other continents, probably due to a different evolution of the same virus (Okamoto, 2007). This work describes the complete genome of Spanish strains of hepatitis E virus and determines their phylogenetic relationship to other genotype 3 strains. Future full-length genome sequences from Europe may contribute to elucidate the origin, evolution and spread of HEV genotype 3 strains. Addendum Since the present paper was submitted for publication, a sequence of genotype 3 subtype 3c of swHEV named swX07-E1 (accession number EU360977) has been reported in Sweden (Xia et al., 2008), which presented an identity of 87–88% with respect to the coding regions of the strains SW626, SW627, SWP6, SWP7 and SWP8. Acknowledgements This study was supported by the research grant AGL2004/06688 from the Spanish government. Bibiana Peralta and Maribel Casas have a fellowship from the Generalitat de Catalunya. Nilsa de Deus has a fellowship from CReSA. We thank Dr A. Bensaid and Dr A. Olvera for useful advice. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.virusres.2008.09.008. References Arankalle, V.A., Paranjape, S., Emerson, S.U., Purcell, R.H., Walimbe, A.M., 1999. Phylogenetic analysis of hepatitis E virus isolates from India (1976–1993). J. Gen. Virol. 80, 1691–1700. Arankalle, V.A., Lole, K.S., Deshmukh, T.M., Chobe, L.P., Gandhe, S.S., 2007. Evaluation of human (genotype 1) and swine (genotype 4)-ORF2-based ELISAs for anti-HEV IgM and IgG detection in an endemic country and search for type 4 human HEV infections. J. Viral. Hepat. 6, 435–445.
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Virus Research journal homepage: www.elsevier.com/locate/virusres
Short communication
Genetic characterization of the complete coding regions of genotype 3 hepatitis E virus isolated from Spanish swine herds夽 Bibiana Peralta a,∗ , Enric Mateu a,b , Maribel Casas a , Nilsa de Deus a , Marga Martín a,b , Sonia Pina a,c a
Centre de Recerca en Sanitat Animal (CRESA), UAB-IRTA, Campus de la Universitat Autònoma de Barcelona, 08193 Barcelona, Spain Departament de Sanitat i d’Anatomia Animal, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain c Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Barcelona, Spain b
a r t i c l e
i n f o
Article history: Received 30 June 2008 Received in revised form 17 September 2008 Accepted 19 September 2008 Available online 14 November 2008 Keywords: Complete coding regions Swine HEV Spain Genotype 3
a b s t r a c t The complete coding regions of five hepatitis E virus isolates of swine origin from two different pig farms and the complete genome sequence of two of these strains were obtained and compared to other full length or partial HEV sequences. Based on the nucleotide sequence, the examined Spanish isolates were 87.1–99.7% similar among them being the closest known strain a Mongolian porcine strain (swMN06C1056) which shares 84.5–86.1% of the nucleotide sequence, and are also close to other HEV porcine strains from Japan. Two isolates from the same farm presented an 87 nucleotide insertion in the polyproline hinge unique among all HEV isolates known so far. Comparison with partial HEV sequenced strains indicates that the isolates described in this study form a cluster containing human and porcine HEV strains from Europe, being the only representatives of the subtype 3f that were completely sequenced. Evolutive pressure analysis indicates that microevolution of HEV seems to be driven by negative selection. Further studies should be carried out in order to clarify the HEV origin and evolution. © 2008 Elsevier B.V. All rights reserved.
