Transcript getics of hepatitis b

GENETICS OF HEPATITIS B
Dr. Osama Hasan
Othman
MBCHB,DM,CABM
FRCP LONDON
KIRKUK MEDICAL COLLEGE
KIRKUK
IRAQ
KIRKUK MEDICAL COLLEGE
KIRKUK
GENETICS = SECTRETES OF LIFE
(5) STATIONS IN HISTORY OF HBV
scientist
1. LURMAN
year
1885
DISCOVERY
SERUM HEPATITIS
2. BLUMBERG
( Nobel prize )
1965
AUSTRALIA ANTIGEN
3. DANE
1970
DANE PARTICLE E.M.
4.
5.
FREDERICK SANGER 1977
(Nobel prize) twice
P. VALENZUELLA
1985
GENOME SEQUENCING
REC. VACCINE DISCOVERY
• HBV GENOME STRUCTURE (5) features
1.The genome of HBV is made of circular DNA
2.It is unusual because the DNA is not fully doublestranded.
3.One end of the full length strand is linked to the
viral DNA polymerase.
4.The genome is 3020–3320 nucleotides long (for the full
length strand)
5.While less 1700–2800 nucleotides long (for the short
length strand).
Unique features of HBV
Although HBV small virion, many unique features make it distinctive
among DNA Viruses:
 It has a partially double stranded DNA with highly complex
genome organization, life cycle and natural history.
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It uses an RNA intermediate called pregenomic RNA (p g RNA)
and Reverse Transcriptase RT for its genome replication.
 Genome replication is accomplished by a complex mechanism of
primer shifting facilitated by direct repeat sequences encoded in the
genome.
 The genome has evolved in such manner that every single nucleotide
of the genome is used for either coding viral proteins or used as
regulatory regions or both.
ULTRASTRUCTURE OF HBV
Sketch of HBV
HVB AS A CIRCLE
GENOME VARIABILITY
 HVB utilizes internal in-frame translation
initiation codons & different reading frames from
the same RNA to generate different proteins
with diverse functions.
 HBV shows considerable genetic variability
which has been related with clinical outcomes,
replication potential, therapeutic responses.
COMPLETION OF VIRION
 The negative-sense, (non-coding) strand is
complementary to the viral mRNA
 The viral DNA is found in the nucleus soon after
infection of the cell.
 The partially double-stranded DNA is rendered
fully double-stranded by completion of the (+)
sense strand by viral polymerase and removal
of a protein molecule from the (-) sense strand
and a short sequence of RNA from the (+) sense
strand.
 Non-coding bases are removed from the ends of the (-)
sense strand and the ends are rejoined.
VIRAL TRANSCRIPTION
 The viral genes are transcribed by the cellular
RNA polymerase II in the cell nucleus from a
covalently closed circular DNA (ccc DNA)
template.
 Two enhancers designated enhancer I (Enh I)
and enhancer II (Enh II) have been identified.
Both enhancers exhibit greater activity in cells of
hepatic origin, they drive and regulate the
expression of the complete viral transcripts.
GENES OF GENOME
Four genes encoded by genome called C, P, S, and X.
The core protein is coded for by gene C (HBcAg), its start codon preceded by
an AUG start codon from which the pre-core protein produced.
HBeAg produced by proteolytic processing of pre-core protein
The DNA polymerase is encoded by gene P.
Gene S ,the gene that codes for the surface antigen (HBsAg).
The HBsAg gene has one long open reading frame, contains three in frame
"start" (ATG) codons that divide the gene into three sections, pre-S1, preS2, and S. Because of multiple start codons, polypeptides of three different
sizes called large, middle, and small (p re-S1 + pre-S2 + S, pre-S2 + S,
or S) produced. The function of the protein coded for by gene X is not fully
understood, but evidence suggests it may function as transcriptional trans
activator.
Several non-coding RNA elements have been identified in the HBV genome.
These include: HBV PRE alpha, HBV PRE beta and HBV RNA
encapsidation signal epsilon
GENOTYPES & SUBTYPES
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Genotypes differ by at least 10% of sequence and have
distinct geographical distributions , has been
associated with anthropological history.
Within genotypes subtypes have been described: these
differ by 5% of the genome.
There are eight known genotypes labeled A through H.
A possible new "I" genotype has been described
Two further genotypes have since been recognised.
The current (2014) listing now runs A though to J ,so (10)
genotypes
Several subtypes also recognized, there are (24)
subtypes.
Different genotypes may respond to treatment in
different ways
Geographical distribution of Individual
genotypes area: blue sky!
 Type F which diverges from other genomes by 14% the most
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divergent type known.
Type A is prevalent in Europe, Africa and South-east Asia,
including the Philippines.
Type B and C are predominant in Asia.
type D is common in the Mediterranean area, the Middle
East and India.
type E is localized in sub-Saharan Africa.
type F (or H) is restricted to Central and South America.
