Download (HBV) in a Case of HBV Infection Acquir

1195
Molecular Evolutionary Analysis of the Complete Nucleotide Sequence
of Hepatitis B Virus (HBV) in a Case of HBV Infection Acquired
through a Needlestick Accident
Fuminaka Sugauchi, Masashi Mizokami, Etsuro Orito,
Tomoyoshi Ohno, Katsuo Hayashi, Takanobu Kato,
Yasuhito Tanaka, Hideaki Kato, and Ryuzo Ueda
Second Department of Internal Medicine/Blood Transfusion, Nagoya
City University Medical School, Nagoya, Japan
To elucidate needlestick transmission of hepatitis B virus (HBV), strains isolated from 1
physician who acquired HBV infection through a needlestick accident and 3 patients with
chronic hepatitis B (donor patients A, B, and C) were tested using molecular evolutionary
analysis based on full-length HBV genomic sequences. Nucleotide sequences of these isolates
were aligned with 55 previously reported full-length genomic sequences. Genetic distances
were estimated using the 6-parameter method, and phylogenetic trees were constructed using
the neighbor-joining method. Strains isolated from patient A and the recipient pair were
clustered within a closer range of evolutionary distances than were strains recovered from the
recipient pair and patients B and C. Furthermore, strains from patient A and the recipient
were also clustered on the S gene sequences of HBV. These results demonstrated that patient
A alone was the source of direct transmission to the recipient. This approach can be used to
investigate the transmission route of HBV.
Infection with hepatitis B virus (HBV) leads to a wide spectrum of liver conditions, including acute self-limited infection,
fulminant hepatitis, and chronic hepatitis with progression to
cirrhosis and hepatocellular carcinoma. Transmission of HBV
from patients to health care workers is a serious problem for
medical institutions. Accidental exposure to HBV by needlestick is a well-known route of transmission in medical staff.
According to previously reported studies, the average risk of
HBV infection after a needlestick accident involving HBVinfected blood ranges from 7% to 30% [1], which is higher than
risk estimates for infection with hepatitis C virus (HCV)
(2%–4%) [2, 3] and HIV infection (0.3%) [4].
Molecular techniques have been used to determine the transmission route of HBV infection. Intrafamilial transmission of
HBV was demonstrated by using sequence analysis of mutant
HBV DNA [5]. Nosocomial spread of HBV in a hemodialysis
unit [6] and in a health care setting [7] and HBV contamination
of a cryopreservation tank [8] were confirmed by comparison
of results of DNA sequencing and serotyping. Molecular bioReceived 22 February 2000; electronically published 7 November 2000.
Financial support: This study was supported by the Ministry of Education,
Science and Culture of Japan (11691222). All nucleotide sequence data reported in this article have been submitted to DNA Data Bank of Japan (DDBJ,
Mishima, Japan), European Molecular Biology Laboratory (EMBL, Hinxton,
UK), and GenBank: National Center for Biotechnology (Bethesda, MD)under
accession numbers AB042282, AB042283, AB042284, and AB042285.
Reprints or correspondence: Dr. Masashi Mizokami, Second Dept. of
Medicine/Blood Transfusion, Nagoya City University Medical School, Kawasumi, Mizuho, Nagoya 467-8601, Japan ([email protected]
.jp).
Clinical Infectious Diseases 2000; 31:1195–201
q 2000 by the Infectious Diseases Society of America. All rights reserved.
1058-4838/2000/3105-0015$03.00
logical techniques including antigenic subtyping (adw, adr, ayw,
and ayr) and oligonucleotide pattern analysis may provide supporting evidence for the transmission source, but it is often
difficult to discriminate between various HBV strains in a country such as Japan where similar subtypes of HBV are predominant [9]. In the present study, we investigated a case of HBV
infection acquired through a needlestick accident by performing
molecular evolutionary analysis using a combination of methods, including determination of genetic distance, phylogenetic
tree analysis, and bootstrap analysis based on full-length nucleotide sequences of HBV.