Hepatitis E virus (HEV) is a small non-enveloped virus belonging to the Hepeviridae family (Emerson et al., 2004). The viral genome consists of a 7.2 kb single-strand positive-sense RNA containing three partially overlapping open reading frames (ORF), a 5 and 3 non-translated regions and a poly-A tract. ORF1 encodes for non-structural proteins such as methyltransferase, helicase and RNA-dependent RNA polymerase; ORF2 encodes the capsid protein, and ORF3 encodes the cytoskeleton-associated phosphoprotein (Reyes et al., 1990; Huang et al., 1992). Four HEV genotypes and several sub-genotypes have been described so far (Lu et al., 2006), which seem to be geographically distributed. Genotype 1 is common in epidemics in Asia, particularly in India (Arankalle et al., 1999). Genotype 2 was originally detected in Mexico but later on has been reported in Africa (Huang et al., 1992; Buisson et al., 2000). Genotype 3 strains are responsible of sporadic infections in humans and also are widespread in swine. This genotype accounts for the largest number of HEV isolates in humans, pig and wild-boars in Europe (Van der Poel et al., 2001; De Deus et al., 2007; Kaci et al., 2008) and have recently
夽 The GenBank accession numbers for the sequences of SW626, SW627, SWP6, SWP7 and SWP8 are EU723512, EU723513, EU723514, EU723515 and EU723516, respectively. ∗ Corresponding author. Tel.: +34 93 5814527; fax: +34 93 5814490. E-mail address: [email protected] (B. Peralta). 0168-1702/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2008.09.008
been described in other animal species in Asia as well (Takahashi et al., 2004; Nakamura et al., 2006). Genotype 4 strains seem to be autochthonous of Asia and have been detected in humans and pigs (Nishizawa et al., 2003). HEV genotypes 3 and 4 are thought to be zoonotic or, at least, have the potential to be transmitted from animals to humans. Thus, porcine, wild-boar and deer strains can be very close to human isolates (Takahashi et al., 2004; Nakamura et al., 2006) and recent reports documented cases of human hepatitis E in which all evidences pointed to the consumption of uncooked or undercooked meat or viscera as the source of infection (Takahashi et al., 2004; Tei et al., 2004). Europe is considered a non-endemic region for human hepatitis E, with few clinical cases, although seroprevalences among healthy population can vary from 1% to 16% (Pavia et al., 1998; Buti et al., 2006; Mansuy et al., 2008). In contrast, recent studies showed that seropositive animals are present in up to 97% of the pig herds (Rutjes et al., 2007; Seminati et al., 2008). In both cases, humans and pigs, European autochthonous isolates belong to genotype 3 (Pina et al., 2000; Clemente-Casares et al., 2003; Buti et al., 2004; De Deus et al., 2007). Up to now, the molecular epidemiology of HEV genotype 3 in Europe, and particularly the relationship existing between human and swine strains, has been studied by comparing very short viral sequences and not a single complete European genotype 3 sequence is available yet. In the present study, five HEV isolates from two pig farms were sequenced, analysed and compared to
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other HEV sequences in order to gain insight in the understanding of the epidemiology and evolution of HEV. Bile samples of five 14-week-old pigs from two different herds (A and B) were selected. Those animals had been submitted to the Veterinary Pathology Diagnostic Service of the Veterinary Faculty of Barcelona for necropsy and suffered subclinical hepatitis as revealed by the histopathological analysis. Total viral RNA was extracted using Nucleospin® RNA virus kit (Macherey-Nagel) and the presence of genomic HEV RNA was determined by means of a seminested RT-PCR as described previously (De Deus et al., 2007). Viral RNA was quantified by real-time PCR as described by Jothikumar et al. (2006) in 2–8 × 106 genomes/ml of bile. The nearly full-length genomes were obtained using the SuperScriptTM III One Step RT-PCR System with Platinum® Taq High Fidelity (Invitrogen) for the cDNA synthesis and first round PCR amplification, and the PfuUltra® II Fusion HS DNA Polymerase system (Stratagene) for the seminested amplification, according to the manufacturer’s instructions. For