Determination of hepatitis B virus genotype G by polymerase chain reaction with hemi-nested primers

Journal of Virological Methods 98 (2001) 153– 159 www.elsevier.com/locate/jviromet

Determination of hepatitis B virus genotype G by polymerase chain reaction with hemi-nested primers Hideaki Kato a, Etsuro Orito a, Fuminaka Sugauchi a, Ryuzo Ueda a, Robert G. Gish b, Sadakazu Usuda c, Yuzo Miyakawa d, Masashi Mizokami e,* a

Second Department of Medicine, Nagoya City Uni6ersity Medical School, Nagoya 467 -8601, Japan Hepatology and Gastroenterology, California Pacific Medical Center, California, CA 94120, USA c Department of Medical Sciences, Toshiba General Hospital, Tokyo 140 -8522, Japan d Miyakawa Memorial Research Foundation, Tokyo 107 -0062, Japan e Department of Laboratory Medicine, Nagoya City Uni6ersity Medical School, Nagoya 467 -8601, Japan b

Received 1 May 2001; received in revised form 23 July 2001; accepted 24 July 2001

Abstract Hepatitis B virus (HBV) has been classified into six genotypes designated A – F by sequence divergence in the entire genome exceeding 8%. Very recently, the seventh genotype was reported and named genotype G. HBV genotype G is distinct from genomes of the other six genotypes in that it possesses an insertion of 36 nucleotides in the core gene, and has been found so far in France and the United States. A method for determining HBV genotype G was developed by polymerase chain reaction (PCR) with primers deduced from the 36-nucleotide (nt) insertion in five isolates of HBV genotype G the sequences of which have been deposited in DNA databases. The validity of this method, for specifically detecting HBV genotype G, was verified on a panel consisting of 142 HBV isolates of six major genotypes and four of genotype G. A total of 540 sera containing HBV in Japan covering symptom free carriers and patients with a spectrum of chronic liver disease were tested by this method, but not a single HBV genotype G sample was found. A possible method for serological determination of hepatitis B surface antigen of genotype G is suggested, without amplification or sequencing nucleotides, which would expand epidemiological and clinical researches on HBV genotype G. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Hepatitis B virus; Genotypes; Subtypes; Polymerase chain reaction

1. Introduction * Corresponding author. Tel.: + 81-52-853-8292; fax: + 8152-842-0021. E-mail address: [email protected] (M. Mizokami).

Hepatitis B virus (HBV) has infected substantial populations throughout the world and there are approximately 350 million carriers of the virus

0166-0934/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 0 9 3 4 ( 0 1 ) 0 0 3 7 4 - 3

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worldwide. HBV induces a spectrum of acute as well as chronic liver diseases ranging from chronic hepatitis through liver cirrhosis to eventual hepatocellular carcinoma (Lee, 1997). HBV has a circular and partially doublestranded DNA genome of approximately 3200 nucleotides (nt). Six genotypes have been proposed based on a nucleotide divergence within the entire genome of greater than 8% (Norder et al., 1994; Okamoto et al., 1988); they are named with capital alphabet letters from A to F. A seventh genotype was introduced very recently, and designated by the next letter, G (Stuyver et al., 2000). HBV genotypes have distinct geographical distributions (Lindh et al., 1997; Magnius and Norder, 1995). Genotypes A and D are common in Europe and the United States, while B and C occur frequently in Asia. Genotype E is restricted to West Africa, and genotype F is found mostly in the Central America. Genotype G has been identified in two of the 18 (11%) HBV samples from France and 11 of the 46 (24%) from Georgia, the United States (Stuyver et al., 2000); its geographical distribution is yet to be described. Nor is it known whether or not genotype G has any virological and clinical relevance in terms of association with replicative activity of HBV and severity of liver disease it induces. Clinical association have been proposed for some of the other genotypes (Kao et al., 2000; Mayerat et al., 1999; Orito et al., 2001). HBV genotype G is unique in that it possesses an insertion of 36 nt in the core gene (Stuyver et al., 2000), which is not shared by HBV isolates of the other six genotypes. Taking advantage of this insertion, a method was developed to amplify specifically HBV genotype G by the polymerase chain reaction (PCR) with primers deduced from its nucleotide sequence. The validity of this method was evaluated by testing 146 HBV samples of diverse genotypes, including the four of genotype G. In addition, the method was applied to 540 HBV samples from Japanese individuals who were infected with or without clinical symptoms.

