Innogenetics NV, Industriepark 7, Box 4, B-9052 Gent, Belgium1
INSERM U271, Lyon, France2
University of North Carolina, Chapel Hill, NC 27599, USA3
Emory University School of Medicine and Veterans Affairs Medical Center, Decatur, GA 30033, USA4
Author for correspondence: Caroline Van Geyt. Fax +32 9 2410 907. e-mail carolvge{at}innogenetics.be
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
By using subtype-specific antibodies against HBsAg, nine different serological subtypes were defined, reflecting the genetic variability of HBV. Of the defined determinants, one is common to all subtypes (a determinant), but also two pairs of mutually exclusive subdeterminants (d or y, and w or r) were commonly found. By using this tool in epidemiological studies, nine serological subtypes have been identified: ayw1, ayw2, ayw3, ayw4, ayr, adw2, adw4, adrq+ and adrq- (Swenson et al., 1991 ; Blitz et al., 1998
).
Genotypically, HBV genomes have been classified into six groups, designated AF, based on an intergroup divergence of 8% or more in the complete nucleotide sequence (Okamoto et al., 1988 ; Norder et al., 1992
; Magnius & Norder, 1995
). These six different genotypes show a characteristic geographical distribution: genotype A is pandemic, but most prevalent in north-west Europe, North America and Central Africa; genotype B is mostly found in Indonesia, China and Vietnam; genotype C is found in East Asia, Korea, China, Japan, Polynesia and Vietnam; genotype D is also more or less pandemic, but is predominant in the Mediterranean area and the Middle East extending into India; genotype E is typical for Africa; and genotype F is found in American natives and in Polynesia (Van Geyt et al., 1998
; Magnius & Norder, 1995
). Some studies have shown that, in certain populations where HBV is endemic, a higher variability of HBV might be expected (Bowyer et al., 1997
; Carman et al., 1997
). However, in areas where HBV is not recognized as endemic, less HBV genotypic data are available. In the US for example, there are an estimated 11·25 million chronically infected persons, but these HBV infections are most often associated with groups at high risk (intravenous drug users, those with a history of other sexually transmitted diseases, prisoners, others; Alter & Shapiro, 1998
). There is a paucity of data concerning the distribution of HBV genotypes in North American infected persons.
In this study, we report the HBV genotype prevalence in Atlanta (Georgia, USA) and Lyon (France) and describe a complete genome sequence of a new human HBV genotype, provisionally named genotype G. This genotype was found in patients chronically infected with HBV.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
HBV DNA extraction and amplification.
HBV DNA was extracted from 100 µl serum samples using the High Pure PCR Template Preparation kit (Boehringer Mannheim) essentially as previously described (Stuyver et al., 1999 ). The complete genome of HBV was amplified using the Expand High Fidelity PCR system (Boehringer Mannheim). The amplification was performed on 5 µl extracted DNA with the primers HBPr108 and HBPr109 (Table 1
). A 45 µl reaction mix was made, containing 5 µl 10x Expand High Fidelity PCR system buffer, 2·6 U Expand High Fidelity PCR system enzyme mix, 200 µM dNTPs, 300 nM of each primer and sterile H2O. Amplification was performed with denaturation at 94 °C for 40 s, annealing (after shifting to 60 °C in 50 s) for 1 min and elongation (after shifting to 72 °C in 15 s) for 4 min, with an increment of 5 s/cycle (Günther et al., 1998
).
|
Sequencing.
Sequencing was performed on an automated DNA sequencer ABI 377 (PE Applied Biosystems), using fluorescence-labelled dideoxynucleotide chain terminators (ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit with AmpliTaq DNA polymerase FS; PE Applied Biosystems). The primers used for sequencing the complete genome are summarized in Table 1. The primers used to sequence the shorter PCR fragments are the same as the amplification primers.
Data analysis.
