1 Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo, Tokyo 113-8519, Japan
2 Department of Gastroenterology and Hepatology, Musashino Red Cross Hospital, Tokyo, Japan
3 Institute for Clinical Research, World Health Organization Collaborating Center for Reference and Research on Viral Hepatitis, National Nagasaki Medical Center, Nagasaki, Japan
4 Division of Viral Hepatitis, Centers for Disease Control and Prevention, Atlanta, USA
5 First Department of Internal Medicine, School of Medicine, University of the Ryukyus, Okinawa, Japan
Correspondence
Nobuyuki Enomoto
nenomoto.gast{at}tmd.ac.jp
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ABSTRACT |
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INTRODUCTION |
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In Japan, chronic HDV infection is relatively rare (Tamura et al., 1993) but is endemic in the Miyako Islands of Okinawa, where the HDV genotype II is prevalent (Sakugawa et al., 1999
). Although the route by which HDV is spread on this island is unclear, our previous studies demonstrated that the severity of liver disease was heterogeneous within this population, despite relatively uniform clinical backgrounds. Thus, a detailed analysis in which the HDV genomes of these patients are correlated with clinical profiles could provide a unique opportunity to define the critical genetic features of HDV that determine liver injury.
To delineate the features of HDV isolates in this area, we determined the sequence of the full-length HDV genome from a large group of patients with chronic HDV infection, the majority of whom were from the Miyako Islands. As a result, we identified a new genetic variant of HDV genotype IIb that was associated with more progressive disease. Subsequently, specific genetic differences among these HDV genotype IIb isolates were correlated with the clinical features in order to reveal the variations in the HDV genome responsible for the progression of liver disease.
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METHODS |
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Sequencing of HDV.
The full-length HDV genome was sequenced in 33 patients. In the other seven patients, the partial genetic sequence enoding the delta antigen (HDAg) was determined. Extraction of RNA from 150 µl of serum by the acid guanidinium thiocyanate/phenol/chloroform method (Chomczynski & Sacchi, 1987) using ISOGEN (Wako, Osaka, Japan) and RT-PCR were performed as described previously (Enomoto et al., 1994
). Four partially overlapping fragments were amplified by nested PCR using the primers shown in Table 1
. These primers were designed and numbered based on HDV genotype II sequences in GenBank. PCR was initially performed with primers designed for HDV genotype II. If HDV cDNA was not amplified with these primers, PCR was performed with primers for HDV genotype I (primer sequences are available on request). Both strands of the PCR products were directly cycle sequenced with the PRISM dye termination kit (Applied Biosystems) and nested PCR primers.
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Statistical analysis.
Categorical data were compared by chi-square or Fisher's exact test. Distributions of continuous variables were analysed by the MannWhitney U-test or Student's t-test using Statview 5.0 software (Abacus Concepts). All tests of significance were two-tailed and P values of less than 0·05 were considered as statistically significant.
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RESULTS |
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DISCUSSION |
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We identified the new HDV genotype IIb variant by phylogenetic analysis of the complete genomes of 33 HDV isolates. Among them, 30 isolates, mostly from the Miyako Islands, were classified as genotype IIb (Wu et al., 1998) or its variant, IIb-M. In previous studies including our own, HDV genotypes in the Miyako Islands have been considered as genotype IIb (Sakugawa et al., 1999
; Ma et al., 2003
; Arakawa et al., 2000
). However, the present detailed phylogenetic analysis using the full genome successfully identified a cluster distinct from the prototype IIb cluster. In fact, the nucleotide homologies between genotype IIb and IIb-M and among genotype IIb-M were clearly different, i.e. 8890 % and 9497 %, respectively. HDV genotype II is divided into two types in Taiwan (i.e. IIa and IIb), with 77 % nucleotide homology between the complete sequences of genotype IIa and IIb (Wu et al., 1998
). Although the criteria for defining identical genotype by homology analysis were not determined, the difference between IIb and IIb-M seems to be less than that between IIa and IIb, as shown by phylogenetic tree analysis. In fact, a IIa variant was recently reported in Siberia (IIa-Yakutia), which in comparison with IIa showed a similar degree of genetic differences (Ivaniushina et al., 2001
). Based on these results, we conclude that IIb-M should be considered as a genetically relevant IIb variant.