2. Materials and methods

2.1. HBV samples Four sera (US15–US18) containing HBV genotype G were obtained from the United States. Full-length sequences of four HBV isolates of genotype G were retrieved from DDBJ/GenBank/ EMBL databases under accession numbers of AB056513 AB056514, AB056515 and AB056516, respectively. Genotypes of 146 HBV samples were deduced from the S-gene sequences (see below). They included 30 samples of HBV genotype A from the United States, 30 each of HBV genotype B and HBV genotype C from Japan, 30 of HBV genotype D from Mongolia and Uzbekistan, 19 of HBV genotype E from Cameroon, three of HBV genotype F and four of HBV genotype G from the United States. They formed a panel in evaluating the PCR method developed for the determination of genotype G. The epidemiology of HBV genotype G in Japan was surveyed on 540 HBV samples collected in and around Nagoya City. Genotypes other than HBV genotype G were determined by enzymelinked immunosorbent assay (ELISA; HBV Genotype EIA, Institute of Immunology, Co., Ltd., Tokyo, Japan); rare samples untypeable by ELISA were sequenced for determination of genotypes. They were obtained from 106 symptom-free carriers, 252 patients with chronic hepatitis, 84 patients with liver cirrhosis and 98 patients with hepatocellular carcinoma. The diagnosis was contingent on clinical symptoms and biochemical tests, as well as ultrasonography and liver biopsy when required. They had the mean9 S.D. age of 43.2914.3 years; 343 (63.5%) were males and 248 (45.9%) were positive for hepatitis B e antigen. They had a mean HBV DNA level of 5.089 1.87 log genomic equivalents (LGE) per ml by determination with a commercial assay kit (DNA Probe Chugai-HBV, Chugai Diagnostics, Tokyo). In this assay, HBV DNA in the test serum was amplified by a transcription-mediated PCR. Thereafter, amplified RNA was captured by microparticles bearing a single-stranded DNA probe labeled with acrydium ester for the determination by electrochemiluminescence.

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The study was approved by Ethics Committees of the institutions, and a written consent was obtained from each of carriers and patients. Sera were stored at −80 °C before tests.

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phase. Since labeled mAb against g does not bind with HBsAg of genotype E, while it binds with a subpopulation of HBsAg samples of genotype D, the cryptic epitope (g) is distinguished from overt g by placing it between parentheses.

2.2. Serum markers for HBV infection Hepatitis B surface antigen (HBsAg) was determined by ELISA using commercial kits (Dade Behring Inc., Delaware, USA). Subtypes of HBsAg were determined by commercial ELISA kits (HBsAg Subtype EIA, Institute of Immunology) involving monoclonal antibodies specific for the four subtypic determinants, i.e. d, y, w and r (Usuda et al., 1986).

2.3. Serological determination of the six HBV genotypes The six major genotypes of HBV (A– F) were determined by ELISA using commercial kits (HBV Genotype EIA, Institute of Immunology). The method was based on five monoclonal antibodies (mAb) directed to the corresponding epitopes on the product of preS2 region which are designated b, m, k, s and u. (Usuda et al., 1999). First, HBsAg particles in the test sera were captured by antibody to HBsAg immobilized on the solid phase. They were then subjected to binding with the five mAb labeled with horseradish peroxidase. The epitope b is shared by HBsAg of all genotypes, and its detection guarantees the preservation of preS-region product that is highly susceptible to proteinases. Because the expression of the other four epitopes depends on HBV genotypes, the six genotypes are distinguished by their combination; bsu for genotype A, bm for B, bks for C, bksu for D as well as E, and bk for F. Since samples of genotypes D and E have the same preS2 serotype, bksu, they were subjected to a sandwich immunoassay with two additional mAb directed to epitopes f and g, respectively (Usuda et al., 2000). Both f and g epitopes are borne by HBsAg samples of genotype E, while either or both are missed by those of genotype D. Furthermore, epitope g on HBsAg samples of genotype E is cryptic in that it is detected only by mAb against g that has been immobilized on the solid

