Influenza Institute, St Petersburg, Russia1
Yakutsk City Hospital, Yakutsk, Russia2
Laboratoire de Bactériologie, Virologie Hygiène, Equipe dAccueil Agents Transmissibles et Hôtes, Signalisation Cellulaire, Oncogenèse, Hôpital Avicenne, UFR Santé Médecine Biologie Humaine, Université Paris 13, 125 route de Stalingrad, 93009, Bobigny cedex, France3
Author for correspondence: Paul Dény. Fax +33 1 48 95 59 11. email paul.deny{at}avc.ap-hop-paris.fr
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Abstract |
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Introduction |
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In Yakutia (Sakha Republic, Russia), a region that encompasses vast areas of Central and Northern Siberia, hepatitis B virus (HBV) infection is a serious health care problem. Indeed, HBV prevalence in Yakutia is 34 times higher than the average for Russia. Furthermore, HDV markers are detectable in 1820% of HBV surface antigen (HBsAg)-positive hepatitis cases (Viazov et al., 1989 ; Alexeeva et al., 1998
), indicating a high level of HDV endemicity in Yakutia. To assess the genetic variations of HDV genomes from this area, we first screened 29 samples from patients who were chronically infected with HDV by restriction fragment length polymorphism (RFLP) analysis of amplified HDV cDNAs. We then focused on 13 specific isolates for sequence and phylogenetic analyses. The complete nucleotide sequences of two isolates, which presented an original RFLP pattern, were obtained. The results indicate that two HDV genotypes (I and II) coexist in this area. Yakutian genotype II isolates form a distinct cluster on HDV phylogenetic trees, possibly representing a specific genotype II subclade.
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Methods |
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RTPCR and RFLP analysis.
Sera from all 29 patients were screened for HDV RNA by RTPCR and analysed by RFLP. HDV RNA was extracted from 100 µl serum by the guanidinechloride method and dissolved in 50 µl DEPC-treated water. For reverse transcription, 5 µl extracted RNA was mixed with random primers (0·4 pM) and dNTP (0·5 mM) and denatured for 3 min at 95 °C. Denatured RNA was added to the reverse transcription mixture (total volume 25 µl) containing 20 U RNasin (Promega) and 100 U SuperScript reverse transcriptase (Life Technologies) in the buffer supplied by the manufacturer. The reaction was incubated at 42 °C for 45 min and stopped by incubation at 94 °C for 5 min. The PCR reaction mixture (total volume 40 µl), containing 0·25 pmol/µl of primers and 1 U AmpliTaq Gold polymerase (PE Applied Biosystems) in the buffer supplied by the manufacturer, was added to 10 µl of the reverse transcription reaction and covered with 50 µl of mineral oil. PCR was carried out in a thermocycler (Gene Amp PCR System 2400) (PE Applied Biosystems) under the following conditions: 9 min at 94 °C, followed by 40 cycles of 45 s at 94 °C, 30 s at 58 °C and 45 s at 72 °C with a final extension step of 5 min at 72 °C. Positive and negative controls were included in each set of reactions. Strict procedures were followed to avoid false-positive results. Results of PCR were analysed by electrophoresis in 1·3% agarose gels. For RFLP analysis, products of amplification with primers 900s and 1280as (Fig. 1) from 50 µl reaction were extracted with phenolchloroform, precipitated with ethanol and dissolved in 8 µl water. Digestion was performed in 10 µl samples with 5 U SmaI (Promega) for 2 h at 25 °C. Digested products were analysed by electrophoresis in 2% agarose gels.
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Secondary structure determination.
Secondary structure determination was performed using the complete genome and antigenome sequences of HDV genotypes I (A20; Italy), IIA (Japan), IIB (Taiwan-TW2b) and III (Peru1) and the Yakutian sequences pt26 and pt62. The mfold program, version 3.1 (Mathews et al., 1999 ; Zuker et al., 1999
), predicts the possible secondary structures for RNA sequences. This program was made available by M. Zuker (http://bioinfo.math.rpi.edu/~zukerm/).
Phylogenetic analysis.
