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Journal of Clinical Microbiology, September 2000, p. 3311-3316, Vol. 38, No. 9
Department of Infectious Diseases, Lund
University Hospital,1 and Division of
Molecular Neurobiology, Wallenberg Neuroscience Center, Lund
University,2 Lund, and Department of
Virology, University of Umeå, Umeå,4
Sweden, and Department of Infectious Diseases, Samara State
Medical University,3 and Laboratory
of Immunology, Samara Medical Institute
"Reaviz",5 Samara, Russia
Received 28 March 2000/Returned for modification 30 May
2000/Accepted 5 July 2000
Until 1991, the Russian city of Samara was largely isolated from
other parts of Russia and the rest of the world. Very recently, Samara
has seen an alarming increase in the incidence of hepatitis. The
proportion of fulminant cases is unusually high. We wanted to assess
the roles of hepatitis B virus (HBV) and hepatitis D virus (HDV) in
acute viral hepatitis in this region by analyzing the prevailing
strains of both and by determining their genotypes and possible origin.
Serum samples were screened for different serological markers and by
PCR followed by direct sequencing. Of the 94 HBV-positive samples (80%
of which were acute infections), 37 (39%) were also HDV positive.
Sixty-seven percent of the patients had anti-HCV antibodies.
Twenty-five percent of all patients in the study had fulminant
hepatitis. Statistically significant sex differences were found among
fulminant cases. For HBV, the core promoter sequences of 62 strains
were determined and all but one were found to be of genotype D. None of
these had any deletions. Only one strain, from a patient with fulminant
fatal hepatitis, showed multiple mutations. The pre-S2 region sequences
of 31 HBV strains were also compared. Phylogenetically, these fell into two distinct groups within genotype D, suggesting different origins. For HDV, part of the region encoding the Hepatitis B virus (HBV) belongs to
the family Hepadnaviridae and has several unique properties.
It has a very compact circular genome, with overlapping reading frames,
and, unlike any other animal DNA virus, replicates through an
intermediate reverse transcription step (3). The mutation
rate of HBV is not known, but the enhanced rate arising from reverse
transcription without proofreading is held in check by the compactness
of the genome, which limits the number of viable mutations possible.
Genetic analysis of HBV has shown there to be six different genotypes,
A to F, based on an intergroup divergence in nucleotide sequence of 8%
or more (19, 20). These genotypes vary in geographical
prevalence and, in part, in clinical and serological outcome.
Approximately half of all acute HBV infections are subclinical. At the
opposite range of the scale, the fulminant cases constitute less than
1% of acute hepatitis B infections. Following reports of outbreaks of
fulminant hepatitis B, it has been suggested that specific mutations in
the HBV genome often detected in these cases are associated with the
development of fulminant disease (10, 14).
An important cause of fulminant viral hepatitis is co- or
superinfection of HBV with hepatitis D virus (HDV) (4, 17). During both acute and chronic infection, HDV infection often leads to a
more severe disease (26). This subviral human pathogen, having an RNA genome of only 1.7 kb, produces only one protein, which
appears in two forms, and is dependent on HBV for packaging (21).
Three phylogenetically distinct genotypes of HDV have been reported.
HDV strains are more heterologous than HBV strains, and strains
differing in more than 20% of their nucleotide sequences constitute
different genotypes (29). Genotypes II and III have only
been isolated in eastern Asia and in northern South America, respectively, whereas genotype I is more widespread geographically (1, 7). Clinically, the disease pattern resulting from
infection with HDV genotype I is very variable, ranging from mild to
severe disease. Genotype II usually gives rise to a milder hepatitis, while genotype III appears to lead more often to fulminant hepatitis (1).
The city of Samara, located in the southeastern part of European Russia
and the fourth largest city of the country, has had an alarming
increase in the incidence of viral hepatitis over the last 2 years.