2.4. Determination of genotypes by HBV DNA sequences Six genotypes of HBV (A–F) were determined by their characteristic nucleotide sequences. DNA was extracted from 100 ml of serum using SmiTest EX-R and D (Sumitomo Metal Industries, Tokyo, Japan). Nucleotide sequences of preS1 and preS2 regions and the S gene were amplified on the extracted DNA by PCR with primers HBHKF3 (sense, 5%-TGG GTC ACC ATA TTC TTG GGA-3% [nt 2813–2833]) and HBVHKR3 (antisense, 5%-GTT GCC GAG CAA CGG GGT AAA-3% [nt 1162-1142]) using AmpliTaq Gold (Applied Biosystems, California, USA). The sample was denatured at 96 °C for 9 min, and subjected to 40 cycles of PCR (95 °C for 1 min; 55 °C for 1 min; 72 °C for 1.5 min [additional 5 min in the last cycle]) in a 96-well cycler (GeneAmp 9600; Perkin–Elmer, Norwalk, USA). Direct sequencing was undertaken on the amplification products with use of the PRISM Ready Terminator Cycle Sequencing kit (Perkin–Elmer). Primers used for sequencing were, HBHKF3, PS1-1 (sense, 5%-CCT CCT GCC TCC ACC AAT CG-3% [nt 3124–3143]) (Surya et al., 1996), HBV58N (antisense, 5%-GAA CTG GAG CCA CCA GCA GG-3% [nt 75-56]), HBV409N (antisense, 5%-AGA TGA GGC ATA GCA GCA GGA TG-3% [nt 431-409]) (Hu et al., 2000), MF2 (sense, 5%-GTC TAG ACT CGT GGT GGA CTT CTC TC-3% [nt 246–271]). MR2 (antisense, 5%AAG CCA NAC AYT GGG GGA AAG C-3% [nt 730-709]) (Mizokami et al., 1999), HBHKF4 (sense, 5%-GTT TCT CCT GGC TCA GTT TA-3% [nt 660–679]) and HBHKR3.

2.5. Determination of HBV of genotype G by PCR with specific primers The five HBV isolates of genotype G retrieved from DNA databases (AF160501, AB056513

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AB056514, AB056515 and AB056516) were aligned within precore region and the C gene (Fig. 1). Most notably, all the five HBV genotype G genomes possessed a 36-nt insertion between the fifth and sixth nucleotides in the core gene, that is the hallmark of this genotype (Stuyver et al., 2000). The sequence of the 36-nt insertion was very well preserved, and identical among the five HBV genotype G genomes. An antisense primer specific for HBV genotype G (HBHKR2) was designed within the 36-nt insert. It had a sequence of 5%-AGC CAA AAA GGC CAT ATG GCA-3% and spanned positions 17–37 in the core gene of HBV genotype G (Fig. 1). Two sense primers were set upstream of it. They were HBHKF1 (5%-ACG GGG CGC ACC TCT CTT TAC-3% [nt 1519 – 1539]) and HBHKF2 (5%-GCA CTT CGT TTC ACC TCT GCA-3% [nt 1581–1601]). The three hemi-nested primers were used for amplifying HBV genotype G sequences by PCR using AmpliTaq Gold. For both the first and second rounds, the sample was denatured at 96 °C for 9 min and then subjected to 40 cycles of PCR (96 °C for 1 min; 60 °C for 1 min; 72 °C

for 1 min [additional 5 min in the last cycle]). An analysis of this PCR method by software ŽAmplify version 1.0 guaranteed no amplification by PCR with the three primers on representative HBV genomes of the major six genotypes from A to F with the full-length sequences known.