Patients and Methods
Study group. Three patients suspected of direct HBV transmission were enrolled in the present study. The first patient (donor
patient A) was a 36-year-old man with chronic HBV infection who
was admitted to our hospital because of acute exacerbation of
chronic hepatitis B (table 1). The second patient (donor patient B)
was a 32-year-old man with chronic HBV infection (table 1). He
was admitted to our hospital at the same time as donor patient A
because of acute exacerbation of chronic hepatitis B. None of the
other inpatients posed a risk of HBV transmission at that time.
The third patient (donor patient C) was a 57-year-old woman with
chronic HBV infection who was the recipient’s mother (table 1).
She was receiving treatment at another outpatient department. She
was in frequent contact with the recipient at his home.
The recipient of HBV infection was a 25-year-old male physician
who had normal results of liver biochemistry tests and was seronegative for all markers of HBV and HCV at the time of the
accidents (figure 1). He sustained an incidental needlestick injury
with a 21-gauge needle on his finger after obtaining a blood sample
from donor patient A. About 1 week after the first accident, he
1196
Sugauchi et al.
CID 2000;31 (November)
Table 1. Serum markers for hepatitis B virus (HBV) for 3 patients suspected of direct transmission of
HBV to a physician.
Donor
patient
A
B
C
a
Age, y/sex
HBsAg
Anti-HBs
HBeAg
Anti-HBe
Anti-HBc
ALT
level U/L
HBV DNA ,
LGE/mL
36, M
32, M
57, F
1
1
1
2
2
2
1
1
2
2
2
1
1 (high titer)
1 (high titer)
1 (high titer)
418
387
25
8.5
8.7
4.6
b
NOTE. ALT, alanine aminotransferase; anti-HBc, antibody to hepatitis B core antigen, anti-HBe, antibody to
hepatitis e antigen; anti-HBs, antibody to hepatitis B surface antigen; HBeAg, hepatitis B e antigen; HBsAg, hepatitis
B surface antigen; 1, positive; 2, negative; LGE/mL, logarithm of the genome equivalent per milliliter.
a
Quantity of HBV DNA was determined by using transcription-mediated amplification and hybridization protection
assay (Fuji Reibo, Tokyo).
b
Inhibition ratio of anti-HBc is 180% by 200 times dilution.
sustained another incidental needlestick injury with a 21-gauge needle on his finger after administering an iv drip infusion to donor
patient B. He bled after the puncture and washed his hand immediately. He did not receive vaccination or iv hyperimmune Ig to
HBV. He developed acute self-limited hepatitis B about 3 months
later (figure 1). The physician had no risk factors for hepatitis B,
including history of blood transfusion, injection drug abuse, and
hemodialysis.
To determine who among the 3 suspected donor patients was
the source of direct HBV transmission to the physician, we obtained
blood specimens from the 3 donor patients and the recipient during
the acute phase of HBV infection (figure 1).
Serological markers for hepatitis B. All serum samples were
tested for the following: hepatitis B surface antigen by reverse passive hemagglutination assay (Fuji Rebio, Tokyo); antibody to hepatitis B surface antigen by passive hemagglutination assay (Fuji
Rebio, Tokyo); hepatitis B e antigen, antibody to hepatitis B e
antigen, antibody to hepatitis B core antigen, IgM antibody to
hepatitis B core antigen, antibody to hepatitis A antigen, and IgM
antibody to hepatitis A antigen by RIA (Dainabbott, Tokyo); and
antibody to HCV antigen by second-generation EIA (Abbott Laboratories, North Chicago, IL). Alanine aminotransferase levels
were also measured in each sample.
Amplification of HBV DNA by PCR analysis. All serum samples were stored at 2807C until assayed. Serum DNA was extracted
from 100 mL of serum by using a DNA extractor kit (SUMITOMO,
Figure 1. Clinical course and serum markers for hepatitis B virus (HBV) infection in a 25-year-old male physician who acquired HBV infection
through a needlestick accident. ALT, alanine aminotransferase; anti-HA, antibody to hepatitis A antigen; anti-HBc, antibody to hepatitis B core
antigen; anti-HBe, antibody to hepatitis B e antigen; anti-HBs, antibody to hepatitis B surface antigen; anti-HCV, antibody to hepatitis C virus;
HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; PHA, passive hemagglutination assay; RPHA, reverse passive hemagglutination
assay; 1, positive; 2, negative.
