Relationship of genotypes of hepatitis B virus to mutations, disease progression and response to antiviral therapy
Journal of Viral Hepatitis
Volume 12 Issue 5 Page 456 - September 2005
A. Kramvis and M. C. Kew
MRC/University Molecular Hepatology Research Unit, Department of Medicine, University of the Witwatersrand, Johannesburg, South Africa
Summary. Phylogenetic analysis has led to the classification of hepatitis B virus into eight genotypes, designated A to H. The genotypes have differences in biological properties and show heterogeneity in their global distribution. These attributes of the genotypes may account not only for differences in the prevalence of hepatitis B virus mutants in various geographic regions, but also be responsible for differences in the clinical outcome and response to antiviral treatment in different population groups.
Hepatitis B virus (HBV), a DNA virus, is a member of the family Hepadnaviridae  that replicate by reverse transcription of the encapsidated pregenomic RNA by the viral encoded polymerase . The viral polymerase lacks proofreading activity and sequence heterogeneity is therefore a feature of HBV.
Phylogenetic analysis has led to the classification of HBV into eight genotypes, defined by an inter-group divergence of >8% in the complete genome sequence [3,4] and of >4% in the S gene . Since the first description of four genotypes (A-D) of HBV in 1988 , four more have been identified, designated E and F , G  and H [6-8]. Moreover, subgenotypes with distinctive sequence characteristics and a divergence in the complete genome of >4% have been found within genotypes A [9-11], B [12-14], C  and F [16,17].
The eight genotypes show a distinctive geographical distribution. Genotype A is prevalent in north-western Europe, North America and Africa [18-20]. Genotypes B and C are characteristic of Asia [4,19,20], whereas genotype D has a worldwide distribution but predominates in the Mediterranean area [19,20]. Genotype E is found in Africans [6,19,21], genotype F in the aboriginal populations of South America [18,22] and genotype H is confined to the Amerindian populations of Central America [8,23]. To date, the isolation of genotype G has been limited to HBV carriers in France and Georgia, USA , UK , Italy  and Germany .
The first instance of genotype-related differences in the biological properties of HBV was the observation that the precore 1896 stop-codon mutant was commonly found in regions where genotype D prevailed and was absent in regions were genotype A occurred . The reason for the association of the 1896 mutant with genotype D was that this mutation enhanced the stability of the encapsidation signal (e) allowing replication, whereas in genotype A it would lead to its destabilization and therefore prevent replication . Subsequently, it has become increasingly evident that the heterogeneity in the global distribution of HBV genotypes may account not only for differences in the prevalence of HBV mutations in the different populations but also be responsible for differences in the clinical outcomes of HBV infections and the response to antiviral treatment.
Genotypes and disease progression
Because disease progression can be affected by a number of factors, such as the age of acquisition and route of the infection , the immune competence of the host, and the influence of environmental factors such as alcohol intake, iron overload and exposure to aflatoxin, care should be exercised when interpreting the role of genotypes in disease progression.
The majority of studies on the effect of genotypes on disease progression have been undertaken in South-east Asia where HBV is hyperendemic and genotypes B and C prevail. A greater frequency and severity of liver dysfunction was initially reported in patients infected with serotype ayr (mainly genotype C) compared with adw (mainly genotype B) [78-80]. Seroconversion from HBeAg- to anti-HBe positivity occurs much earlier in genotype B than genotype C carriers [42,45,46,61,78,81-85]. Higher HBV-DNA levels have been detected in patients infected with genotype C compared with those infected with genotype B in some studies [44,81,86], but not in others [45,46]. The difference might be attributed to the HBeAg status of the patients. Genotype C was found to have lower HBV DNA levels than genotype A, B and D in the HBeAg-positive phase . In south-western Japan carriers of genotype D were younger and exhibited earlier anti-HBe seroconversion than carriers with genotype C .
Patients infected with genotype B are more likely to have a sustained biochemical remission after spontaneous HBeAg seroconversion than patients infected with genotype C , who are more likely to develop chronic and advanced liver disease [44,86]. Genotype C is more prevalent in patients with fibrosis or cirrhosis [43,46,82] and is associated with more severe histological liver damage than genotype B  or genotype D . Patients infected with genotype C have higher scores of histological activity and fibrosis [41,42] and higher alanine aminotransferase (ALT) levels relative to those infected with genotype B [44,83], genotype A or D .
The majority of studies in Far Eastern countries have shown a greater risk of HCC development with genotype C than with genotype B [43,68,90,91]. However, patients infected with genotype B exhibit earlier HBe seroconversion and progress to liver fibrosis and HCC at a slower rate than those infected with genotype C, and it has been suggested that the life-long risk of progression to advanced fibrosis and development of HCC may not differ among genotype B- and C-related chronic liver disease [46,61]. Because most studies have been cross-sectional, it will be helpful if prospective, longitudinal studies are undertaken to determine whether the genotypes influence the incidence of disease in the long term.