3. Results

3.1. Specificity and sensiti6ity of PCR for the determination of HBV genotype G The amplification by the PCR with hemi-nested primers was attempted on seven representative sera covering HBV of seven distinct genotypes (Fig. 2). The products of 357 base pairs (bp) were amplified on HBV genotype G as expected; no amplifications were observed for HBV samples of the other six genotypes. When this PCR method was applied to the 142 HBV samples of genotypes other than G (genotype A, 30; B, 30; C, 30; D, 30; E, 19; F, 3), no amplification occurred; genotypes

Fig. 1. Nucleotide sequences of precore region and the core gene in the five HBV isolates of genotype G. Translation start sites of precore region and core gene are indicated by arrows. A 36-nt insertion is shown in white letters in a black background. Two stop codons in the precore region are boxed. Dashes represent the same nucleotides as in AF106501 (Stuyver et al., 2000).

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determined by ELISA (Usuda et al., 1999, 2000), genotype B was found in 40 (7.4%) and genotype C in the remaining 500 (92.6%); the other four genotypes (A, D, E and F) were not detected in any of the sera.

3.3. Serological determination of genotype G Fig. 2. Amplification by PCR with hemi-nested primers specific for HBV genotype G. Products are shown for HBV samples representative of the seven genotypes. Markers with known sizes in base pairs were run in parallel in the right.

in these samples were determined by sequencing nucleotides in the S gene. Combined, these results indicate that the PCR method with hemi-nested primers including the one characteristic of the genotype G (HBHKR2) would be able to detect HBV genotype G with high specificity. The sensitivity of PCR for the determination of HBV genotype G was evaluated on serial 10-fold dilutions containing known numbers of HBV copies of this genotype (Fig. 3). The PCR could detect ten copies of HBV genotype G.

3.2. Tests for HBV genotype G in Japanese HBV samples The PCR for the detection of HBV genotype G was applied to sera from 540 Japanese individuals, including 106 who carried HBV without symptoms and 434 patients with liver diseases (chronic hepatitis, 252 cases; liver cirrhosis, 84 cases; hepatocellular carcinoma, 98 cases). Genotype G was not detected in any of the patients. When genotypes of HBV in these samples were

Fig. 3. Sensitivity of the PCR method for detecting HBV genotype G. Serial 10-fold dilutions of a serum containing known number of HBV copies of genotype G were tested by the PCR method. Markers with known sizes in base pairs were run in parallel in the right.

There is a possibility that HBV genotype G would be distinguished serologically by combination of HBsAg subtypes and preS2 serotypes determined by ELISA (Usuda et al., 1999, 2000). The four HBsAg samples of genotype G examined (US15– US18) had a preS2 serotype of bksu(g) that is characteristic of genotype D; (g) defines the cryptic expression of epitope g that is detectable by immobilized but not labeled mAb against epitope g (Usuda et al., 2000). Subtypes of the four HBsAg samples of genotype G were invariably adw, which was corroborated by corresponding nucleotide sequences deposited in DNA databases (AB056513 AB056514, AB056515 and AB056516). Based on the amino acid sequence of HBsAg in the reported isolate of genotype G (AF160501), its subtype was deduced to be adw, also, with lysine as amino acid 122 and lysine as amino acid 160 (Okamoto et al., 1987). In remarkable contrast, all the 30 HBsAg samples of genotype D examined were of subtype ayw. Hence, genotype G may be determined serologically by the combination of preS2 serotype by ELISA compatible with genotype D (Usuda et al., 1999, 2000) and subtype adw.