the analysis of the fragment comprising the entire ORF1 and the 5 -end of the ORF2, primers F25 (5 -GGTCGAYGCCATGGAGGCCC-3 ) and R5500 (5 -GVGGGGCGCTGGGACTGGTC-3 ) were used for the first round, and primers F25 and R5470 (5 -TGGGACTGGTCRCGCCAAG3 ) for the second round of the PCR. For the amplification of an overlapping fragment containing the 3 -end portion of the ORF1 and the complete ORF2 and ORF3, including the 3 -NTR, primers F5000 (5 -AATGTKGCKCAGGTYTGTG-3 ) and R7260 (5 -TTTTTTTTTTTTTCCKGGGRGCGCG-3 ) were used for the first round of amplification, and the primers F5160 (5 MGGSTRGAATGAATAACATG-3 ) and R7260 were used for the seminested amplification. The products were analysed by electrophoresis and stained with SYBR Gold® (Molecular Probes). The amplified products of about 5400 and 2100 bp respectively, were gel purified using the NucleoSpin® Extract II (MachereyNagel) following manufacturer’s instructions and cloned into pCR® II-Blunt-TOPO® (Invitrogen). Recombinant plasmids were used to transform competent E. coli DH10B (Invitrogen). Positive clones were cultured and recombinant plasmids were extracted with the NucleoSpin® Plasmid kit (Macherey–Nagel). Sequencing of both strands was performed from five colonies of each fragment using the ABI PRISM 3700 DNA analyzer (Applied Biosystems, Foster City, USA), using specific primers designed along the genomes. The 5 -NTR of one strain of each farm was determined using the 5 RACE System for Rapid amplification of cDNA Ends, Version 2.0 (Invitrogen) following manufacturer’s instructions. Sequence assembly was accomplished using the programs Phred (Ewing and Green, 1998; Ewing et al., 1998), Phrap and Consed (Gordon et al., 1998), and Bioedit (Hall, 1999). Homology and identity search with respect to HEV strains available at the GenBank was performed by using the BLAST utilities (http://www.ncbi.nlm.nih.gov/BLAST). Alignments were carried out with the ClustalX 1.8 program (ftp://ftpigbmc.ustras/pub/clustalX) (Thompson et al., 1997) and the phylogenetic trees for the entire genome or partial ORFs were constructed with the neighbour-joining method using MEGA4.0 (Tamura et al., 2007). Bootstrap test of 1000 replicates was done to evaluate the reliability of the different groups (Felsenstein, 1985). Selection pressures along the genome were assessed by calculating the difference between non-synonymous (dN) and synonymous (dS) rates (dN − dS) for each ORF, using the SNAP (http://www.hiv.lanl.gov/content/sequence/SNAP/SNAP.html) and DataMonkey (http://www.datamonkey.org) web servers. Sequence assembly yielded a consensus sequence of 7216 nucleotides for the completed sequenced strain from farm A (SW626) and 7304 nucleotides for the sample completely
sequenced from farm B (SWP6). For the rest of the samples the 5 NTR was not determined, thus sequence assembly yield a consensus sequence of 7192 nt for the strain from farm A (SW627), and 7279 nt for the strains from farm B (SWP7 and SWP8). In farm B isolates, ORF1 had an in-frame insertion of 87 nucleotides in the poly-proline hinge, unique among all of the HEV isolates known so far. To confirm this feature an RT-PCR analysis was performed in bile from both herds. The sequencing of the amplicons was in perfect agreement with those of the sequences showing a correct assembly. Over the entire genome, nucleotide similarity for samples obtained within the same herd ranged from 96.6% to 99.7%; in contrast, nucleotide sequences from herd A and B were only 87.1–89.5% similar. The closest HEV full-genome sequence available in GenBank was swMN06-C1056, a genotype 3 porcine isolate from Mongolia that shared 84.5% and 86.1% of the nucleotide sequence when compared to the Spanish farm A or B isolates, respectively. Identities with other genotype 3 strains ranged between 79.3% and 85.1%. Similarity to strains of other genotypes was below 75%. When considering individual ORFs, the examined Spanish isolates have a nucleotide identity among them of 85.8–99.7% for ORF1; 90.1–99.9% for ORF2 and 95.0–100% for ORF3. Comparison of the predicted translated frame displayed aminoacid sequence identities were 95.2–99.9% for ORF1; 97.7–100% for ORF2 and 96.4–100% for ORF3, indicating that most of the changes corresponded to synonymous mutations. The comparative