In contrast to genotype B found in Taiwan  and China , which is associated with the development of HCC at a young age, in Japan, the mean age of HCC patients infected with genotype B is significantly older than those infected with genotype C [46,81,92]. Although it has been suggested that this discrepancy between Chinese and Japanese HCC patients, could be a result of host factors and the intake of aflatoxin in Taiwan , the difference is probably the consequence of the different subgenotypes found in mainland Asia (Ba) and Japan (Bj) . Further studies are required to resolve this issue. When matched HBV carriers were compared, HBeAg-positivity occurred in a significantly lower proportion of those infected with subgenotype Bj compared with Ba or genotype C (and loss of HBeAg occurs earlier in carriers of Bj) [13,47]. Subgenotype Ba occurred more frequently in acute than in chronic hepatitis patients .
Genotypes A and D were found to be prevalent in the Indian subcontinent. In one study genotype D was associated with more severe liver disease and with HCC in young patients , whereas in another study, where the majority of patients were infected with genotype D, it was concluded that genotype D did not influence the clinical outcome of infection .
There have been fewer studies on the effect of genotype on disease progression in western countries. The long-term outcome of HBV infection was found to be different in patients infected with different genotypes in Europe. Chronic infection with genotype A is more frequent than when individuals are infected with genotype D . Genotype A was more prevalent in HBeAg-positive chronic hepatitis patients, whereas genotype D was more prevalent in those positive for anti-HBe [20,49,50,96]. HBeAg-positive and HBeAg-negative carriers infected with genotype D were found to have higher levels of HBV-DNA when compared with genotype A, B and C [20,41]. The prognosis of chronic hepatitis B may be better in patients infected with genotype A than in those infected with either genotypes D or F because concomitant sustained biochemical remission and decrease in HBV-DNA levels occurred at a higher rate in genotype A- than in genotype D- or genotype F-infected patients . Genotype D was also found to be associated with severe recurrent disease post-transplantation . In a single study, genotype F-infected individuals showed a higher mortality rate than those infected with genotype A or D . However, this does not agree with other reports that showed a low pathogenicity of genotype F [22,98].
Very few isolates of genotype G have been characterized making it difficult to draw any conclusions regarding the influence of this genotype on disease progression. Nevertheless a trend is observable. Chronic hepatitis patients infected with genotype G are characterized by high HBV-DNA and HBeAg levels [20,24,99-101] and elevated ALT levels . However, coinfection with genotype A may account for these attributes [101,102]. One patient infected with genotype G had cirrhosis  and two were found to lack an anti-HBc response [24,99].
Genotypes and response to antiviral therapy
Although mass vaccination programmes have begun to control the spread of HBV infection, therapeutic intervention is the only option for those with established chronic HBV-associated disease.
One of the more effective therapies available is treatment with interferon (IFN)-alpha, a naturally occurring cytokine primarily produced by B lymphocytes, null lymphocytes and macrophages [103-105]. IFN has anti-viral, anti-proliferative and immunomodulatory effects . The molecular virological factors that contribute to the responsiveness of HBV infection to IFN treatment and may play a significant role in predicting whether IFN can be used effectively for treatment are largely unknown. The ability to predict responsiveness is important in the clinical setting, considering the fact that IFN treatment is expensive, is administered by injection, and can have side-effects and be poorly tolerated.
To date, the most important viral factor that has convincingly been shown to determine the response to IFN is the pretreatment HBV-DNA titre, the lower the titre the better the response . Other viral factors that may play a role include the presence of BCP and precore mutations. It has been proposed, although not proven, that the BCP mutations together with a low HBV-DNA level and elevated ALT may be favourable factors of response in IFN-induced anti-HBe seroconversion . The data on the relevance of precore mutants and their influence on the long term response to IFN is also inconclusive. In some studies precore mutants were considered to be necessary for response to IFN [107,109,110]. These data differ from other studies that showed that the precore mutants do not have prognostic value for virus elimination following IFN therapy in HBeAg-positive or -negative patients [111-113]. These differences in responsiveness to IFN treatment may possibly be the result of different genotypes of the virus and therefore an analysis of how various mutations influence the therapeutic response to IFN also requires knowledge about the genotype .
