| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Animal: DNA Viruses |
Department of Clinical Virology, Göteborg University, Guldhedsgatan 10B, 413 46 Göteborg, Sweden1
Department of Virology, Swedish Institute for Infectious Disease Control, Stockholm, Sweden2
Author for correspondence: Magnus Lindh. Fax +46 31 827032. e-mail magnus.lindh{at}microbio.gu.se
 |
Abstract |
|---|
 TOP Abstract Introduction Methods Results Discussion References |
|---|
|
Introduction |
|---|
|
TOPAbstractIntroduction Methods Results Discussion References |
|---|
). Phylogenetic tree analyses later confirmed these genotypes, and two further genotypes, E and F, were described (Norder et al., 1992 , 1994 ). These six genotypes have a relatively distinct geographical distribution (Lindh et al., 1997 ; Norder et al., 1993 ). Recently, a putative seventh genotype with a unique core insertion was observed in HBV carriers from southern France and Georgia, USA (Stuyver et al., 2000 ).
In East Asia, where the majority of the world’s HBV carriers live, genotypes B and C prevail. However, considering that few strains from this part of the world have been studied, in particular in relation to the large number of carriers, as yet undiscovered genotypes might exist. In previous studies of HBV genotypes we observed strains with aberrant features in Vietnamese carriers. One group of strains appeared from pre-S and S region restriction fragment length polymorphism (RFLP) to be of genotype A (Lindh et al., 1997 ), and sequencing of the S region supported this classification. This finding was surprising because, with the exception of the Philippines, genotype A is rarely observed in East Asia. Moreover, these strains had thymine at nt position 1858, while essentially all genotype A strains described so far have cytosine at position 1858. This co-variation has been recognized because in genotype A strains C1858 effectively prevents the emergence of mutations at nt 1896 in the precore region, explaining the low prevalence of these mutations in geographical areas like north-western Europe, where genotype A prevails (Li et al., 1993 ).
The second group of strains was investigated because it could not be genotyped by pre-S or S region RFLP analysis. Sequencing of the pre-S region indicated that these strains belonged to genotype C, but they also showed similarities with genotype A (Lindh et al., 1998 ).
By analysing five complete genomes representing the two groups of aberrant strains we investigated whether any of them might represent a new genotype. Considering that our previous analyses had showed similarities with genotype A we thought that the study might also provide information about the origin and phylogenetic relatedness of genotype A.
|
Methods |
|---|
|
TOPAbstractIntroduction Methods Results Discussion References |
|---|
Patients and samples.
Sequencing.
PCR amplifying eight overlapping fragments of the HBV genome was done as described previously (Hannoun et al., 1999 ). Cycle sequencing was done by the chain-termination method using fluorescent dye terminators (ddNTPs) and the same primers as in PCR, analysing each amplicon in both sense and antisense direction. The sequence was read in an ABI Prism 310 automated capillary sequence reader (Applied Biosystems) and then processed using the Sequence Navigator software (Applied Biosystems).
Database sequences.
The following complete genomes (represented by their accession numbers) were used in SimPlot and phylogenetic tree analyses: genotype A, Z35717, X72478, S50225, X02763, X70185 and L13994; genotype B, D00329, D50521, D50522 and D23678; genotype C, AB014374, AB014376, D23681, D23683, D23684, D12980 and X75665; genotype D, J02203 and M32138; genotype E, X75664 and X75657; genotype F, X75663. In addition, sequences representing southern African and East Asian genotype A strains were used in trees based on S region comparison.
Phylogenetic analysis.
Phylogenetic trees were constructed by maximum likelihood analysis by quartet puzzling (Strimmer & Haeseler, 1996 ) using TreePuzzle, available at http://www.tree-puzzle.de. The following settings were used: 1000 puzzling steps, no clock-like branch lengths, nt Hasegawa-Kishono-Yano substitution model, transition/transversion parameter estimated from data, and both uniform and gamma distributed (alpha parameter from data set) rate heterogeneities. Trees were also constructed by distance matrix and parsimony analysis using the DNAdist, Neighbor and DNApars software of PHYLIP 3.5c (Felsenstein, 1989 ; phylogenetic inference package, distributed by the author, J. Felsenstein, at http://evolution.genetics.washington.edu/phylip.html). Bootstrapping of 1000 replicates was then done by using Seqboot (PHYLIP). Recombination was investigated using SimPlot (Lole et al., 1999 ; distributed by the author, S. Ray, at http://www.welch.jhu.edu/), and by bootscanning (Salminen et al., 1995 ).