analysis of the individual ORFs among the Spanish strains and other 27 genotype 3 strains available at Genbank, showed nucleotide identities of about 77.9–84.3% for ORF1, 83.5–88.5% for ORF2 and 91.1–97% for ORF3, respectively. As before, comparison in terms of the predicted aminoacid sequence produced higher identity scores: 91.1–96% for ORF1 polyprotein, 95.3–97.7% for ORF2 capsid protein, and 91.1–99.1% for ORF3 protein. In ORF3, however, nucleotide identity was often higher than aminoacid identity suggesting that non-synonymous mutations may occur more frequently in this fragment (Supplementary Table 1). The 5 NTR was 25 nt long and the homology was 96–100% with other genotype 3 strains and between 92% and 96% with sequences of other genotypes. In the 3 NTR, an identity of 73.3–98.3% was observed between the Spanish isolates, and 58–75% with respect to other genotype 3 strains, and 49.1–62.9% when compared to genotypes 1, 2 and 4. In order to determine the genetic relationship of the examined Spanish strains with other known European sequences, phylogenetic trees using the neighbour-joining method based on short fragments were constructed. A phylogenetic tree based on a 168 nucleotide fragment located between positions 6083 and 6250 of the capsid gene was done (Fig. 1A). In that tree, the Spanish isolates reported in this study were grouped in the subtype 3f according to the classification proposed by Lu et al. (2006). Closest sequences were other European pig or human genotype 3 HEV strains reported previously in Spain and Netherlands (Pina et al., 2000; Van der Poel et al., 2001; Fernandez-Barredo et al., 2006; De Deus et al., 2007; Martín et al., 2007) although this grouping was not supported by significant bootstrap values. Selection of a region of 232 nucleotides in the 5 -end of the ORF1 permitted the inclusion of more genotype 3 strains (Fig. 1B). The resulting phylogenetic tree grouped the isolates of the present study again within subtype 3f with the Spanish human isolates VH1, VH2 and VH3 (Pina et al., 2000; Buti et al., 2004), and also with sequences from Greece (Schlauder et al., 1999), the Netherlands (Van der Poel et al., 2001) and Japan. A third phylogenetic tree was constructed based on the alignment of all coding regions of 79 HEV strains from human or animal origin available at GenBank and the Spanish strains SW626, SW627, SWP6, SWP7 and SWP8 (Fig. 2). The analysis showed
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Fig. 1. Phylogenetic trees constructed by the neighbor joining method using: (A) 168 bp sequence located in the capsid gene of HEV and (B) 232 bp sequence located in the 5- end of the ORF1. Bootstrap values of >70% are indicated for the major nodes. Sequences reported in this study are marked as (䊉).
that swine strains of this study clustered in genotype 3; being the only representatives of the subtype 3f that were completely sequenced, and closely related to the yet unclassified Mongolian strain swMN06-C1056 (Lorenzo et al., 2007) and to the Japanese strains HE-JA04-1911, SWJ12-4 and SWJ8-5 (Inoue et al., 2006) of subtype 3e and OSH205 from Kyrgyzstan (Lu et al., 2004) representing subtype 3 g (Lu et al., 2006). The results obtained in this work showed that each examined herd had its own HEV variant of which one had a unique insertion in the poly-proline hinge of ORF1. The significance of that insertion in terms of virulence or survival is unknown and should be further investigated. The fact that the insertion was present in the virus infecting different animals suggest that this could be a target for an epidemiological marker, and could be useful for monitoring the spreading of the HEV infection in the same farm and between farms. Also this study shows that most changes in ORF1 and ORF2 correspond to synonymous mutations, while changes in ORF3 were mainly non-synonymous ones. Although in ORF1 mean dN − dS was −0.31, positive selection was observed in the poly-proline hinge (dN − dS = 0.171). Also, ORF2, where mean dN − dS was −0.35 positive selection was predominant in the N-terminal region. Then, in the ORF3, a mean value dN − dS = 0.017 indicated that ORF3 is subjected to positive selection. Therefore, these results showed that microevolution of HEV seems to be driven by negative selection (dN < dS) except for ORF3 (Fig. 3 summarizes these results). This would be consistent with the expected behaviour of a small genome