Genotype II is confined to East Asia (mainly Siberia, Japan and Taiwan), in contrast to the ubiquitous global distribution of genotype I (Gerin et al., 2002). Genotype IIb was first identified in Taiwan (Wu et al., 1998
) and we subsequently reported it among patients from the Miyako Islands (Sakugawa et al., 1999
). However, the origin of clusters of IIb-M cannot be precisely determined. If in the future the precise evolution rate of the HDV genome can be determined, then the temporal estimation of the spread of HDV using a molecular clock might be possible.
One of the most important findings in the present study is that the clinical pictures differ between genotype IIb and IIb-M. Our previous studies demonstrated that HDV genotype II is predominant in this area and that these patients show heterogeneous clinical pictures ranging from ASC to HCC (Sakugawa et al., 1999; Nakasone et al., 1998
); however, the reason for this diversity could not be explained based on the known clinical and virological factors of HBV. In the present study, all of the patients with chronic HDV genotype IIb infection were ASC or CH and none were at the LC or HCC stage. In contrast, 55 % and 45 % of patients with genotype IIb-M were in the CH and LC stages, respectively, and none of them was ASC. These findings indicate that patients with genotype IIb-M are more likely to progress to LC and HCC than those with genotype IIb and that differences in HDV genotype could cause the different clinical pictures observed in this population.
The main cause of the difference in liver disease between patients with IIb and IIb-M seems to be the diversity of HDV itself. Although the severity of liver disease in hepatitis D can be influenced by a variety of host factors including genetic backgrounds as well as HBV status, no apparent differences were found between patients with genotype IIb and IIb-M. In particular, in most patients, serum HBV DNA levels were below 105 copies ml-1 with negative HBeAg, which were too low to cause HBV-related liver injury (Sakugawa et al., 2001; Lok et al., 2001
). Similarly, the HBV genotype, which is also known to cause diversity of liver disease (Kao et al., 2000
; Orito et al., 2001
), was genotype B in all of the patients from the Miyako Islands. Differences in HDV genotype are known to affect the pathogenesis and diverse clinical pictures of HDV infection (Casey et al., 1993
; Wu et al., 1995a
; Ivaniushina et al., 2001
). Genotype III, exclusively found in the northern part of South America, is associated with fulminant hepatitis (Casey et al., 1993
). On the other hand, genotype II in Taiwan is generally associated with a more favourable outcome than genotype I, which causes liver disease with diverse clinical presentation from asymptomatic carrier to rapidly progressive CH (Wu et al., 1995a
). A IIa variant recently reported in Yakutia, Siberia, Russia, also causes a severe hepatitis comparable with genotype I in this cohort (Ivaniushina et al., 2001
). These findings strongly suggest that the genetic structure of HDV can profoundly influence the pathogenesis of liver injury in HDV infection. However, the genetic structure responsible for such clinical features could not be readily determined because the genetic differences between the different genotypes are too diverse, as seen in Fig. 2
. In contrast, despite the different clinical pictures between IIb and IIb-M, the genetic differences are small enough to enable the definition of the genetic features of HDV pathogenesis and replication in vivo.
By comparative analysis between the genotype IIb and IIb-M genomes, the highest difference was found in the hypervariable region (nt 1598657) and moderately high in HDAg (nt 9571597), whereas the autocatalytic regions encoding ribozyme activity were well conserved (Wu & Lai, 1989). The hypervariable region was markedly variable even within the same genotype, supporting the notion that this region cannot confer any relevant biological function aside from the formation of the rod structure of HDV RNA required for RNA synthesis by RNA polymerase II (Modahl & Lai, 2000
). On the other hand, the requirement for strict secondary or tertiary structure of the autocatalytic domain seems to be so crucial for full activity of the ribozyme needed for the rolling-circle mechanism of HDV replication that divergence of this region could not exist among isolates. Therefore, HDV genetic regions other than the hypervariable region or the autocatalytic domain, i.e. the HDAg coding region, confer the clinical difference between IIb and IIb-M. In the HDAg coding region, we found that the most prominent differences are in the RNA editing site and the packaging signal in the C terminus of the large HDAg (Modahl & Lai, 2000
). Although the coiled-coil domain (Wang & Lemon, 1993
) also showed modest differences, the leucine zipper motif (Chen et al., 1992
) was preserved, and the nuclear localizing signal (Xia et al., 1992
) and RNA binding domain (Lin et al., 1990
) were identical in IIb and IIb-M, indicating that these regions are not responsible for liver damage.