|
Results |
|---|
|
TOPAbstractIntroduction Methods Results Discussion References |
|---|
) and bootscanning (not shown) indicated recombination, because the part of the genome ranging from nt 1801 to 2865 (33% of the genome) was more similar to genotype C than to the other genotypes, while the rest of the genome (nt 2866–1800, 67% of the genome) appeared to be unique, resembling neither of the known genotypes. In agreement, in phylogenetic trees (Fig. 2) based on nt 1801–2865 these sequences always clustered with genotype C, while in 70% of trees based on nt 2866–1800 they were placed on a unique branch close to genotype A, and in the remaining 30% were just proximal of the node dividing genotypes A and B, or, in a few cases, close to the base of the genotype C branch. Comparison of these trees (i.e. nt 1801–2865 versus nt 2866–1800) using TreePuzzle (option: tree comparison, user defined trees) showed that the topology was significantly different (P<0·05; Kishino & Hasegawa, 1989 ). This was not found when the group I sequences were excluded from the analysis, indicating that the difference in topology was indeed due to the divergent placement of the group I strains, thus supporting the presence of recombination. Trees as analysed by DNA distance matrix and neighbour joining (not shown) produced nearly identical topologies to those based on maximum likelihood (TreePuzzle). In trees created by maximum likelihood analysis of amino acid sequences of the polymerase, which represents about 75% of the genome (nt 2307–1620), the group I strains were placed between genotype A and C.
|
|
Fig. 3 shows the HBsAg amino acid sequences of representatives of the group I and II strains in comparison with database sequences of genotypes A–F. All five genomes carried I110, T126 and K160, which are characteristic for serotype adw, and thus differed from most genotype C strains which are of serotype adr. The three sequences representing the putative recombinant showed no unique amino acids, but a unique combination of residues at genotype-related positions, including K24, I110, S114, T126, N131, S143, K160, Y161, A168, V194, N207 and L209.
|
shows the genetic difference between group I and II strains and the sequences of all genotypes, for the segment between nt 1801 and 2865, as well as the segment between nt 2866 and 1800. In the former segment the difference between group I sequences and genotypes A and C was 10·6% and 5·2%, respectively, while in the latter segment it was 6·6% and 7·7%, respectively. The table also shows the nt differences between genotypes A–F (consensus sequences) and the sequences Z35717 (genotype A) and D12980 (genotype C), which by tree analysis were the sequences most similar to the nt 2866–1800 and 1801–2865 segments (respectively) of the group I genomes. Thus, in relation to the intra-genotype variability, the nt 2866–1800 part is more distant from published genotype A sequences than the nt 1801–2865 part is from genotype C. The S regions of group I strains (included in the nt 2866–1800 segment) were also compared to further, non-European, S region sequences (not available as complete genomes), showing that they were distant from genotype A from southern Africa and East Asia (Fig. 4).
|
|
|
Discussion |
|---|
|
TOPAbstractIntroduction Methods Results Discussion References |
|---|
To some extent the assessment of recombination was hampered by the lack of available databases of genotype A and C sequences from Vietnam or the surrounding areas. Both the recombinant, genotype C segment (nt 1801–2865) of the group I sequences and the two group II isolates were placed between Japanese and New Caledonian sequences on the genotype C branch. The absence of C1858 and a 6 nt insertion in the core region, both of which are typical for genotype A, agrees with the interpretation that the nt 1801–2865 segment of the group I strains originates from genotype C.