Prior to 1991, this city was relatively isolated, both from other
countries and from other parts of Russia. As there appears to be an
unusually large proportion of fulminant hepatitis cases, we set out to
assess the roles of principally HBV and HDV in acute viral hepatitis in
this region. Having found HDV to be prevalent among HBV-infected
individuals, we attempted to determine the genotype and possible origin
of the HDV and HBV strains circulating in Samara between 1997 and 1999.
Patients.
Consecutive patients admitted with acute hepatitis
to the Department of Infectious Diseases, University Clinics of Samara, Samara, Russia, between October 1997 and May 1999 were included in the
study. The patients were bled as part of the routine clinical procedure, and an aliquot of serum was separated and frozen pending further testing for hepatitis markers and nucleic acid analysis in
Sweden. After excluding 15 cases lacking sufficient patient data and 7 cases with no biochemical and/or serological signs of viral hepatitis,
105 patients remained in the study. There were 84 men and 21 women, and
the mean age was 24 years (range, 15 to 76). The vast majority of the
patients were intravenous drug users (IVDUs). All patients but one had
elevated alanine aminotransferase levels. Twenty-seven patients had
fulminant hepatitis, as defined by encephalopathy and coagulopathy
accompanied by deteriorated liver function tests, including elevated
bilirubin levels. There were limited volumes of serum available from
some patients. In these cases, priority was given to testing for HBV
DNA by PCR, followed by anti-HCV antibody, HBsAg, and HDVAg and/or
anti-HDV antibody.
Serological tests.
HBsAg, anti-HBc immunoglobulin M (IgM),
HBeAg, anti-HBe antibody, anti-HCV antibody, and anti-HAV IgM were
tested by AXsym (Abbott Laboratories, North Chicago, Ill.). HDVAg and
anti-HDV antibody were tested by an in-house radioimmunoprecipitation
assay (5).
Extraction and amplification of HBV DNA.
HBV DNA was
extracted from serum by the phenol-chloroform method as previously
described (15). Two sets of primers (KL28-KL6 and KL12-KL33;
Table 1), amplifying the core
promoter-precore region and the pre-S-S region, respectively, were
used in the PCR. The amplification followed a protocol described
earlier, with slight modifications of cycling temperatures (94°C for
1 min, 45°C for 1 min, and 72°C for 2 min) (15).
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Copyright © 2000, American Society for Microbiology. All rights reserved.
Recent High Incidence of Fulminant Hepatitis in Samara, Russia:
Molecular Analysis of Prevailing Hepatitis B and D Virus
Strains
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-antigen was sequenced from
four strains. All proved to be of genotype I and were similar to Far
Eastern and Eastern European strains. The contribution of intravenous
drug use to the sharp increase in viral hepatitis in this unique
setting is discussed.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Primers used for amplification and sequencing of HBV
and HDVa
Extraction and amplification of HDV RNA.
HDV RNA was
isolated using the RNAWIZ RNA Isolation Reagent (Ambion, Austin, Tex.).
A total of 140 µl of serum was incubated in 280 µl of RNAWIZ for 5 min. After the addition of 84 µl of pure chloroform, the samples were
incubated for another 10 min and then centrifuged for 15 min in a
microcentrifuge. The upper aqueous phase was transferred to a new
microcentrifuge tube, to which 210 µl of sterile double-distilled
water (ddH2O) was also added. An addition of 420 µl of
isopropanol was followed by a 10-min incubation and then centrifugation
for 15 min. The RNA pellet was washed in 420 µl of cold 75% ethanol,
centrifuged for 5 min, and subsequently air dried for 10 to 15 min. The
pellet was resuspended in 80 µl of sterile ddH2O and, if
necessary, the suspension was stored at 
20°C. All steps were
performed at room temperature, and mixing was achieved by inverting the
tubes a few times. To amplify the purified HDV RNA, the Access RT-PCR System Kit (Promega, Madison, Wis.) was used according to the manufacturer's instructions, with 20 µl of RNA suspension serving as
template for the reverse transcription. Primers EF3 and A
(1) (Table 1) were used in the ensuing PCR, amplifying a
400-nucleotide (nt)-long region of the HDV genome proposed for the
classification of HDV genotypes.