Editing of raw sequence data was carried out with Sequence Navigator, version 1.0.1 (PE Applied Biosystems). Sequences were aligned with CLUSTAL W 1.8 (Thompson et al., 1994 ) using the fast option and different levels of gap extension penalty (2, 5, 10). Minimal manual corrections were performed with SeqPup. Neighbour-joining (NJ) analyses of the sequences were carried out using the DNADIST and NEIGHBOR programs of the PHYLIP package, version 3.572 (Felsenstein, 1989
), or PAUP 4.0b3 (Swofford, 1998
) with pairwise distances estimated through Kimura 2-parameter or maximum-likelihood (ML) options. Maximum parsimony (MP) analyses were carried out using PAUP 4.0b3 with branch-and-bound (for less than 20 taxa) or heuristic searches. All characters were weighted equally and tested as unordered. ML analyses were performed using PAUP 4.0b3 with base frequencies, proportion of invariable sites, shape parameter for gamma distribution of variable sites and substitution rate-matrix estimated through ML analyses from the data. Due to computational limits imposed by the ML algorithms, the number of sequences analysed was limited, as described in Results, and bootstrapping was performed on a limited number of replicates (n=100). Otherwise, we analysed the robustness of different branches by bootstrapping (103104 replicates). All trees were visualized with TreeView (Page, 1996
).
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Results |
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The estimation of nucleotide sequence similarity among the Yakutian isolates showed that they form two distinct groups, in accordance with the RFLP results. Given the R1 genomic region, the first group was more closely related to the reference genotype I sequences than to the genotype II and III sequences. Yakutian type I isolates had inter-strain genetic distances (d), estimated through Kimura 2-parameter analysis, ranging from 7·4 to 11·2%. Their genetic distances from the reference strain A20 range between 8·4 and 12·3%. Inside the group with the new restriction pattern, virus sequences were less distantly related to each other, as divergence between isolates ranged only from 1·9 to 5·6%. These novel Yakutian sequences had a strong level of similarity with genotype II sequences characterized from Japan (d=8·8%) (Imazeki et al., 1991 ). In contrast, Yakutian type II sequences were more distantly related to Taiwan-TW2b (d=15·9%), described recently as the prototype of subtype IIB (Wu et al., 1998
). The prototype genotype I sequence (strain A20) was more distantly related to the Yakutian type II cluster (d=20·2%) and, consistent with most HDV sequences characterized so far, the genotype III reference sequence (strain Peru1) remains the most distant (d=40·0%). This result encouraged us to consider genotype III sequences as an outgroup for phylogenetic analyses.
Phylogenetic analyses
Phylogenetic analyses were performed on three sets of data. For R0, R1 and complete HDV genomes (see Methods), each set included 19, 15 and 16 reference sequences from databases and 8, 11 and 2 sequences obtained from Yakutian isolates, respectively. For these three regions, results of distance analyses followed by NJ tree reconstructions (distances estimated through ML) are shown in Fig. 2 as radial representations. NJ tree reconstructions resulted in phylogenetic trees of similar topology. Genotype I, II and III sequences defined previously are easily distinguishable. Based on analysis of regions R0 and R1 (Fig. 2a
, b
), five samples (pt8, pt12, pt30, pt51 and pt724) with the type I SmaI restriction profile were scattered among type I sequences. As proposed previously, genotype II appears to be subdivided into subtype IIA and Taiwan-TW2b (Wu et al., 1998
). Type IIA corresponds to type II described previously and includes the Japan (Imazeki et al., 1991
) and Taiwan3 sequences (Lee et al., 1996
). All Yakutian HDV sequences with the new original restriction pattern (pt6, pt13, pt26, pt29, pt62, pt63, pt245 and pt704) form a monophyletic group on each data set analysed, the branch of which arises between Taiwan-TW2b and type IIA nodes (Fig. 2a
, b
). Furthermore, when the complete sequences are analysed (Fig. 2 c
), pt26 and pt62 form, together with Japan and Taiwan3, a distinct group that excludes Taiwan-TW2b. These analyses indicate that Yakutian isolates consist of two distinct profiles corresponding to two different genotypes. Yakutian type I sequences are distantly related to each other and scatter among the different type I reference sequences studied, while Yakutian type II sequences form a more closely related, well-defined subgroup on tree reconstructions.