CID 2000;31 (November)
Molecular Evolutionary Analysis of HBV
1197
Table 2. HBV (hepatitis B virus) DNA oligonucleotide primers used for PCR analysis and
sequencing in a study of direct transmission of HBV from suspected patients to a physician.
Primer type, name
For detection of HBV DNA
a
HBS-1
HBS-2
HBS-3
HBS-4
For sequencing
EN-C
EN-B
EN-D
EN-E
EN-A
P-A
P-B
P-C
P-D
Co-A
Co-B
Co-C
Co-D
P-E
P-F
EN-F
EN-G
Nucleotide sequence
50-TTCCTCTTCATCCTGCTGCT-30
50-CAAGGTATGTTGCCCGTTTG-30
50-ACTGAACAAATGGCACTAGT-30
50-CTGAGGCCCACTCCCATAGG-30
50-CTCTGCWAGATCCCAGAGT-30
50-GAACTGGAGCCACCAGCAGG-30
50-CATAGAGGTTCCTTGAGCAG-30
50-TGACATACTTTCCAATCAAT-30
50-GGTCACCATATTCTTGGGAA-30
50-GCCAAGTCTGTACAACATCTTGAG-30
50-AGTTGGCGAGAAAGTGAAAGCCTG-30
50-ATGCCTTTRTATGCATGTAT-30
50-CGGGACGTAGACAAAGGACGT-30
50-TTGTYTACGTCCCGTCGGCG-30
50-AACAGACCAATTTATGCCTA-30
50-GAGACCACCGTGAACGCCCA-30
50-CCTGAGTGCTGTATGGTGAGG-30
50-TATCGGGAGGCCTTAGATCTCCG-30
50-GGATAGAACCTAGCAGGCAT-30
50-CGCAGAAGATCTCAATCTCGG-30
50-GGGTTGAAGTCCCAATCTGGATT-30
Nucleotide position
Polarity
401–420
455–474
698–679
656–637
Sense
Sense
Antisense
Antisense
21–39
75–56
553–534
992–973
2821–2840
760–783
1107–1084
1054–1073
1434–1414
1421–1440
1803–1784
1611–1630
2072–2048
2012–2035
2654–2635
2417–2437
2987–2965
Sense
Antisense
Antisense
Antisense
Sense
Sense
Antisense
Sense
Antisense
Sense
Antisense
Sense
Antisense
Sense
Antisense
Sense
Antisense
NOTE. Definition of nucleotides: R, A or G; W, A or T; Y, C or T.
a
This primer was also used for sequencing.
Tokyo). HBV DNA was detected by nested PCR analysis with use
of the primers listed in table 2. PCR analysis was initiated by means
of the hot-start technique. The amplification reaction was done in
a 96-well cycler (GeneAMP9600, Perkin-Elmer Cetus, Norwalk,
CT). The first round of PCR analysis was performed with an outer
primer set for 35 cycles (947C for 1.5 min, 557C for 1 min, and
727C for 1 min), which was followed by an extension reaction at
727C for 7 min. The second round was performed with an inner
primer set for 30 cycles and was also followed by an extension
reaction. The PCR products were analyzed by electrophoresis on
2.0% agarose gels stained with ethidium bromide; an ultraviolet
transilluminator was used for visualization. The standard precautions for avoiding contamination during PCR analysis were observed. A control serum sample negative for HBV was also included
in each run to ensure specificity. The quantity of HBV DNA was
determined by using transcription-mediated amplification and hybridization protection assay (Fuji Rebio, Tokyo).
Sequencing of full-length genomic sequences. PCR analysis with
use of the primers listed in table 2 was also performed to amplify
the full-length genomic sequence of HBV. PCR-amplified HBV
DNA was sequenced directly according to the dideoxy method by
means of a Taq Dye Deoxy Terminator cycle sequencing kit (Perkin-Elmer Applied Biosystems, Foster City, CA) with a fluorescent
373A DNA sequencer (Applied Biosystems, Foster City, CA).