Analysis and sequencing of viral DNA. The products of the HBV and HDV PCRs were first analyzed on a 1.8% agarose gel and then directly sequenced as previously described (11), using the primers in Table 1.
Phylogenetic analyses. Nucleotide sequences of both HBV and HDV were compared to previously reported sequences and aligned by the Clustal method in the program MEGALIGN of the Lasergene99 software package (DNASTAR, Madison, Wis.). CLUSTALX version 1.8 (28) was then used to generate the data files to be used in a combination of the programs DNADIST and FITCH. This produced phylogenetic trees using the distance matrix method, which estimates the pairwise genetic similarity (or distance) between strains. The method starts by grouping the two most homologous strains, then adding strains of increasing variability one by one. The resulting trees were finally visualized with DRAWTREE. In a radial tree based on a distance matrix, the genetic distance between any two strains is displayed by the combined length of the branches connecting them. The programs DNADIST, FITCH, RETREE (for layout modifications), and DRAWTREE are all part of the Phylogeny Inference Package (PHYLIP) version 3.5c (2) (http://evolution.genetics.washington.edu/phylip.html).
Statistical methods. The chi-square test with Yates' correction was used.
Nucleotide sequence accession numbers. The 31 Samara pre-S2 sequences can be found in GenBank under accession no. AF247933 to AF247963. The four new HDV sequences have been deposited in GenBank under accession no. AF247964 to AF247967.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Serological testing.
Serum samples from 105 patients from
Samara with signs of acute viral hepatitis were analyzed. Due to
limited volumes of serum, all markers could not be analyzed in every
sample. Of the 105 patients, 94 had an HBV infection as defined by
positive HBV DNA PCR and/or HBsAg and/or anti-HBcIgM (Table
2). Eighty samples were HBV DNA PCR
positive. Of the 84 samples tested for anti-HBc IgM, the majority
(80%) were positive, indicating acute HBV infection.
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Molecular analysis of HBV. All samples were tested for HBV DNA by PCR using two separate sets of primers. Eighty samples were positive with primer pair KL28 and KL6. A 159-nt long region (positions 1742 to 1900 according to the numbering of Okamoto et al. [20]), spanning the core promoter region and precore gene, was directly sequenced. There were no deletions in the core promoter region and only one strain (no. 13) belonged to HBV genotype A. The remaining strains were all of genotype D. Commonly seen mutations in the core promoter (nucleotide positions 1762 and 1764, AGG to AGA or TGA) and in the precore region (G-to-A mutation at position 1896 leading to a translational stop) were found in some strains. HBeAg and anti-HBe status was not tested in all HBV-positive samples, but in 13 of 17 samples with anti-HBe antibody and lacking HBeAg, both the core promoter and precore region had the wild-type sequences. One strain (no. 98) had multiple mutations in both the core promoter and the precore gene, including G-to-A mutations at precore positions 1896 and 1899. The patient infected with this strain had fulminant fatal hepatitis.
The PCR using primer pair KL12 and KL33 amplified the whole pre-S region and most of the S gene, and 78 samples were positive in this PCR. No major deletions were found in these samples, as judged by the size of the bands in the agarose gels. Thirty-one of these samples were randomly chosen for further analysis by direct sequencing. The whole of the pre-S1 and pre-S2 genes and a region of the S gene spanning codon 118 to 170, encompassing the a-determinant, were sequenced. No insertions or deletions were found, and there were no mutations in the a-determinant. The sequences of the S gene confirmed that all strains characterized but one belonged to genotype D. The pre-S2 region, being the most variable region sequenced, was chosen for phylogenetic analysis. The sequences from the 30 Samara genotype D samples were compared to those from 12 other, geographically different strains of the same genotype (8, 9; three unpublished strains). The resulting radial tree clearly shows that the prevailing strains from Samara fall into two distinct clusters which are separate from strains with other geographic origins (Fig. 1).