Phylogenetic trees were created and analysed by distance matrix comparison using DNADIST, Neighbor and Drawgram software programs of PHYLIP version 3.5c (Felsenstein, 1993 ). Statistical analysis (t-test for the mean) was performed using the MedCalc program (Mariakerke). A P value of 0·05 was considered to be statistically significant. Complete genome sequences representing the different genotypes were retrieved from GenBank; their accession numbers are indicated in Fig. 2
. In addition, the sequences from Tran et al. (1991)
describing the core (HBcAg) and surface antigen (HBsAg) of an atypical variant of HBV were also included (M74499 and M74501).
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
In order to compare the genetic relatedness of the FR1 strain with 36 other complete HBV genomes, homology percentages, as well as phylogenetic distances, were calculated. The FR1 sequence showed an average homology of: 87·1% (min. 86·2%, max. 87·7%) with genotype A (five sequences); 86·6% (min. 86·5%, max. 86·6%) with genotype B (four sequences); 86·5% (min. 86·0%, max. 87·0%) with genotype C (14 sequences); 86·9% (min. 86·3%, max. 87·3%) with genotype D (eight sequences); 88·3% (min. 88·2%, max. 88·4%) with genotype E (two sequences); and 84·7% (min. 84·5%, max. 84·8%) with genotype F (three sequences). Phylogenetic distances between the six recognized HBV genotypes (36 sequences) were compared to each other and to the FR1 strain (Fig. 1). A clear difference emerged between the phylogenetic distances (i) within one genotype (distance range of 0·010·06) and (ii) between different genotypes (distance range 0·080·17). Using a t-test for the mean and a distance of 0·08 as a border value between genotypes, FR1 was found to be significantly different from genotypes AF (range 0·110·17; P<0·019). Phylogenetic trees of the complete genome sequences (Fig. 2A
), as well as of the individual ORF [Fig. 2B
for the surface region (preS1, preS2, HBsAg); data not shown for the other ORFs] were constructed, illustrating that FR1 is indeed located on a separate branch. Fig. 2(B)
further illustrates that the HBV samples from the US and France, including the B1 isolate, are closely related to each other. Based on these calculations and illustrated by means of phylogenetic trees, FR1 and the ten other virus strains shown belong to a new HBV genotype (called genotype G).
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In addition to the prominent prevalence of genotype G virus in the US samples, this genotype was also found in samples originating from France. The prevalence in other geographical areas needs further exploration. The complete HBV genome sequence that is presented in this study was determined from a sample taken from a chronic but asymptomatic carrier living in the Lyon area, France. The homology of this FR1 strain with the previously published B1 sequence (another French isolate), which covers the complete S gene as well as the preCore and Core genes, is surprisingly high (Tran et al., 1991 ; Fig. 2B
). This atypical HBV strain was already detected and partially sequenced in 1991, but never recognized as a new genotype. In order to study the genotype G sequence with respect to recombination events (as described by Tran et al., 1991
), the complete FR1 genome was inspected for such events at the nucleotide level and phylogenetically for co-segregation with other known genotypes; evidence for recombination was not found. Despite the absence of recombination in FR1, recombination events in HBV were presented as a more common event than previously thought (Tran et al., 1991
; Bollyky et al., 1996
).
The expression of HBeAg in HBV genotype G needs additional attention. In all isolates studied here (geographically unrelated samples were included), a virus variant with two preCore translational stop codons (TAA at codon 2, TAG at codon 28) was found. This observation suggests that the dual variant is a naturally occurring configuration for genotype G. Due to the presence of an insert in the Core, the stability of the encapsidation signal (Lok et al., 1994 ) might be altered. There might be a need for a compensatory change, possibly resulting in selection for this dual variant. As a consequence, however, this finding makes all these viruses incapable of expressing the HBeAg. Paradoxically, HBeAg was found in at least one French patient (FR2). Similar observations were also made for the B1 isolate, although codon 2 was not mutated (Tran et al., 1991
). The mutation at codon 2 was previously described (Lindh et al., 1996
), but not in the context of a genotype G virus. If these two stop codons are naturally existing in this viral genotype, alternative strategies for HBeAg expression should exist. The insert might than play an important role in helping the newly translated proteins either towards a secretion pathway (for HBeAg) or capsid formation. More research is needed to investigate and explain HBeAg expression, the stability of the encapsidation signal, and the serological and structural importance of this 12 aa insert in both the HBcAg and HBeAg proteins.