virus in which most parts would probably be essential for viral viability. On the other hand, as occurs with all the sequences described, the aminoacid sequence of the capsid gene of the Spanish isolates was very similar (91–97%) to that of other isolates whatever its origin or genotype. Since most of the available ELISAs are based on entire or N-terminal truncated recombinant ORF2 proteins of genotypes 1 and 2 cross-reactivity has been demonstrated (Engle et al., 2002; Arankalle et al., 2007). Thus, the selective pressure map presented here pinpoints some of the regions of interest for further epidemiological studies and virus evolution. The phylogenetic tree based on short nucleotide fragments showed that the Spanish isolates grouped with European genotype 3f strains according to the classification by Lu et al. (2006), while Japanese and American isolates formed different clusters within the same genotype. A similar analysis was obtained using the RNAdependent RNA polymerase region which have demonstrated to be the one who better correlates with the complete genome (Zhai et al., 2006) (not shown), but any other European sequences was available to be included in that analysis. Nevertheless, partial regions used for the analysis usually showed inconsistency in the grouping although Spanish isolates always grouped in genotype 3f (Fig. 1). Otherwise, when comparing the nearly full-length genome, the position of the Spanish strains was confirmed with a significant bootstrap value. The closest isolate was a Mongolian strain of porcine origin, but this finding must be considered a bias, since no other European
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Fig. 2. Phylogenetic tree constructed by the neighbour joining method using the HEV complete coding region. Bootstrap values >70% are indicated for the major nodes.
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Fig. 3. Differences between non-synonymous and synonymous (dN − dS) rates plot for HEV ORF1, ORF2 and ORF3. ORF1 domains are numbered as follows: (1) methyltransferase domain, (2) Y domain, (3) papain-like protease, (4) poly-proline hinge, (5) X domaine, (6) RNA helicase, (7) RNA-dependent RNA polymerase. Arrow indicates the insertion in the poly-proline hinge. * indicates positions with significant positive selection (p < 0.1).
sequences are available. Previous reports suggested that despite the diversity of HEV genotype 3, a geographical pattern can be observed, each continent having autochthonous HEV strains different but related to the ones from other continents, probably due to a different evolution of the same virus (Okamoto, 2007). This work describes the complete genome of Spanish strains of hepatitis E virus and determines their phylogenetic relationship to other genotype 3 strains. Future full-length genome sequences from Europe may contribute to elucidate the origin, evolution and spread of HEV genotype 3 strains. Addendum Since the present paper was submitted for publication, a sequence of genotype 3 subtype 3c of swHEV named swX07-E1 (accession number EU360977) has been reported in Sweden (Xia et al., 2008), which presented an identity of 87–88% with respect to the coding regions of the strains SW626, SW627, SWP6, SWP7 and SWP8. Acknowledgements This study was supported by the research grant AGL2004/06688 from the Spanish government. Bibiana Peralta and Maribel Casas have a fellowship from the Generalitat de Catalunya. Nilsa de Deus has a fellowship from CReSA. We thank Dr A. Bensaid and Dr A. Olvera for useful advice. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.virusres.2008.09.008. References Arankalle, V.A., Paranjape, S., Emerson, S.U., Purcell, R.H., Walimbe, A.M., 1999. Phylogenetic analysis of hepatitis E virus isolates from India (1976–1993). J. Gen. Virol. 80, 1691–1700. Arankalle, V.A., Lole, K.S., Deshmukh, T.M., Chobe, L.P., Gandhe, S.S., 2007. Evaluation of human (genotype 1) and swine (genotype 4)-ORF2-based ELISAs for anti-HEV IgM and IgG detection in an endemic country and search for type 4 human HEV infections. J. Viral. Hepat. 6, 435–445.
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