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Fulminant hepatitis.
One quarter of the 105 consecutive
hepatitis patients included in this study had fulminant hepatitis
(25.7%). Table 3 shows the viral
etiology of the fulminant cases. Of the 27 fulminant cases, 13 were
infected with HDV. A significant sex difference between HDV-positive
and HDV-negative patients with fulminant hepatitis was seen; males were
HDV positive more often (P < 0.05). This skewed sex
distribution was also seen in the total proportion of males versus
females acquiring fulminant hepatitis, as females were seen to have a
statistically significant higher risk of fulminant disease
(P < 0.005). When the 14 HDV-negative patients with
fulminant disease were studied more closely, 7 of the 14 were also
anti-HCV positive. With the exception of sample no. 98, commonly
occurring mutations in the core promoter and precore region were not
more prevalent in the HBV strains infecting these patients (Table
4).
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Molecular analysis of HDV. In order to determine the genotype of the HDV strains prevailing in Samara, the genomes of four randomly chosen strains were isolated, reverse transcribed, amplified by PCR, and then sequenced using primers EF3 and A. The 257-nt sequence from nt 934 to 1190 (numbering according to Makino et al. [18]) was then used for BLAST searches in GenBank, revealing that Samara HDV belongs to genotype I. The Samara strains also clustered with other genotype I sequences in the subsequent phylogenetic analysis and not with either of the two other genotypes (data not shown).
The Samara HDV sequences were compared to 47 previously reported HDV genotype I sequences representing different geographical regions. The resulting radial phylogenetic tree is shown in Fig. 2. The tree shows two major clusters, with the Samara HDV strains located between them. Some homology to Far Eastern and Eastern European strains is seen. Interestingly, there is also a close match with another Russian strain. Unfortunately, there is no further epidemiological information about this strain (24).
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In Samara, a city in the southeastern part of European Russia, there has been a sudden and sharp rise in the incidence of viral hepatitis over the last 2 years. Compared to the figures for the whole country, which have been stable over the last 4 years, the incidence of HBV infection has doubled and that of HCV has tripled in Samara between 1998 and 1999 (23; unpublished observations). When an epidemiological phenomenon like this is observed, it is often caused by a change either in the population or in the infecting agent. Until the beginning of the 1990's, Samara and its surrounding region were isolated and foreign travel into the area was not permitted. After this ban was lifted, increased travel brought an increase in illicit drug commerce and the number of IVDUs (mainly young) increased. It is possible, therefore, that the rising incidence of viral hepatitis in Samara is the result of the increasing illicit intravenous drug use.
The situation is similar to that seen in southern Sweden several decades ago, when HDV infection was introduced in 1973 and spread among the IVDUs, with a yearly rise in incidence (5). An unusually large proportion of HDV infections among the IVDUs were fulminant (16), an observation also made in Ireland (25). Fulminant HDV hepatitis has also been described from South America (4, 17). Later studies investigating fulminant strains from the South American studies have shown that HDV genotype III appears to be associated with more aggressive disease (1).
In the present study, as many as 39% of the HBV-positive patients were also infected with HDV, and one-third of these HDV-positive patients had fulminant hepatitis. In order to assess the prevailing HDV genotype in Samara, four strains isolated from patients with different age and sex patterns were genotyped, and they were all of genotype I. The sequences used for HDV genotyping in this study were slightly shorter than those used by some other groups (1, 29), and after analyzing the sequences, we found that alignment with the shorter ones still gives a good representation of the comparison of the entire genome (data not shown).
The possibility of mutations in HBV DNA giving rise to more aggressive disease and being another cause of the high proportion of fulminant cases seen in this study was then investigated. The specific mutations that have been pinpointed most often as being associated with fulminant disease are located in the core promoter at nucleotide positions 1762 and 1764 and in the precore gene at nucleotide position 1896 (6, 22, 27). Not all HBV strains from fulminant non-HDV cases could be sequenced in this study, but in 7 of 10 strains, both the core promoter and the precore regions were of the wild type. This supports the study by Laskus et al. (13), in which fulminant hepatitis B (of mainly genotype A or D) in the United States could not be correlated with core promoter or precore mutations.
