Hepatitis B Virus Genotypes :

Clinical Implications
Subrat Kumar Acharya, Yogesh Batra

87
Professor, Department of Gastroenterology, All India Institute of Medical Sciences,
New Delhi 110 029

Introduction ‘D’ and ‘E’. Where as adr and ayr subtypes are encountered with
Hepatitis B virus (HBV) infection is a global health problem. genotype ‘C’.
Approximately 400 million people in the world are chronic HBV The clinical relevance of such genotype is yet unclear. However,
carries.1 In India, 10% (40 million) of the total carrier population because the HBV-induced disease is the resultant of virus-host
resides.2 Liver injury in HBV infection is predominantly interaction, the disease characteristics may be influenced by the
immune-mediated and therefore virus-host interaction is the genotypes of the virus. Interaction between hepatocyte genome
major determinant of HBV-induced liver disease.3 Host immune and HBV genome may also vary according to the prevalent
system varies from individual to individual and is dependent on genotype (It is established that prolonged HBV infection may
many factors, such as, age of infection, genetic susceptibility, alter or effect the proto-oncogenes and tumor suppressor genes
inherent immunological competence and environmental in hepatocytes).7
factors.4 The virus characteristics may also vary and therefore
events determined by host-virus interaction may be extremely Classification and Geographical
heterogeneous causing heterogeneous disease characteristics. distribution
HBV is the smallest DNA virus with 3200 base pairs, which The gold standard for classifying any virus is the sequence
contains four overlapping genes (Open Reading Frames or ORFs) divergence in the entire genome. In 1988, based on sequence
encoding the viral envelope (S and pre S), necleocapsid (Pre- divergence of more than 8% in 18 HBV isolates, four genotypes
core and Core), polymerase with reverse transcriptase enzyme of HBV were proposed (A-D).8 In 1994, two more HBV
and X proteins.1 HBV is also known to replicate through RNA genotypes (E,F) satisfying the same sequence divergence criteria
intermediates on which reverse-transcriptase enzyme acts to were added to the list of HBV genotypes9 and Genotype G and
produce complementary DNA (c-DNA) for further replication H were recently identified after 2000.10,11 The ORFs among
through DNA-polymerase enzyme.1 Reverse transcriptase is a these genotypes (particularly among A-D) have been found
poor proofreader and therefore nuclear substitution during HBV to be different; the inter-typing difference being maximum
replication is a spontaneous event. Approximately 1.4 – 3.2 x in Pre S/S region and minimal in PreC/C region. Among all
10 -5 nucleotide substitution per year occurs in the HBV genome genotypes, genotype F has the maximal divergence in which
during its replication.5 Due to such substitutions the antigenic nucleotide divergence upto 15.5% have been documented.6 The
characteristics of HBV peptides (predominantly documented in recently described genotype G has a peculiar difference from
the envelope region) may change. Therefore, after a long time the other genotypes. This genotype has a 36 bp insertion in the
of evolution four major HBV serotypes (adw, adr, ayw, ayr) core gene and two stop codon in the precore gene causing arrest
with subtypes have been described.3-4 The major group specific of translation of HBeAg. However, genotype A invariably co-
antigen ‘a’ remains similar in all the serotypes, however, the infects along with genotype G5 and is responsible for presence
minor variation in the amino acid profile of envelope protein of HBeAg in sera of such patients.6 Such observation have raised
leading to different serotypes of the virus have been associated the possibility whether, genotype G is replication in completent
with epidemiological or distribution characteristics of the virus. and requires coinfection of other HBV genotypes?
Such change in the envelope peptides has been associated with
nucleotide changes in HBV genome. Sequence analysis of full Partial correlation between HBV genotypes and serotypes have
length HBV genome of different isolates by now have revealed at been established, however discrepancy between genotypes and
least eight different HBV isolates which has variation from each serotypes have been observed, which is due to change of serotypes
other in more than 8% of genomic sequences.6 These isolates with from d to y or w to r by single point mutation from A to G at nt
> 8% variation in nucleotide sequence homology are called ‘HBV 365 and nt 479 respectively,6 which converts codon 122 and 160
genotypes’ and have been alphabetically named from ‘A’ through from lysine to arginine.6
‘H’. The relationship between serotypes and genotypes has been The geographical distribution, serotypic correlation, and types of
elucidated.6 The serotype ayw occurs in all genotypes except in clinically recognized mutated HBV among various genotypes are
‘C’. Serotype adw have been associated with all genotype except summarized in Table 1.