Type G has been found in France and Germany.
Genotypes A, D and F are predominant in Brazil and all genotypes
occur in the United States with frequencies dependent on ethnicity.
The E and F strains appear to have originated in aboriginal
populations of Africa and the New World, respectively
Further geographical divisions
 Type B hans two distinct geographical distributions: Bj/B1
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('j'—Japan) and Ba/B2 ('a'—Asia). Type Ba has been further
subdivided into four clades (B2–B4).
Type C has two geographically subtypes: Cs (C1) in Southeast Asia and C e (C2) in East Asia. The C subtypes have
been divided into five clades (C1–C5). A sixth clade (C6)
has been described in the Philippines but only in one
isolate to date.
Type C1 is associated with Vietnam, Myanmar and Thailand;
type C2 with Japan, Korea and China;
type C3 with New Caledonia and Polynesia;
C4 with Australia;
and C5 with the Philippines. A further subtype has been
described in Papua, Indonesia.
subdivisions
 Type D has been divided into 7 subtypes (D1–D7).
 Type F has been subdivided into 4 subtypes (F1–F4). F1
has been further divided into 1a and 1b.
In Venezuela subtypes F1, F2, and F3 are found in East
and West Amerindians.
 Among South Amerindians only F3 was found. Subtypes
I a, III, and IV exhibit a restricted geographic
distribution (Central America, the North and the South
of South America respectively) while clades I b and II
are found in all the Americas except in the Northern
South America and North America respectively.
Life cycle of HBV
 The life cycle of hepatitis B virus is complex. Hepatitis B is
one of a few known non-retroviral viruses which
use reverse transcription as a part of its replication
process.
1. Attachment: The virus gains entry into the cell by
binding to a receptor on the surface of the cell and enters
it by clathrin-dependent endocytosis. The cell surface
receptor has been identified as the Sodium/Bile acid
cotransporting pepetide SLC10A1 (also named NTCP).
2. Penetration: The virus membrane then fuses with the
host cell's membrane releasing the DNA and core
proteins into the cytoplasm.
LIFE CYCLE OF HBV
3. Uncoating: the virus multiplies via RNA made by host enzyme, the
viral genomic DNA transferred to the cell nucleus.
The capsid transported to microtubules via nuclear pore.
The core proteins dissociate from the partially double
stranded viral DNA & fully double stranded DNA formed and
transformed into covalently closed circular DNA (cccDNA) that
serves as a template for transcription of four viral mRNAs.
4. Replication: The largest mRNA, ( longer than the viral genome),
used to make the new copies of the genome and make
the capsid core protein viral DNA polymerase.
5. Assembly: These four viral transcripts undergo additional
processing and form progeny virions then released from the cell or
returned to the nucleus and re-cycled to produce even more copies.
6.Release: The long mRNA then transported back to the cytoplasm
where virion P protein synthesizes DNA via its reverse
transcriptase activity.
Genome Diversities Related to Clinical
Outcome
 Naturally occurring mutations on HBV genome are located at
the CORF. The dual BCP mutation A1762T + G1764A results
in a down regulation of HBeAg synthesis, and the PC
G1896A stop-codon mutation prevents the expression
of HBeAg.
 Another PC mutation A1899G has been reported in
combination with G1896A, which stabilizes the lower stem of
the “ϵ” encapsidation signal and enhances the replication.
The presence of both BCP and PC variants has been reported
in severe liver cirrhosis and HCC.
 Several mutations, including deletions and insertions or
stop codons have been reported in the SORF of the HBV
genome .
CLINICAL OUTCOME OF MUTATIONS
Deletions or missense mutations in Pre-S2 region
can abolish the synthesis of the protein and alter
B and T cell epitopes.
HBsAg insertion or deletion or missense
mutations can help the virus to evade host immune
response.
Truncated HBV surface proteins contribute to
chronicity of HBV.
a pattern of hotspot mutations located at the X region
of the HBV genome (xI127T, xK130M, xV131I and
xF132Y) are known to be associated with
transactivating function and HCC development.
Genome Variations Related to FHF
 It has been hypothesized that both viral and host
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factors play a role in the pathogenesis and clinical
outcome of HBV infection.
FHF develops when there is an overwhelming
immune-mediated lysis of infected hepatocytes
It has been suggested that FHB can be explained by
three main virologic markers :
(i) increase in viral replication fitness.
(ii) change in viral gene expression.
(iii) alteration of B and T cell epitopes .
mutations in overlapping envelope & polymerase genes of HBV
 1.The potential for (HBV) to alter its genome is
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considerable.
2.This potential occurs as the virus utilizes a reverse
transcription step in replicating it s genome.
3.Like HIV virus, the reverse transcriptase of HBV is error
prone and as a consequence of specific selection pressures
within a host a population of viral quasi species emerges.