In this study, evidence was provided for the existence of a seventh HBV genotype, called genotype G. The virus structure is essentially identical to that of the other genotypes, but has some unique features (like an insert of 36 nt in the core region). The prevalence of this strain was found to be more than 11% in the Georgia area, USA, but needs to be further determined for other geographical regions. These findings may have an impact on the immunological and genetic diagnosis of HBV, as well as on the treatment of the ubiquitous disease it causes.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
b Present address: Pharmasset Inc., 1860 Montreal Road, Tucker, GA 30084, USA.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alvarado-Esquivel, C., Wyseur, A., Herrera-Ortiz, F., Ruiz-Maya, L., Ruiz-Astorga, R., Zarate-Aguilar, A., Carrillo-Maravilla, E., Herrera-Luna, R., Moralers-Macedo, M., Maertens, G. & Stuyver, L. (1998). Hepatitis B and C virus infections in Mexico: genotypes and geographical distribution in blood donors and patients with liver disease. In Therapies for Viral Hepatitis, pp. 35-41. Edited by R. F. Schinazi, J.-P. Sommadossi & H. C. Thomas. London, UK: International Medical Press.
Attia, M. A. (1998). Prevalence of hepatitis B and C in Egypt and Africa. In Therapies for Viral Hepatitis, pp. 15-24. Edited by R. F. Schinazi, J.-P. Sommadossi & H. C. Thomas. London, UK: International Medical Press.
Bartholomeusz, A., Schinazi, R. F. & Locarnini, S. A. (1998). Significance of mutations in the hepatitis B virus polymerase selected by nucleoside analogues and implications for controlling chronic disease. Viral Hepatitis Reviews 4, 167-187.
Blitz, L., Pujol, F. H., Swenson, P. D., Porto, L., Atencio, R., Araujo, M., Costa, L., Monsalve, D. C., Torres, J. R., Fields, H. A., Lambert, S., Van Geyt, C., Norder, H., Magnius, L. O., Echevarria, J. M. & Stuyver, L. (1998). Antigenic diversity of hepatitis B virus strains of genotype F in Amerindians and other population groups from Venezuela. Journal of Clinical Microbiology 36, 648-651.
Bollyky, P. L., Rambaut, A., Harvey, P. H. & Holmes, E. C. (1996). Recombination between sequences of hepatitis B virus from different genotypes. Journal of Molecular Evolution 42, 97-102.[Medline]
Bowyer, S. M., van Staden, L., Kew, M. C. & Sim, J. G. M. (1997). A unique segment of the hepatitis B virus group A genotype identified in isolates from South Africa. Journal of General Virology 78, 1719-1729.[Abstract]
Carman, W. F., Van Deursen, F. J., Mimms, L. T., Hardie, D., Coppola, R., Decker, R. & Sanders, R. (1997). The prevalence of surface antigen variants of hepatitis B virus in Papua New Guinea, South Africa, and Sardinia. Hepatology 25, 1658-1666.
Carrilho, F. J. & Corrêa, M. C. J. M. (1998). Magnitude of hepatitis B and C in Latin America. In Therapies for Viral Hepatitis, pp. 25-34. Edited by R. F. Schinazi, J.-P. Sommadossi & H. C. Thomas. London, UK: International Medical Press.
Felsenstein, J. (1993). PHYLIP (Phylogeny Inference Package) version 3.5c. Distributed by the author. Department of Genetics, University of Washington, Seattle, USA.