The similarity of the nt 2866–1800 segment with genotype A agrees with our previous finding that S region RFLP classified these strains as genotype A (Lindh et al., 1997 ). However, in the SimPlot analysis the nt 2866–1800 segment appeared to differ from all genotypes, with genotype A being only slightly less different than the other genotypes, supporting the fact that this segment represents a new genotype rather than genotype A. This agrees with the finding that in phylogenetic analysis the S region of group I strains was clearly distinct from available genotype A sequences of Asian and African origin. Accordingly, a detailed analysis of the HBsAg region of the group I strains revealed similarities with all genotypes, in particular A, B and C. These similarities as well as the tree topology indicate that this putative new genotype (or subgroup of genotype A) may be a link between the European/African genotype A and the Asian genotypes B and C, suggesting that all these genotypes may have a common ancestor, as previously suggested (Norder et al., 1996 ).
The fact that one of the three genomes in group I was 1·2% different from the other two but also contained the recombined segment suggests that the recombination may not be recent. Analysis of more sequences from southeast Asia (and in particular from Vietnam) is essential for investigating whether this recombinant is widespread and for identifying complete genomes representing the putative new genotype or subgroup of genotype A. Such studies could possibly also identify the genotype C strains from which the recombined segment originates. Moreover, further study of recombination should be undertaken, in particular of isolates from this geographical region, where the HBV prevalence is high and co-infection with different genotypes may exist.
Recombination of segments from different genotypes of HBV has so far not been much discussed. However, signs of recombination have been found in integrated HBV DNA (Georgi-Geisberger et al., 1992 ), and analysis of HBV genomes from databases suggests that recombination may be relatively frequent (Bollyky et al., 1996 ; Bowyer & Sim, 2000 ). These studies describe genotype B strains that by recombination probably have acquired a portion of the core gene from genotype C. Of interest, this segment largely overlaps with the recombined (genotype C) segment described in the present study, possibly indicating that this part is particularly prone to recombination. It is unclear at what stage of replication recombination is most likely to occur. Recombination during reverse transcription of the pregenomic RNA, a process taking place simultaneously with encapsidation, appears unlikely because it would require two pregenomic RNA molecules to be encapsidated, and such phenomena have not been observed in studies of HBV particle sedimentation rates. It seems more likely that recombination would take place in the nucleus where co-existing covalently closed circular DNA copies derived from different genotypes could exchange genomic segments. Regardless of mechanism, recombination requires co-infection with different strains (genotypes) and this has not been well documented either. It is possible that co-infection is more frequent than previously thought, but that it is rarely detected because infection with a second strain is suppressed or merely cannot be established due to the very high virus load of the first strain. In such cases, co-infection may become visible only when the first strain disappears. Such shift in genotypes (from A to D) has indeed been observed during HBe seroconversion in European children (Gerner et al., 1998 ).
Further study of East Asian HBV sequences might clarify the detailed relatedness between these genotypes and is required for establishing the existence of the putative new genotype and to further evaluate recombination events in HBV.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
|
References |
|---|
|
TOPAbstractIntroduction Methods Results Discussion References |
|---|
Bowyer, S. M. & Sim, J. G. M. (2000). Relationships within and between genotypes of hepatitis B virus at points across the genome: footprints of recombination in certain isolates.Journal of General Virology81, 379-392.
Felsenstein, J. (1989). PHYLIP: phylogeny inference package (version 3.2).Cladistics5, 164-166.
Georgi-Geisberger, P., Berns, H., Loncarevic, I. F., Yu, Z. Y., Tang, Z. Y., Zentgraf, H. & Schroder, C. H. (1992). Mutations on free and integrated hepatitis B virus DNA in a hepatocellular carcinoma: footprints of homologous recombination.Oncology49, 386-395.[Medline]
Gerner, P. R., Friedt, M., Oettinger, R., Lausch, E. & Wirth, S. (1998). The hepatitis B virus seroconversion to anti-HBe is frequently associated with HBV genotype changes and selection of preS2-defective particles in chronically infected children.Virology245, 163-172.[Medline]
Hannoun, C., Horal, P. & Lindh, M. (1999). Long-term mutation rates in the hepatitis B virus genome.Journal of General Virology81, 75-83.
Kishino, H. & Hasegawa, M. (1989). Evaluation of the maximum likelihood estimate of the evolutionary tree topologies from DNA sequence data, and the branching order in hominoidea.Journal of Molecular Evolution29, 170-179.[Medline]
Li, J. S., Tong, S. P., Wen, Y. M., Vitvitski, L., Zhang, Q. & Trepo, C. (1993). Hepatitis B virus genotype A rarely circulates as an HBe-minus mutant: possible contribution of a single nucleotide in the precore region.Journal of Virology67, 5402-5410.