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Hepatitis B Virus Genotypes: Clinical Implications
 Table 1 : HBV Genotypes: Distributions, Serotypes, Mutant Forms
Frequency of Mutation
Genotypes Subtypes Geographical Distribution
Pre-core Mutants Core Promoter Mutants
A adw2, ayw1 Uncommon Common India, Western Europe, USA, Central Africa
B adw2, ayw1 Common Common South East Asia, Japan, USA, China
C adr, ayr, adw2 Common Common Same as above
D ayw Common Common India, Mediterranean, USA
E ayw Not known Not known West Africa
F adw, ayw Uncommon Not known Central and South America, Polynesia
G adw Common Not known Europe, USA
H adw Common Not known Central America

Methods used for genotyping result in amplification of particular genotype of HBV

Genotyping of viruses by nucleotide sequencing and subsequent depending upon the corresponding specific primer.
homology comparison of phylogenetic tree analysis is cumbersome d. Genotype Specific Probe assay15
and labor intensive and therefore cannot be applied to large In this method nucleotide sequences corresponding to
number of samples. With recent advances in molecular techniques Pre S region are amplified by PCR with a predesigned
several novel genotyping methods have been described. These Pre S1 primer labeled with Biotin. These PCR
methods can be divided into two broad categories. amplicons are then delivered into wells on a microplate
on which complementary sequences specific to one or
1. Genotyping based on study of nucleotide sequences other genotypes have been immortalized. Thereafter
2. Serological methods for genotyping HBV hybridized HBV DNA amplified from test samples
These methods usually targets to analysis of preS / S region in these wells are identified by colorimetric method.
(as it is most heterogenous among the genotypes) and This test kit is available commercially (Smitest HBV
pre C/C region (as it is least heterogenous and analysis of genotyping Kit, Genome Science, Fukushima, Japan)
sequence in this region identifies core promoter or pre-core
mutation which is correlated with the genotypes. The above
2. Serological method for HBV genotyping
(ELISA)16,17
mentioned mutants have been associated with occurrence
of liver cancers and progressive liver disease. Therefore such Usuda et al16,17 identified seven distinct antigenic epitopes on
Pre S2 envelope peptides (Pre S2 is the most divergent region
analysis have tried to associate genotype, mutant HBV and
in HBV genome) against which monoclonal antibodies were
severity of liver disease).6
developed. These epitopes have been named as, a (common
1. Genotyping by nucleotide sequence analysis :- antigenic determinant), b (specific for Pre S2 and present in
all genotypes), k, m, s, u, g (these epitopes in various distinct
There are four described methods for nucleotide sequence
combinations for each genotype are present on Pre S2).
study in HBV.
In the first step, HBsAg present in test sera sample are
a. PCR-RFLP12 (polymerase chain reaction – restriction immobilized on a well of microlitre plate coated with
fragment length polymorphism) antibody to common antigenic determinant of HBsAg (a).
This method was described by Mizokami et al12 in Captured HBsAg then is tested binding with monoclonals
which he identified the restriction site in the genome of against b, k, m, s and u. The epitope b is ubiquitously present
‘S’ region for each genotype, and developed RFLP for on Pre S2 across all genotypes and ensures presence of Pre S2
each genotype. In this method, first PCR amplification on the microtitre wells. Genotypes are then distinguished
for the S region is performed. The PCR product is then by presence of combinations of various epitopes: bsu for
subjected restriction enzyme (endonucleases) digestion genotype A, bm for genotype B, bks for genotype D and
specific for each genotype and then the digested product E and bk for F. To distinguish between genotype D and
is subjected to agar-gel electrophoresis, and staining. E monoclonals against another cryptic epitope ‘g’ which is
Digested nucleotide specific for each genotype is then expressed with HBsAg is used. For this, on a solid phase
identified. monoclonals to ‘a’ common antigenic determinant and ‘g’
epitope labeled with an enzyme are immobilized, to which
b. Line probe assay13 test sera is added. HBsAg along with monoclonals against
Sequence specific oligomers for each genotype are g epitope (sandwich ELISA) detect presence of g epitope
immortalized on a paper strip, to which PCR amplified indicating presence of bks (g), i.e. genotype D.
test samples are hybridized (reverse hybridization). Genotype G described recently is determined by the
c. PCR using type Specific Primers14 combination of Pre S2 serotype for genotype D i.e. bks (g)
In this method genotype specific primers for Pre along with HBsAg-serotype adw (it is the characteristics of
S/S region are used on the test sample, which would this genotype). The results of ELISA genotyping have been

