HBV subgenotype misclassification expands quasi-subgenotype A3


Corresponding author: M. Van Ranst, Laboratory of Clinical Virology, Rega Institute for Medical Research, University of Leuven, Minderbroedersstraat 10, BE-3000 Leuven, Belgium


Clin Microbiol Infect 2011; 17: 947–949


Recently, we proposed a new classification for ‘subgenotype A’ of hepatitis B virus (HBV), in which the novel ‘quasi-subgenotype A3’ group comprising HBV ‘subgenotype A3’, ‘tentative A4’, and A5 was introduced. Newly ‘Tentative subgenotype A7’ strains from Cameroon were introduced by Hubschen et al. However, our meticulous phylogenetic analysis demonstrated that these isolates should also be classified into ‘quasi-subgenotype A3’. Such misclassification can be avoided by following established principles for HBV subgenotyping. Moreover, their close evolutionary relationship with A3 highlights our hypothesis that geographical origin may be an important factor in further classification of HBV subgenotypes.

We read with great interest the recent article by Hubschen et al. [1] published in Clinical Microbiology and Infection (the journal), which presented HBV genotypes and subgenotypes from Cameroon and Nigeria. The authors introduced a novel HBV subgenotype, ‘tentative A7’, originating from Cameroon, where previously subgenotype A3 had been isolated [2]. The molecular survey provided evidence for the circulation of HBV A3, A5 and A7 in Cameroon. The authors constructed a phylogeny tree based on ten tentative A7 isolates (full-length genome) and a restricted number of other subgenotypes of HBV genotype A as reference genes. Within genotype A, the tentative A7 strains formed a monophyletic cluster that was well supported by 96% bootstrap analyses (96%). The authors also stated that the mean inter-subgenotype distance of the proposed A7 subgenotype met the subgenotype definition (Figure 2 and Table 1 in [1]).

In our recent article entitled ‘Are hepatitis B virus subgenotypes defined accurately?’ a detailed phylogenetic analysis based on all-available hepatitis B virus (HBV) complete genome sequences of genotype A was conducted [3]. We demonstrated that particular HBV strains that were mainly of African origin (A3, A4 and A5) could not be classified as ‘definite’ subgenotypes. Interestingly, the mean genetic distance (complete genome sequence) for these isolates ranged between values that were defined for ‘clades’ and ‘subgenotypes’. We therefore designated these as ‘quasi-subgenotype A3’, whereas three definite subgenotypes, A1, A2 and A6, were perfectly compatible with the subgenotype definition [3]. We predicted that introducing more African strains of HBV genotype A would increase the population of quasi-subgenotype A3 and we proposed that geographical origin might have a key role in further classification of HBV subgenotypes. Based on our work, we strongly recommended that each HBV phylogenetic study intending to introduce a ‘novel’ genotype and particularly subgenotype should consider certain definitions [3]. At the same time, Schaefer et al. [4] expressed similar concerns.

The limited number of genotype A taxa in the phylogeny tree and the lack of consistency with subgenotype assignment rules in terms of genetic distances [5,6] encouraged our team to reanalyse these data presented by Hubschen et al. [1]. To investigate the relation of these new strains with quasi-subgenotype A3, we analysed all ‘tentative A7’ full genome strains with almost all previously described genotype A strains as reference strains [3]. Of note, all analyses were done using the same methods and software that were applied by Hubschen et al. [1].

Our phylogenetic analysis showed that the introduced ‘tentative A7’ indeed clustered with similar bootstrap support within quasi-subgenotype A3. This cluster was obtained by 80% bootstrap support. Our nucleotide divergence calculation, however, showed remarkable differences with the study of Hubschen et al. (Table 1). Tentative A7 strains showed less than 4% nucleotide divergence when compared with A1, and particularly with A3, A4 and A5. Similar results were obtained when tentative A7 was compared with quasi-subgenotype A3 (according to the recent proposed classification). We believe that a few selected strains as reference sequences are not representative of the nucleotide divergence because we obtained different results when analysing all available strains. Subgenotypes A1, A2 and A4 (previously known as A6) can be considered as definite ‘subgenotypes’, because they form clear phylogenetic clusters supported by high bootstrap values (Fig. 1), with considerable divergence in mean genetic distances.

Table 1.   Mean percentage of nucleotide divergence between HBV full genomes from tentative A7 and members of quasi-subgenotype A3. (a) Between new proposed groups. (b) These data confirm that HBV subgenotype A7 does not meet a clear subgenotype definition
A7 (Hubschen)5.1 ± 0.375.34 ± 0.334.24 ± 0.243.99 ± 0.253.81 ± 0.245.1 ± 0.35
A7 In this study3.9 ± 0.034.7 ± 0.033.7 ± 0.023.5 ± 0.033.10 ± 0.034.6 ± 0.03
 A1A2Q-S-A3A4 (previous A6)
A73.9 ± 0.034.7 ± 0.033.1 ± 0.024.6 ± 0.03
Figure 1.

 The neighbour-joining phylogenetic tree constructed based on HBV full-length genomes of almost all complete genomes of subgenotype A strains available in GenBank (A1, A2, quasi subgenotype A3, A4 and tentative A7). The numbers represent the bootstrap value (1000 replicates) in the main nodes. HBV genotype C was used as an out-group. HBV genotype A1 and A2 strains were collapsed. As illustrated, tentative HBV subgenotype A7 clustered within HBV quasi-subgenotype A3 branch.

In conclusion, our analysis demonstrated that the introduced strains ‘tentative subgenotype A7’ did not meet the ‘subgenotype’ definition according to genetic distance and topology of phylogeny tree. Therefore, we propose that these strains should be also considered as new members of quasi-subgenotype A3. Moreover, their close evolutionary relationship with A3 is consistent with the geographical origins of the strains [2]. This finding highlights our hypothesis that geographical origin may have a key role in further classification of HBV subgenotypes [3]. In addition, it underscores the arguments of Schaefer et al. [4] on the roles of the International Committee on Taxonomy of Viruses (ICTV) as well as those of experts in HBV subgenotyping. Finally, we would like to stress again that the introduction of a new HBV genotype and especially a subgenotype should adhere to clear rules to avoid misclassification and confusion.

Transparency Declaration

This study was co-funded by the institute for the Promotion of Innovation by Science and Technology in Flanders (strategic basic research project SIMID). All authors declare that they have no conflicts of interest.