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Discussion |
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Since 1993, it has been known that three major HDV genotypes exist (Wang et al., 1986 ; Imazeki et al., 1991
; Casey et al., 1993
). Because of the absence of trans-complementation between HDV types I and III, Casey & Gerin (1998)
argue that these viruses are not only of a different genotype but also of a different type. Using RFLP and genome sequencing associated with phylogenetic analyses, we demonstrated that at least two HDV genotypes, I and II, are currently present in Yakutia. It was predictable to find genotype I in Yakutia, because all Russian isolates described so far (Ryzhova et al., 1997
; Flodgren et al., 2000
) are affiliated with this genotype. In contrast, the discovery of genotype II in Yakutia was both unexpected and surprising, since the novel restriction profile observed among 15 isolates (Table 1
) might be more likely derived from type I isolates (acquisition of one SmaI site) than from type II isolates (acquisition of two SmaI sites). HDV genotype II is described classically as being restricted to Far East Asia and is found only in Taiwan and Japan (Imazeki et al., 1990
; Lee et al., 1996
; Wu et al., 1998
; Sakugawa et al., 1999
). Our findings indicate that HDV genotype II should be considered to be more widespread. Unlike in East Asia, where type II prevails over type I (Wu et al., 1995
), an equal proportion of both genotypes was observed among hospitalized patients in Yakutia. Interestingly, in this area, type II isolates are less distantly related to each other than type I isolates (Fig. 2
). This observation may suggest that different type I isolates might have been introduced repeatedly into this area during the last century when the migration of individuals from the European part of Russia to Yakutia increased significantly. Another possible explanation is that type I isolates have been present in this area for a very long time and that type II isolates were introduced recently. The first hypothesis is probably supported by the fact that all of the Russian patients in our study (4 out of 29) were infected with type I viruses. The coexistence of two genotypes in this area might represent a ground for HDV inter-type recombination; however, we could detect in this preliminary study neither mixed infections (Wu et al., 1999b
) nor virus recombination between type I and II viruses.
The pathogenesis of HDV liver disease remains intriguing and complex. It might involve specific HDV features (expression and replication, genetic variability, ribozymes, etc.) and specific HBV features (genotypes, mutants, level of replication, etc.) in the context of host genetic background, immune response and superimposed cofactors (such as alcohol and other viruses). Wu et al. (1995) and Sakugawa et al. (1999)
discovered that in Taiwan or in the Miyako islands, HDV genotype II seemed to be associated with a lower level of pathogenicity than HDV genotype I. In hospitalized patients from Yakutia, we observed that both genotype I and genotype II infections could lead to severe infections.
Other factors could account for the differences in the different cohorts of patients studied. First, in Taiwan, the difference of pathogenesis between HDV types I and II is significant for fulminant hepatitis associated mainly with HDV type I. Interestingly, a comparison of the secondary structures obtained suggests that base pairing surrounding the editing site seems to be less strong for type II than for type I sequences (Fig. 4). Studies from type I sequences indicate that conservation of these complementary base pairs is crucial for editing efficiency (Casey et al., 1992
; Polson et al., 1996
). HDV fulminant hepatitis might be associated with the efficiency of HDV type I editing, leading to swift virus dissemination during acute hepatitis. The characterization of Yakutian HDV viruses linked to HDV fulminant hepatitis and in vitro studies might help to test such a hypothesis. Second, the distribution of HDV genotypes may be connected to the distribution of HBV genotypes. In the south eastern part of European Russia, a high incidence of acute and fulminant HBV genotype D has been observed recently and is associated with infection with HDV genotype I in 29% of cases (Flodgren et al., 2000
). Whether or not a specific link exists for Yakutian HDV type II isolates with HBV genotype B or C isolates that are predominant in Asia (Theamboonlers et al., 1999
; Orito et al., 2001
) requires further study.
The original population of Yakutia is believed to have been present there for the past 40000 years. The characterization of HDV lineages might be a useful tool to follow population migration, as it has been suggested for HBV, human T-lymphotropic virus type I and JC viruses (Agostini et al., 1997 ; Blitz et al., 1998
; Van Dooren et al., 1998
). HDV phylogenetic studies have been based almost exclusively on the analysis of the 3'-terminal part of the HD gene (Fig. 2a
). The low similarity of this region might lead to erroneous HDV lineage reconstruction. Indeed, while being functional for assembly, LHDAg is not necessary for RNA replication, but inhibits it. Recombinations (Wu et al., 1999a
) or deletions (P. Dény, unpublished) of parts of the genome have also been characterized. In contrast to LHD, the product of the sHD gene is essential for RNA replication and seems also to be type-specific (at least for studies involving complementation between types I and III). We suggest, therefore, that the coding sequence for sHD is a more appropriate marker for evolutionary genetics than the region encoding the carboxy-terminal extension. By characterizing new HDV isolates from Yakutia and using extensive phylogenetic approaches on various genomic regions, we demonstrated that all type II Yakutian sequences might be derived from a common ancestor and that all of these viruses had probably evolved from a common Asian and Siberian HDV type II prototype. Although our results based on the sHD gene and complete genome phylogenetic studies are preliminary (Fig. 6
), they could also suggest that Taiwan-TW2b (Wu et al., 1998
) (proposed to be the prototype of HDV subtype IIB) might no longer be affiliated with HDV clade II. This suggestion reflects a wider HDV variability than thought previously.
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Acknowledgments |
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Received 2 April 2001;
accepted 23 July 2001.