In a study of German patients a higher rate of HBeAg seroconversion following IFN treatment was found in those infected with genotype A than those with genotype D (37%vs 6%) . The rate of HBeAg loss was also significantly higher in patients with genotype B compared with those with genotype C in a Taiwanese study (41%vs 15%) . In the former study, additional factors besides genotype, including the number of BCP mutations and low DNA levels, were found to be related to a better response and in the latter study; young age was found to be an additional positive predictive factor. Because genotype B-infected patients have a high HBeAg seroconversion rate, Wai et al.  compared treated and untreated Chinese patients with chronic hepatitis B. They showed that, in addition to low pre-treatment HBV-DNA levels and elevated ALT levels, genotype B was associated with a higher antiviral response to IFN treatment. The response to IFN treatment of genotype A-infected Chinese patients was found to be better than those infected with genotype D/E (70%vs 40%) . In contrast, in a study performed in Japan, IFN was given to seven patients with chronic HBV infection. Of the four responders, one was infected with HBV genotype B and three with genotype C. HBsAg persisted in the remaining three patients, all of whom were infected with genotype A, and HBeAg remained positive in one of them . In a longitudinal study, no difference was reported in the rates of sustained seroconversion to anti-HBe in IFN-treated patients compared with those that were untreated, and this was not affected by the HBV genotype with which the patient was infected. However, the cumulative probability of HBsAg clearance was greater in patients infected with genotype A than those infected with genotype D . These studies involved a small number of patients, which may be the reason for the conflicting observations.
Genotype switching has also been observed after IFN treatment indicating infection with a mixture of genotypes prior to treatment [65,119,120]. The minor populations, however, were not detected by either standard genotyping assays or direct sequencing and were detected using either a genotype-specific PCR plus RFLP or cloning.
Thus the results of studies from one geographical region cannot be extrapolated to other regions, without a thorough knowledge of the HBV strains circulating in each of the regions.
Lamivudine, a nucleoside analogue, has been approved for the treatment of chronic hepatitis B. In addition to reducing inflammatory activity in liver, lamivudine reduces HBV-DNA levels in most patients. A drawback of this treatment, however, is the appearance of lamivudine-resistant mutants.
Genotype B has a better virological response to lamivudine than genotype C in Taiwan. However both genotypes have a similar risk of developing lamivudine resistance after 1 year of therapy . On the contrary, adw has been shown to have a 20-fold higher risk of lamivudine resistance than ayw infections . Emergence of resistance was also found to be more rapid in adw carriers. The appearance of the lamivudine-resistance mutations was predicted to result in a change in hydrophilicity in the S region of the ayw subtype but not in the adw subtype  and this could explain the reduced risk of developing resistance in ayw subtype. The genotypes can be deduced to be genotype A (adw) and genotype D (ayw) because these are the genotypes predominant in Germany and the serotypes associated with them. When comparing patients infected with genotype A and D isolates, it was found that although the risk of emergence of lamivudine resistance mutations was higher during the first year of treatment in genotype A-infected patients, there was no difference when the time of treatment was prolonged to 2 or 3 years. In other words, lamivudine resistance mutations took longer to develop in genotype D . In a large-scale study in Japan, the emergence rate of lamivudine resistance was independent of genotype A, B and C. On the contrary, the emergence rate was significantly higher in subgenotype Ba than in Bj . This study also suggested that the risk of HBeAg-positivity on the development of lamivudine resistance may differ between the genotypes and that the risk of severe breakthrough hepatitis may be higher in patients infected with genotype C .
Adefovir dipivoxil, an oral prodrug of adefovir, has been tested in phase III trials and was approved recently for the treatment of chronic hepatitis B in the USA . There was no significant difference in the antiviral response between patients infected with the different genotypes of HBV .
Relationship of mutations
1896 stop codon mutation
The precore-core region of the HBV genome codes for the precore-core fusion protein that is post-translationally modified to give rise to hepatitis B e antigen (HBeAg) [27,28]. Although HBeAg is not required for viral replication or infectivity [29,30], its exact function is not known. It is thought to play a role in immune modulation and can alter the host response to core protein [31,32]. Thus, the emergence of the 1896 G to A stop-codon mutation [33,34] that prevents expression of HBeAg may be a means of immune evasion. The occurrence of the 1896 mutation is restricted by the secondary structure of the encapsidation signal (e) [26,35-37], which is transcribed from the same region of the HBV genome coding for HBeAg (Fig. 1). Destabilization of this structure by the disruption of the G-C base pair between positions 1858 and 1896 (that would result from a G-to-A mutation at 1896) would be detrimental to viral replication , as has been shown by transfection experiments [25,35]. Thus the development of the 1896 mutation depends on the presence or absence of C or T at position 1858 and shows geographic variation that is related to the distribution of the various genotypes . Genotypes B, D and E have T1858, whereas A and H have C1858. Genotype C and F isolates can have either C1858 or T1858: genotype F strains in Central America have a T1858  and Japanese genotype C strains have T1858 exclusively [38,39], whereas C1858 is confined to carriers of genotype C in South-east Asia [19,40-44]. Some studies found no difference in the prevalence of 1896 mutants between genotypes B and C [45,46]. This is to be expected because both these studies were carried out in Japan where genotype C with T1858 predominates. It is of interest that although both subgenotypes of genotype B, identified in Asia (Ba and Bj) have T1858, the 1896 mutation was found to occur more often in subgenotype Bj . The 1896 mutation is found most frequently in anti-HBe positive patients infected with genotype D [25,26,48-51] and E  and is rarely found in genotype A [25,50,51], genotype H  and in a minority only of genotype C [19,41,42] and genotype F strains [6,17,19,22].