Günther, S., Sommer, G., Von Breunig, F., Iwanska, A., Kalinina, T., Sterneck, M. & Will, H. (1998). Amplification of full-length hepatitis B virus genomes from samples from patients with low levels of viraemia: frequency and functional consequences of PCR-introduced mutations. Journal of Clinical Microbiology 36, 531-538.
Lindh, M., Horal, P., Dhillon, A. P., Furuta, Y. & Norkrans, G. (1996). Hepatitis B virus carriers without precore mutations in hepatitis B e antigen-negative stage show more severe liver damage. Hepatology 24, 494-501.[Medline]
Lok, A. S. F., Akarca, U. & Greene, S. (1994). Mutations in the pre-core region of hepatitis B virus serve to enhance the stability of the secondary structure of the pre-genome encapsidation signal. Proceedings of the National Academy of Sciences, USA 91, 4077-4081.[Abstract]
Magnius, L. O. & Norder, H. (1995). Subtypes, genotypes and molecular epidemiology of the hepatitis B virus as reflected by sequence variability of the S-gene. Intervirology 38, 24-34.[Medline]
Norder, H., Couroucé, A.-M. & Magnius, L. O. (1992). Molecular basis of hepatitis B virus serotype variations within the four major subtypes.Journal of General Virology 73, 3141-3145.[Abstract]
Norder, H., Couroucé, A.-M. & Magnius, L. O. (1994). Complete genomes, phylogenetic relatedness, and structural proteins of six strains of the Hepatitis B virus, four of which represent two new genotypes. Virology 198, 489-503.[Medline]
Okamoto, H., Tsuda, F., Sakugawa, H., Sastrosoewinjo, R. I., Imai, M., Miyakawa, Y. & Mayumi, M. (1988). Typing hepatitis B virus by homology in nucleotide sequence: comparison of surface antigen subtypes.Journal of General Virology 69, 2575-2583.[Abstract]
Stuyver, L., De Gendt, S., Cadranel, J. F., Van Geyt, C., Van Reybrouck, G., Dorent, R., Gandjbachkc, I., Rosenheim, M., Opolon, P., Huraux, J. M. & Lunel, F. (1999). Three cases of severe subfulminant hepatitis in heart transplanted patients after nosocomial transmission of a mutant hepatitis B virus. Hepatology 29, 1876-1883.[Medline]
Swenson, P. D., Riess, J. T. & Krueger, L. E. (1991). Determination of HBsAg subtypes in different high risk populations using monoclonal antibodies. Journal of Virological Methods 33, 27-28.[Medline]
Telenta, P. F. S., Poggio, G. P., Lopez, J. L., Gonzalez, J., Lemberg, A. & Campos, R. H. (1997). Increased prevalence of genotype F hepatitis B virus isolates in Buenos Aires, Argentina. Journal of Clinical Microbiology 35, 1873-1875.[Abstract]
Tran, A., Kremsdorf, D., Capel, F., Housset, C., Dauguet, C., Petit, M.-A. & Brechot, C. (1991). Emergence of and takeover by hepatitis B virus (HBV) with rearrangements in the pre-S/S and pre-C/C during chronic HBV infection. Journal of Virology 65, 3566-3574.[Medline]
Van Geyt, C., De Gendt, S., Rombout, A., Wyseur, A., Maertens, G., Rossau, R. & Stuyver, L. (1998). A line probe assay for hepatitis B virus genotypes. In Therapies for Viral Hepatitis, pp. 139-145. Edited by R. F. Schinazi, J.-P. Sommadossi & H. C. Thomas. London, UK: International Medical Press.
Xu, Z.-Y., Zhao, S.-J., Mahoney, R. T., Deng, X.-Q., Lin, X. & Ding, D. (1998). Epidemiology and control of hepatitis B and C in eastern Asia. In Therapies for Viral Hepatitis, pp. 9-14. Edited by R. F. Schinazi, J.-P. Sommadossi & H. C. Thomas. London, UK: International Medical Press.
Received 28 June 1999;
accepted 23 August 1999.