Molecular analysis of HBV DNA. The nucleotide sequences
of these isolates were then aligned with 55 previously reported fulllength genomic sequences and 91 S gene sequences of HBV that
were obtained from international DNA databases (DNA Data
Bank of Japan [DDBJ]; European Molecular Biology Laboratory
[EMBL], Hinxton, UK; GenBank, Bethesda, MD). Genetic distances were estimated by using the 6-parameter method [10], and
phylogenetic trees were constructed by means of the neighbor-join-
ing method [11]. To further confirm the reliability of phylogenetic
tree analysis, bootstrap resampling and reconstruction were carried
out [12]. These analyses were performed by using the ODEN program of the National Institute of Genetics (Mishima, Japan). HBV
genotypes were classified as A to F according to previous reports
[13, 14].
Results
All 4 strains that had a nucleotide length of 3215 bases and
belonged to genotype C were analyzed. A precore-stop mutation at codon 28 (TGG to TAG) was found only in the strain
recovered from donor patient B. Within the core gene, mutation
E to G at codon 182 was found in the strains isolated from
donor patient A and the recipient, and mutation S to P at codon
183 was found in the strain recovered from donor patient C.
The sequence homology between strains recovered from the
recipient and donor patient A was 99.8%, the recipient and
donor patient B was 97.7%, and the recipient and donor patient
C was 95.9%. A phylogenetic tree was constructed based on 55
full-length genomic sequences of HBV that were obtained from
DDBJ, EMBL, and GenBank, including 45 genotype C sequences (figure 2). The strains isolated from donor patient A
and the recipient pair were clustered together within a closer
range of evolutionary distances than were the strains recovered
from the recipient pair and the other donor patients.
Bootstrap analysis was also performed to estimate the reliability of the phylogenetic tree. The aligned sequences were
resampled 1000 times, and the phylogenetic trees were recon-
Figure 2. Phylogenetic trees of genomic sequences of hepatitis B virus (HBV) in a study of direct transmission of HBV from 3 suspected
patients (donors A–C) to a physician (recipient); the trees were constructed by the neighbor-joining method [11]. Bootstrap analysis for evaluation
of the statistical reliability of the trees revealed that values for all clusters for genotypes A–F were 100%. The accession numbers of sequences
obtained from the DNA Data Bank of Japan (DDBJ), European Molecular Biology Laboratory (EMBL), and GenBank are indicated on the
trees. Phylogenetic tree of full-length genomic sequences of HBV strains from the study cases and 55 full-length genomic sequences of HBV that
were obtained from DDBJ, EMBL, and GenBank.
1198
Figure 3. Phylogenetic trees of genomic sequences of hepatitis B virus (HBV) in a study of direct transmission of HBV from 3 suspected
patients (donors A–C) to a physician (recipient); the trees were constructed by the neighbor-joining method [11]. Bootstrap analysis for evaluation
of the statistical reliability of the trees revealed that values for all clusters for genotypes A–F were 100%. The accession numbers of sequences
obtained from the DNA Data Bank of Japan (DDBJ), European Molecular Biology Laboratory (EMBL), and GenBank are indicated on the
trees. Phylogenetic tree of S gene sequences of HBV strains from the study cases and 91 S gene sequences of HBV that were obtained from DDBJ,
EMBL, and GenBank.
1199
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Sugauchi et al.
structed for all these alignments. These results suggested that
there was a close evolutionary relationship between the strain
isolated from donor patient A and the strain recovered from
the recipient. However, there was also a close evolutionary relationship between the strain isolated from donor patient C and
the strain recovered from the recipient pair, because there was
only a 1-strain difference between them. To confirm close relationships, we included 91 genotype C nucleotide sequences
of the S gene of HBV in our analysis that were obtained from
DDBJ, EMBL, and GenBank (including 81 genotype C sequences) and aligned them to maximize homology, and another
phylogenetic tree was constructed based on the S gene sequences of HBV (figure 3). The strains recovered from donor
patient A and the recipient were also closely clustered. On the
other hand, with regard to the strain isolated from donor patient C and the strain isolated from the recipient pair, there was
a 6-strain difference between them. These results demonstrated
that the source of direct transmission of HBV to the recipient
was donor patient A, not the other donor patients.