T1762 A1764 basic core-promoter mutations
An adenine (A) to thymine (T) transversion at position 1762 together with a guanine (G) to A transition at 1764 in the basic core promoter (BCP) were first described in HBV isolates from Japanese patients [53,54]. The presence of the mutations precedes seroconversion from HBeAg to anti-HBe in genotype A strains but not in genotype D [48,55]. Their presence results in reduced levels of precore mRNA and HBeAg expression in transfection studies [56-59]. The T1762A1764 mutations develop more frequently in genotypes A and H with C1858, but in a minority of genotype C with C1858  and more often in subgenotype Ba than in Bj or genotype C . However, in other analyses, they are equally distributed among the HBV genotypes [49,51]. Yet other studies show a higher frequency of the T1762A1764 in genotype C compared with genotype B [41,42,45,46,60,61], and this does not correlate with HBeAg status [45,46]. Only 25% of carriers infected with genotype E possess the T1762A1764 mutations and this finding is independent of HBeAg status . These mutations are found to be significantly associated with more severe liver disease [liver cirrhosis with or without hepatocellular carcinoma (HCC)] and an older age (>35 years) [45,62,63].
Various mutations in the pre-S region have been described. These range from point mutations and small deletions and insertions  to very large deletions [65,66] and deletions that prevent the expression of the pre-S2 protein . A few studies have analyzed the relationship between genotypes and the occurrence of pre-S mutants. In two independent studies, pre-S deletion mutants were found to occur more frequently in genotype C than in genotype B isolates  and in adr (corresponding to genotype C) than in adw (corresponding to genotype B) isolates . In contrast, in another study, although these mutants are found more frequently in genotypes B and C than in the other genotypes, no statistical difference was found between their incidence in genotypes B and C . This discrepancy may be explained by the different geographic distributions of the isolates or the fact that the latter study was not a case-control study. Moreover, these deletion mutants are more frequently detected in isolates from patients with severe liver disease (liver cirrhosis and HCC) than other patients [68,70].
Deletions within the pre-S region can lead to impaired viral clearance without affecting HBV binding to hepatocytes and their subsequent penetration, and therefore could contribute to chronicity of infection . In keeping with this possibility is the observation that pre-S deletion mutations are more frequently detected in isolates from patients with cirrhosis or HCC [68,70].
Mutation clustering regions within the core region
Ehata et al.  have identified mutation clustering regions (MCR) within the core region of HBV isolates from individuals with liver disease and speculated that these may be immunological targets for cytotoxic T lymphocytes . MCRs have been mapped to different positions in the different genotypes. Genotypes B (most) and C (adr subtype) have mutations clustering at positions 84 to 99 of the core gene and genotypes A, B and D (adw subtype) at positions 48 to 60 . The MCR of genotype F residues was mapped between positions 57 and 68 of the core protein .
These HBV variants arise from the encapsidation and reverse transcription of spliced pregenomic RNA . Using in vitro tranfection studies, there is no difference in the dominant splice variant produced by genotypes D, C and E, whereas the minor splice variants synthesized by isolates belonging to the different genotypes vary . Splicing in HBV may contribute to the pathogenicity and/or persistence of the virus [75-77].
The variation in the presence and the development of mutations in the different genotypes of HBV may be a contributing factor to the influence of genotypes in disease progression.
It is becoming increasingly evident that the genotype of HBV may have a role to play in predicting the response to various therapies and that this should be taken into account as a variable before initiating any treatment. However, differences in host and environmental factors make it difficult to extrapolate findings from one geographical region to another. Therefore larger, in-depth studies are necessary, in various regions of the world, especially in regions where HBV is hyperendemic such as South-east Asia and sub-Saharan Africa. Although some studies have shown that HBV serotypes may influence the efficacy of HBV vaccination , to our knowledge, no studies have been undertaken to find the relationship between genotypes of HBV in response to vaccination and/or the emergence of vaccine-escape mutants. This is another area that requires further research. As most studies to date have been cross-sectional, more longitudinal prospective studies could provide more information on the relationship of HBV genotypes to the severity of liver disease and therefore clinical outcome. Moreover, the advent of new molecular antivirals makes it paramount that the genotypes are well characterized, so that the drugs can be tailor-made to the sequence of the virus prevalent in the different regions of the world.