4.HBV mutants with survival advantages over the wild
type virus appear within the selective in vivo environment.
5.Some of these viruses include HBV vaccine escape and
anti-viral resistant mutants that have changes in the
envelope (S) and polymerase genes, respectively
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Pre core & core GENE VARIATION & FHB
Many evidences suggest that FHB strongly associated
with HBV strains not producing HBeAg due to
BCP or PC mutations.
HBV DNA clones were propagated from FHB patients
and sequenced within the BCP and PC region.
Interestingly, a significant number of clones carried the
PC G1896A stop-codon and G1899A mutations &
dual BCP A1762T + G1764A mutation .
The precursor protein of HBeAg decreases the
encapsidation of the pregenomic RNA.
Sequences of mutations
Absence or decrease of HBeAg synthesis can lead
to enhanced viral replication and consequently
increased host immune response The double
A1762T + G1764A mutation can slightly increase
viral DNA replication
The C1766T + T1768A mutational pattern exhibits a
10-fold higher replication capacity than a wild
type strain .
Mutations can synergistically influence HBV
replication. For instance, BCP and PC can further
enhance the replication of G145R and
other“a-determinant” mutants
Overlapping mutations
The genome of HBV is organized in to overlapping reading
frames. The S gene is completely overlapped by the
polymerase gene. As a consequence, mutations in the gene
may produce changes in the overlapping polymerase
gene.
mutations in the polymerase gene may produce changes in the S
gene.
Certain mutations in either the S or polymerase gene produce
functionally significant changes in the respective overlapping
gene.
Treatment of chronic hepatitis B carriers with long-term
lamivudine (LMV) results in the selection of HBV
mutants that are resistant to this nucleoside analogue.
LMV resistance & changes in Genome
The polymerase mutations associated with LMV resistance produce changes
in the overlapping S gene and in its envelope protein ( HBsAg) that
results in a reduced antigenicity of the HBsAg protein.
The selection of vaccine escape mutants by HBV vaccination or
hepatitis B immune globulin is associated with changes in the S gene
that are accompanied by mutations in the fingers sub-domain
of the polymerase protein.
When combined with polymerase mutations that are associated with
resistance to LMV the changes within the fingers sub-domain of the viral
enzyme behave as compensatory mutations that are able to restore the
replication of LMV resistant HBV.
The ability to change a viral protein by mutations in an
overlapping but unrelated viral gene may produce HBV mutants
with altered antigenicity and/or replication and a natural
history that may be distinctly different to wild type HBV.
Genotypes & pathogenesis + outcome
 As HBV genotypes and sub-genotypes have distinct
geographical distributions, abundant evidence has shown
that different genotypes and sub-genotypes are
associated with distinctive pathogenesis and
outcomes of HBV infection.
 Generally, HBV genotype C has been associated with an
increased risk of liver inflammation, flares of
hepatitis, liver fibrosis and HCC.
 Genotypes D, C, and F1 are more likely to develop
complications such as liver cirrhosis and HCC.
 HbeAg seroconversion occurred much later in
patients infected with genotype C compared to other
genotypes.
MUTATIONS IN SPECIFIC SITES
Mutations in the pre S/S region are associated with:
1.vaccine failure
2.immune escape
3.occult HBV infection
4. HCC
Mutations in the P region may cause drug resistance
to NA antivirals.
Mutations in the preC/C region are related to HBeAg
negativity, immune escape, and persistent hepatitis.
Mutations in the X region play critical roles in promoting
HCC.
Clinical significance of mutations
Surface gene mutations were initially noted as
vaccine escape mutants in HBV endemic regions
receiving HBV immuno prophylaxis at birth.
These mutations have subsequently been noted in
patients after liver transplantation who
exhibit HBV recurrence despite receiving
hepatitis B immune globulin ( immune
escape mutant).
Surface gene mutations in the pre-S2 region
have been associated with fulminant hepatitis
Mutation & disease activity
Mutations in the precore, core, and surface
region have been associated with a broad range of
disease activity, varying from asymptomatic HBV
carriage to fulminant hepatitis.
Mutations in the basal core promotor have
been associated in vitro with enhanced viral
replication.
Detailed genomic studies have sought to correlate
distinct mutations with enhanced virulence,
however, it remains difficult to dissect the role
of viral versus host factors in this issue.
Polymerase variants & Antivirals
A variety of polymerase mutations have been shown after
HBV therapy with antiviral nucleosides inhibitors.
Treatment with either lamivudine or famciclovir has been
associated with the development of polymerase
variants in HBV isolates.
Most patients developing polymerase YMDD variants
continue to derive clinical benefit from prolonged
lamivudine therapy, which may be secondary to the
diminished viral replication conferred by these nucleotide
changes.
Patients on extended lamivudine therapy harboring these
variants may be identified by the presence of persistent
HBeAg and moderate elevations of ALT and DNA..
THANK YOU Dr. Gene ..