Only one strain (no. 98) showed multiple changes in both the core promoter and precore region. No larger deletions could be seen in the pre-S and S genes. The patient fatally infected by this strain was HCV and HDV negative, and no other explanation for the fulminant course of illness could be found.
The most notable finding in the group of patients with fulminant hepatitis was the significant difference in sex distribution. That a larger proportion of males were infected with HDV and acquired fulminant HDV infection can be explained by intravenous drug use being by far more common among young males in Samara (unpublished observation). The disproportionately high number of females with non-HDV fulminant viral hepatitis, however, is not explained by differences in social behavior. To our knowledge, such a difference in sex distribution has not been described before.
Taking into account the HBV sequences from both the core promoter-precore region and the pre-S-S region in this study, there were remarkably few changes when comparing them to a consensus sequence from strains of the same genotype. No deletions, and only a limited number of point mutations, were found. This is in line with the view that some of these commonly occurring mutations arise from selection through immunological pressure in the chronic carrier (13). Eighty percent of the patients in this study who were tested for anti-HBc IgM were positive, and thereby had, by definition, acute HBV infections. This, taken together with the observed sharply rising incidence, implies that HBV (and HDV) have recently gained access to this community.
A phylogenetic analysis of prevailing strains in a community together with strains isolated from other regions can serve two main purposes. Firstly, it will demonstrate whether many different strains circulate in the community. Secondly, it may indicate the geographic origin of the prevailing community strains. The advantage of using the distance matrix method and displaying the results as a radial tree, as we chose to do in this study, is that it demonstrates very clearly how far apart different strains are from each other phylogenetically.
Phylogenetic analysis of the HBV pre-S2 gene demonstrated two clear clusters of genotype D strains present in Samara that were clearly separate from strains of the same genotype isolated from other regions of the world. It thus appears that two main HBV strains circulate in the Samara region and that they are distinct from strains from the other regions studied.
In contrast, the phylogenetic tree emerging from comparisons of four Samara HDV strains with 47 HDV strains from other regions, showed that the Samara strains were similar to each other, but did not cluster close together. Such clusters could be seen among strains isolated in the United States. The similarity between HDV strains in the United States and also between strains in some regions of Greece have been suggested to represent the recent introduction of HDV into these populations (24). The Samara HDV strains appeared to be phylogenetically closest to Far Eastern and Eastern European strains.
In summary, this study has shown that the abruptly increasing number of cases of viral hepatitis in the previously isolated Russian city of Samara is caused by HBV, HCV, and HDV, with the majority of patients having an acute HBV infection. Although many of the patients studied were also infected with HCV, acute HCV infection is often subclinical. Acute HBV infection, on the other hand, more frequently leads to the kind of severe disease seen in many of the patients in Samara. An unusually large proportion of these patients have fulminant cases, and a disproportionately large number of non-HDV fulminant cases in females has also been demonstrated. Two main HBV strains of genotype D appear to circulate in the community, whereas the HDV strains are phylogenetically more distinct from each other. There is a potentially serious situation in a community with such a rising incidence of HBV, HCV, and HDV if other blood-borne viruses, such as human immunodeficiency virus, are introduced.
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ACKNOWLEDGMENTS |
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This work was supported by the Swedish Society of Medicine and the Swedish Medical Research Council grant no. K98-16X-11592-03A. Erik Flodgren is a recipient of a grant from the Segerfalk Foundation.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Infectious Diseases, Lund University, SE-221 85 Lund, Sweden. Phone: 46-46-171858. Fax: 46-46-137414. E-mail: Karin.Kidd{at}infek.lu.se.
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Discussion
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Journal of Clinical Microbiology, September 2000, p. 3311-3316, Vol. 38, No. 9
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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