Discussion
Molecular evolutionary techniques including determination
of genetic distance and phylogenetic tree analysis have been
used recently to determine the transmission route of viral infection. Molecular evolutionary analysis of the route of infection was first used in a study of the transmission pattern of
HIV in one dental practice [15]. After that study, HCV transmission through needlestick accidents [16] and vertical transmission [17] was investigated by means of this method. Transmission of HBV in health care settings also was analyzed by
using this method [7, 18]. The occurrence of genetic variation
is necessary for studying the molecular epidemiology of infectious diseases. Although HBV is a DNA virus, the reverse transcriptase activity of this virus may be responsible for its high
rate of mutation. In fact, the average annual evolutionary rate
of HBV strains is 1025–1026 mutations per site [19]. For a virus
with substantial genomic variation, the identification of strains
with a high degree of genetic relatedness may be considered
demonstration of a molecular epidemiological linkage between
different patients infected with these strains [20]. To our knowledge, this is the first study demonstrating HBV transmission
through a needlestick accident by molecular evolutionary analysis (by use of a combination of methods, including estimation
of nucleotide substitutions, phylogenetic tree analysis, analysis
of nucleotide sequence diversity, and bootstrap resampling)
with full-length genomic sequences of HBV.
The complete nucleotide sequence of the HBV genome contains 4 open reading frames designated S, C, P, and X. The
rate of synonymous substitutions in the S gene is less than in
other open reading frames [19]. Because the P region partially
overlaps with the S gene, it encodes a DNA polymerase, including a functional domain of reverse transcriptase. In a previously reported study [14], we demonstrated that results of
CID 2000;31 (November)
HBV genotyping with use of the S gene sequence were consistent with those of genetic analysis with use of the full-length
genomic sequence. Our results of phylogenetic analysis with use
of the S gene sequence of HBV for investigating the relationship
between strains isolated from donor patients and the recipient
were also consistent with those of genetic analysis with use of
the full-length genomic sequence. These findings indicate that
the S gene contains nucleotide sequences with sufficient variability to distinguish between strains and therefore provides a
feature essential for establishing direct transmission of HBV.
Furthermore, the HBV sequence database contains a relative
abundance of sequences for comparative purposes.
These results demonstrate that the nucleotide sequence of S
gene is a useful tool for analyzing routes of HBV transmission
by molecular evolutionary analysis. It is often difficult to discriminate between various HBV strains on the basis of shortlength nucleotide sequences. The S gene sequence (with its sufficient length) and a large number of HBV strains for comparison
with other strains are recommended for use in molecular evolutionary analysis, because the S gene is a comparatively conserved region as described previously [19].
A core and precore variant of HBV has been found to cause
fulminant hepatitis in various instances such as HBV outbreaks
[21]. A precore-stop mutation at codon 28 (TGG to TAG) was
found only in the strain isolated from donor patient B. Within
the core gene, mutation E to G at codon 182 was found in the
strains recovered from donor patient A and the recipient, and
mutation S to P at codon 183 was found in the strain isolated
from donor patient C (data not shown). These mutations are
noteworthy because their association with fulminant hepatitis
has been suggested [22]. In the present study, the clinical course
of acute hepatitis in the recipient was severe. A relationship
between the severity of his clinical course and the core gene
variant was suspected.
In conclusion, we report a case of HBV infection acquired
through a needlestick accident in which direct transmission was
determined by molecular evolutionary analysis (via a combination of methods, including determination of genetic distance,
phylogenetic tree analysis, and bootstrap analysis based on the
full-length nucleotide sequences of HBV). This approach is considered useful for analyzing routes of HBV transmission.
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