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Medicine Update 2005
 validated by RFLP. ELISA kits for serological genotyping Based on this observation, the authors suggested to classify
are commercially available (HBV genotype EIA, Institute of genotype B further to Bj (Japan) and Ba (Asia) respectively.
Immunology, Tokyo, Japan) As mentioned earlier genotype ‘G’ does not possess ability
Clinical Relevance of HBV to produce HBeAg, due to presence of two stop codons in
the pre core region. Therefore, genotype G is usually co-
genotypes: infected with genotype A which is competent to synthesize
1. HBV genotype and Pre core mutants HBeAg. A/G recombination in few patient have also been
reported.6 It is by now believed that 10% of HBV isolates
G 1896 A mutation at nucleotide 1896 in the Pre core region
may have inter genotypic recombination, which obviously
of HBV genome converts codon 28 from tryptophan to a stop
cannot occur without mixed genotypic infection.6
codon, as a result of which translation of HBeAg precuser
is aborted with a consequent HBeAg-ve, yet replicating Superinfection of one genotype over other may hepatitis flare
HBV which is classically known as pre core mutant (PCM). has also been reported,5 and may account for 2% of the flare
HBV DNA, is present in a double helix form with base documented among patients with chronic HBV infection.5
pairing. The nucleotide 1896 in HBV DNA, pairs with the
3. Disease and virological characteristics
nucleotide 1858. Usually base pairing such as A-T or C-G
are stable and viable. Interestingly, genotype ‘B’ and C are highly prevalent
in hyperendemic countries for HBV, such as in China,
Nucleotide variability (T or C) at position 1858 has been
Taiwan, Vietnam, Indonesia and rarely in low/intermediate
commonly observed. The relationship between HBV
endemic countries like Japan. In contrast, genotype A and
genotype, type of Pre core mutation and nucleotide
D are distributed in low or intermediate endemic countries
variability at position 1858 has been studied5,6,18. Genotypes
like USA. Europe, and India. Even though, occurrence of
other than ‘A’ usually have a T at nucleotide 1858 (T-1858),
HCC has not been documented to be associated with any
which makes a wobble or loose pairing with G-1896, in the
particular genotype, it is more prevalent in the countries
stem of the ‘ε’ encapsidation signal. The mutation of G to A
with genotype B or C HBV infection in comparison to
in 1896 resulting in A 1896 (Pre-core stop codon mutant)
similar prevalence in countries with genotype A and D HBV
tighten the stem structure by making a T-A pair between
infection.
nucleotide position of 1858 and 1896. In contrast, genotype
‘A’ possesses a C 1858 making a stable C-G pair with G Reports from Japan, Taiwan and China indicates that5: a)
1896. If G 1896 A mutation occurs in genotype A HBV, HBeAg positivity and HBV DNA load among genotype
then A 1896 would pair with C 1858 making a C-A woobly B infected individual may be lower than in genotype C
base pair which would not provide encapsidation signal infection, b) Immune clearance phase in genotype C may
and therefore would be non-viable. G 1896 A mutation in be longer than in genotype B, therefore liver damage among
genotype A HBV then would be possible, if a simultaneous former group may be more than in the latter patients, c) Due
mutation T 1858C would occur. Therefore pre core mutant to the latter fact, histological severity among genotype C
only occurs in HBV strains with T1858 and rarely occurs patient may be greater than genotype B, d) Hepatitis flares
with C1858 HBV strains. On the other hand core promoter have been more frequently documented among genotype
mutation such as A 1762T and G1764A, associated with ‘C’ patients than in genotype B patients, and e) genotype B
reduced production of Pre core transcripts and reduced patients responds better to antiviral treatment in comparison
HBeAg production resulting in HBeAg -ve HBV infection to genotype C patients. Therefore initial reports suggest that
with replication have been well documented among genotype genotype ‘C’ infection may be associated with more severe
A HBV infection.5,6,18 liver disease with poor response to therapy than in patients
with genotype ‘B’ virus infection.
2. Recombination of genomes of distinct HBV Studies from Taiwan have documented that genotype
genotype and coinfection of HBV genotypes ‘C’ was significantly more prevalent among patients
Mixed infections of distinct genotype have been well with cirrhosis and hepatocellular cancer (HCC) among
recognized.6 During replication of HBV of each genotype population older than 50 years of age, in comparison to age-
in the same host, genome recombination of two or more matched asymptomatic carriers, whereas, genotype ‘B’ was
HBV genotype is possible. Indeed, recombination of B and documented more frequently among younger HCC patients
C,19 A and D,20 A and G21 have been reported. Interestingly, (age < 50 years) in comparison to age-matched asymptomatic
sites of recombination among genotype B and ‘C’ have been carriers.22 The latter patients (particularly < 35 years) did not
constant and located between nucleotides 1731 to 1838 and have cirrhosis.23 In Japan, where both genotype B and C are
2437 to 2486.6 These insertion sites belonged to Pre core / prevalent, genotype ‘C’ rather than genotype ‘B’ was found
Core region of HBV DNA genome.19 Further, more often to be associated more often with cirrhosis and HCC and
combination of core region of ‘C’ genotype with B genotype unlike Taiwan, none of the younger patients (<35 years)
have been described.19 One recent study19 which analysed 70 with HCC had genotype ‘B’ inection.24
HBV sequences from Japan and South east Asian countries, Reports on genotype A and D have predominantly been
reported that genotype ‘B’ prevalent in Japan was pure from Europe, USA and India. One report has documented
without any recombination, where as genotype B isolated preferential association of genotype D with acute hepatitis
from the latter countries had recombination of genotype ‘C’. and A with chronic hepatitis.24 Thakur et al from India

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Hepatitis B Virus Genotypes: Clinical Implications
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