In genotype IIb-M, there was particular disruption of the base-pairing structure two bases upstream of the editing site, resulting in a characteristic structure in this region distinct from genotype IIb and IIa (Fig. 3). There is a possibility that the unique structure of the RNA editing site of genotype IIb-M may affect the observed difference in pathogenesis between genotype IIb and IIb-M. RNA editing is a pivotal event during the HDV replication cycle (Casey et al., 1992
), where initially in HDV infection, small HDAg transactivates HDV RNA synthesis by RNA polymerase II (Wu et al., 1995b
). Large HDAg, which has 19 additional amino acids (the packaging signal sequence) at the C terminus of small HDAg, is produced in the late stage of infection by RNA editing of the umber stop codon (UAG) to a tryptophan codon (UGG) in the small HDAg gene by the host adenosine deaminase (Modahl & Lai, 2000
). Large HDAg suppresses HDV RNA replication and promotes virion assembly by extranuclear export of the HDAgRNA complex and binding to HBsAg. The regulatory mechanism of this RNA editing is not fully understood, but the secondary structure of the antigenomic region corresponding to the 3' end of the small HDAg gene influences the editing efficiency (Casey et al., 1992
; Wu et al., 1995b
; Casey, 2002
). A recent in vitro mutational study clearly demonstrated that the base-pairing structure surrounding the RNA editing site profoundly influences RNA editing efficiency (Hsu et al., 2002
). In genotype I, the base pairing surrounding this site is particularly strong (four base pairs on each side), whereas a weaker secondary structure is found within genotype II that is associated with milder liver disease. In addition, the distinct structure of genotype III is thought to be involved in fulminant hepatitis (Casey, 2002
). Collectively, the specific differences in the base-paired structure of the RNA editing site might explain to some extent the difference in virulence among HDV genotypes. Therefore, although in vitro confirmation is necessary, it appears that the loose structure around the RNA editing site found in genotype IIb-M might influence the editing efficiency in comparison with genotype IIb, leading to the observed clinical differences.
In addition to the difference in the RNA editing site, there are four characteristic amino acid differences (codons 198, 200, 201 and 203) in the packaging signal sequence of the large HDAg between genotype IIb and IIb-M (Fig. 5). This region is almost completely conserved among IIb-M isolates. As mentioned above, addition of this packaging signal reverses the property of HDAg (Modahl & Lai, 2000
; Chang et al., 1993
). The exact molecular mechanism of this phenomenon is not completely understood, but, as shown in Fig. 4
, a sequence of 19 amino acids was highly genotype specific. In vitro analysis demonstrated that swapping the packaging signal sequence of genotype IIa with that of genotype I HDAg decreases the virus replication of genotype I, while the replication of genotype II was intensified, indicating that this region directly regulates HDV RNA replication (Hsu et al., 2002
). Thus, the structural characteristics of this region in IIb-M can profoundly influence virus replication. In particular, two of the four amino acid differences found in IIb-M were located in the proline residues, which are implicated in the assembly process by extranuclear export of the HDAgRNA complex. In fact, in a recent study with cultured cells, mutation of the proline residue in this region attenuated the extranuclear export of large HDAg (Lee et al., 2001
). However, these data did not directly prove that the C-terminal domain structure of HDAg influences the pathogenesis. In the future, in vitro mutational studies should be performed to verify the hypothesis that differences in the packaging signal sequence in genotype IIb-M can modulate HDV replication and lead to progressive disease.
In conclusion, we have identified a new genetic subclass of HDV genotype IIb in the Miyako Islands, Okinawa, Japan. This HDV variant is associated with more aggressive liver disease and has specific genetic changes in the C-terminal packaging signal of large HDAg as well as the RNA editing sequence. These findings should prompt further investigation into the relationship between HDV genetic structures and their function and pathogenesis. This study provides valuable information for molecular epidemiology and diagnosis, contributes to a better understanding of HDV biology and offers the potential for new therapies for HDV a disease for which no effective therapy has yet been established.
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ACKNOWLEDGEMENTS |
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Received 11 July 2003;
accepted 16 September 2003.