4. Discussion A remarkable characteristic of the original HBV strain of genotype G (AF160501) is an insertion of 36 nt in the core gene (Stuyver et al., 2000). The insertion was conserved in an additional four HBV genotype G isolates retrieved from DNA databases (AB056513 AB056514, AB056515 and AB056516). HBV variants with insertion or deletion of nucleotides have been reported, and rearrangements would incorporate excessive nucleotides into the HBV genome

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Table 1 PreS2 epitopes detected on HBsAg in the four sera containing HBV genotype G Seraa

US15 US16 US17 US18

PreS2 epitopes (optical density in ELISAb)

Serotype

b

m

k

s

u

f

g Overt

(g) Crypticb

\3 \3 \3 \3

0.009 0.035 0.009 0.013

\3 \3 \3 \3

\3 \3 \3 \3

\3 \3 \3 2.172

0.003 0.004 0.005 0.025

0.015 0.015 0.012 0.014

\3 \3 \3 \3

bksu(g) bksu(g) bksu(g) bksu(g)

a

Four sera (US15–US18) contained HBV isolates of genotype G. Their nucleotide sequences are deposited in DNA databases under respective accession numbers of AB056513 AB056514, AB056515 and AB056516. b Five preS2 epitopes (b, m, k, s and u) were determined by a commercial ELISA kit (HBV Genotype EIA, Institute of Immunology). Two preS2 epitopes ( f and g) were determined by the method of Usuda et al. (2000). Cryptic expression of epitope (g) is detected by mAb to g immobilized on the solid phase and antibody to the common determinant a of HBsAg labeled with enzyme. It is placed between parentheses for distinguishing it from overtly expressed g that is detectable by free mAb to g labeled with enzyme.

(Georgi-Geisberger et al., 1992; Gerken et al., 1991). They are not consistent mostly and observed only occasionally. There have been no insertions of this size, which were restricted to certain genotypes of HBV. Search of DNA databases could not identify any sequences with a similarity to the 36-nt insertion. Hence the 36-nt insertion would be specific for HBV genomes of genotype G, and enable the PCR method with primers based on it for the detection of this genotype. Taking advantage of the 36-nt insertion in the core gene, a method was developed for the specific detection of HBV genotype G. It involved amplification by PCR with a single antisense primer deduced from the sequence of 36-nt insertion (HBVHKR2 [nt spanning positions 17 – 37 in the core gene of HBV genotype G]) and two nested sense primers (HBHKF1 [nt 1519– 1539] and HBHKF2 [nt 1581– 1601]). The validity of this method was confirmed on 146 HBV samples that had been genotyped by sequencing; they cover seven genotypes (A– G). This method could detect ten copies of HBV genotype G (Fig. 3), with a sensitivity comparable to that for detecting the other six genotypes in our hands. When this method was applied to 540 HBV samples from Japan including symptom-free carriers and patients with various liver diseases, none were positive for HBV genotype G. They all

possessed a preS2 serotype of B or C (Usuda et al., 1999); mixed infection with HBV genotype G was not identified in any of the samples. From these results, HBV genotype G would appear to be very rare in Japan. Considering the small number of HBV samples obtained in and around Nagoya City, however, more samples from the other areas are needed to be tested before concluding the absence of HBV genotype G in Japan. Up to present, HBV genotype G is reported only from France and the United States (Stuyver et al., 2000). Taken together with the detection of HBV genotype G only in the four sera from the United States in the present study, it appears to have restricted geographical distribution. All the four HBV genotype G isolates examined had a preS2 serotype of bksu(g) by ELISA which is compatible with genotype D (Usuda et al., 1999, 2000). Unlike HBV genotype D that has invariably subtype ayw (Sugauchi et al., 2001), however, the four examples of HBV genotype G as well as the reported one (AF160501) possessed subtype adw. Hence, the combination of a preS2 serotype for genotype D (bksu(g)) and the HBsAg subtype of adw would be able to identify HBV genotype G serologically. Should such a serological combination be extended to additional HBV genotype G isolates, it will streamline the detection of genotype G for accelerated clinical studies and epidemiological surveys Table 1.

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Acknowledgements We thank Dr Ruzibakiev, Dr Oyunsyuren and Dr Hayami for providing us with valuable serum samples. This work is supported in part by a grant from the Japanese Ministry of Education, Science, Sports and Culture (Grant-in-Aid for Scientific Research 12670506 and 11691222), the Japanese Ministry of Health and Welfare, Health Science Research Grant (Non-A, Non-B Hepatitis Research Grant) and grants from the Japanese International Cooperation Agency and the Viral Hepatitis Research Foundation of Japan.

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