Lindh, M., Andersson, A. S. & Gusdal, A. (1997). Genotypes, nt 1858 variants, and geographic origin of hepatitis B virus – large-scale analysis using a new genotyping method.Journal of Infectious Diseases175, 1285-1293.[Medline]
Lindh, M., Gonzalez, J. E., Norkrans, G. & Horal, P. (1998). Genotyping of hepatitis B virus by restriction pattern analysis of a pre-S amplicon.Journal of Virology Methods72, 163-174.[Medline]
Lole, K. S., Bollinger, R. C., Paranjape, R. S., Gadkari, D., Kulkarni, S. S., Novak, N. G., Ingersoll, R., Sheppard, H. W. & Ray, S. C. (1999). Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination.Journal of Virology73, 152-160.
Norder, H., Hammas, B., Löfdahl, S., Couroucé, A. M. & Magnius, L. O. (1992). Comparison of the amino acid sequences of nine different serotypes of hepatitis B surface antigen and genomic classification of the corresponding hepatitis B virus strains.Journal of General Virology73, 1201-1208.
Norder, H., Hammas, B., Lee, S.-D., Bile, K., Couroucé, A.-M., Mushahwar, I. K. & Magnius, L. O. (1993). Genetic relatedness of hepatitis B viral strains of diverse geographical origin and natural variations in the primary structure of the surface antigen.Journal of General Virology74, 1341-1348.
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.Virology198, 489-503.[Medline]
Norder, H., Ebert, J. W., Fields, H. A., Mushahwar, I. K. & Magnius, L. O. (1996). Complete sequencing of a gibbon hepatitis B virus genome reveals a unique genotype distantly related to the chimpanzee hepatitis B virus.Virology218, 214-223.[Medline]
Okamoto, H., Tsuda, F., Sakugawa, H., Sastrosoewignjo, 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 Virology69, 2575-2583.
Salminen, M. O., Carr, J. K., Burke, D. S. & McCutchan, F. E. (1995). Identification of breakpoints in intergenotypic recombinants of HIV type 1 by bootscanning.AIDS Research and Human Retroviruses11, 1423-1425.[Medline]
Strimmer, K. & Haeseler, A. (1996). Quartet puzzling: a quartet maximum-likelihood method for reconstructing tree topologies.Molecular Biology and Evolution13, 964-969.
Stuyver, L., De Gendt, S., Van Geyt, C., Zoulim, F., Fried, M., Schinazi, R. F. & Rossau, R. (2000). A new genotype of hepatitis B virus: complete genome and phylogenetic relatedness.Journal of General Virology81, 67-74.
Received 14 March 2000;
accepted 24 May 2000.
This article has been cited by other articles:
|
|
 |
|
  K. Tatematsu, Y. Tanaka, F. Kurbanov, F. Sugauchi, S. Mano, T. Maeshiro, T. Nakayoshi, M. Wakuta, Y. Miyakawa, and M. Mizokami A Genetic Variant of Hepatitis B Virus Divergent from Known Human and Ape Genotypes Isolated from a Japanese Patient and Provisionally Assigned to New Genotype J J. Virol., October 15, 2009; 83(20): 10538 - 10547. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. W. Bloomquist, K. S. Dorman, and M. A. Suchard StepBrothers: inferring partially shared ancestries among recombinant viral sequences Biostat., January 1, 2009; 10(1): 106 - 120. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z.-L. Fang, C. A. Sabin, B.-Q. Dong, S.-C. Wei, Q.-Y. Chen, K.-X. Fang, J.-Y. Yang, J. Huang, X.-Y. Wang, and T. J. Harrison Hepatitis B virus pre-S deletion mutations are a risk factor for hepatocellular carcinoma: a matched nested case-control study J. Gen. Virol., November 1, 2008; 89(11): 2882 - 2890. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Kurbanov, Y. Tanaka, A. Kramvis, P. Simmonds, and M. Mizokami When Should "I" Consider a New Hepatitis B Virus Genotype? J. Virol., August 15, 2008; 82(16): 8241 - 8242. [Full Text] [PDF]
|
|
|
|
|
|
T. T. T. Huy, T. T. Ngoc, and K. Abe New Complex Recombinant Genotype of Hepatitis B Virus Identified in Vietnam J. Virol., June 1, 2008; 82(11): 5657 - 5663. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Yang, K. Xing, R. Deng, J. Wang, and X. Wang Identification of Hepatitis B virus putative intergenotype recombinants by using fragment typing. J. Gen. Virol., August 1, 2006; 87(Pt 8): 2203 - 2215. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Sakamoto, Y. Tanaka, E. Orito, J. Co, J. Clavio, F. Sugauchi, K. Ito, A. Ozasa, A. Quino, R. Ueda, et al. Novel subtypes (subgenotypes) of hepatitis B virus genotypes B and C among chronic liver disease patients in the Philippines J. Gen. Virol., July 1, 2006; 87(7): 1873 - 1882. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Zhang, M.-y. W. Yu, R. Venable, H. J. Alter, and J. W.-K. Shih Neutralization epitope responsible for the hepatitis B virus subtype-specific protection in chimpanzees PNAS, June 13, 2006; 103(24): 9214 - 9219. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B.-F. Chen, C.-J. Liu, G.-M. Jow, P.-J. Chen, J.-H. Kao, and D.-S. Chen Evolution of Hepatitis B virus in an acute hepatitis B patient co-infected with genotypes B and C J. Gen. Virol., January 1, 2006; 87(1): 39 - 49. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Simmonds and S. Midgley Recombination in the Genesis and Evolution of Hepatitis B Virus Genotypes J. Virol., December 15, 2005; 79(24): 15467 - 15476. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Suwannakarn, P. Tangkijvanich, A. Theamboonlers, K. Abe, and Y. Poovorawan A novel recombinant of Hepatitis B virus genotypes G and C isolated from a Thai patient with hepatocellular carcinoma J. Gen. Virol., November 1, 2005; 86(11): 3027 - 3030. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Wang, Z. Liu, G. Zeng, S. Wen, Y. Qi, S. Ma, N. V. Naoumov, and J. Hou A new intertype recombinant between genotypes C and D of hepatitis B virus identified in China J. Gen. Virol., April 1, 2005; 86(4): 985 - 990. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. C. Kimbi, A. Kramvis, and M. C. Kew Distinctive sequence characteristics of subgenotype A1 isolates of hepatitis B virus from South Africa J. Gen. Virol., May 1, 2004; 85(5): 1211 - 1220. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Flodell, J. Schleucher, J. Cromsigt, H. Ippel, K. Kidd-Ljunggren, and S. Wijmenga The apical stem-loop of the hepatitis B virus encapsidation signal folds into a stable tri-loop with two underlying pyrimidine bulges Nucleic Acids Res., November 1, 2002; 30(21): 4803 - 4811. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Cui, J. Shi, L. Hui, H. Xi, Zhuoma, Quni, Tsedan, and G. Hu The dominant hepatitis B virus genotype identified in Tibet is a C/D hybrid J. Gen. Virol., November 1, 2002; 83(11): 2773 - 2777. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Arauz-Ruiz, H. Norder, B. H. Robertson, and L. O. Magnius Genotype H: a new Amerindian genotype of hepatitis B virus revealed in Central America J. Gen. Virol., August 1, 2002; 83(8): 2059 - 2073. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Kidd-Ljunggren, Y. Miyakawa, and A. H. Kidd Genetic variability in hepatitis B viruses J. Gen. Virol., June 1, 2002; 83(6): 1267 - 1280. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Kalinina, H. Norder, S. Mukomolov, and L. O. Magnius A Natural Intergenotypic Recombinant of Hepatitis C Virus Identified in St. Petersburg J. Virol., March 19, 2002; 76(8): 4034 - 4043. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Alestig, C. Hannoun, P. Horal, and M. Lindh Phylogenetic Origin of Hepatitis B Virus Strains with Precore C-1858 Variant J. Clin. Microbiol., September 1, 2001; 39(9): 3200 - 3203. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
