1

Research Report

epatitis B

Authors: Rizwan (2014-EE-323)

Rehan (2014-EE-325)
Omer (2014-EE-332)
Mubashir (2014-EE-338)
Dept. of Electrical Engineering, University
of Engineering and Technology, Lahore,
Pakistan

verview

I. Introduction
Hepatitis B (HBV) is a virus that
damages the liver. Please refer to Fig. 1 below.

Fig. 1 HBV viral particle

Adopted from [1]

The word hepatitis indicates the

presence of inflammation in the liver. Once this
inflammation begins, scar tissue (called
fibrosis) may form. In 20-30% of patients who
carry the hepatitis B virus, this scar tissue
leads to cirrhosis and/ or liver cancer.
II. Transmission
The virus is transmittable and can be
spread through:
Mother-to-child infection
Sexual contact
Blood-borne infection

Having a tattoo or body piercing done

with dirty tools that were used on
someone else
Sharing drug needles
Sharing a toothbrush or razor with an
infected person
Getting pricked with a bloodcontaminated needle or sharp object
Having a blood transfusion with blood
that has not been screened

III. Key facts

About 240 million people are
chronically infected with hepatitis B.
More than 780,000 people die every
year due to complications of hepatitis
B, including cirrhosis and liver
cancer.

IV. Geographical distribution

Hepatitis B prevalence is highest in
Sub-Saharan Africa and East Asia, where
between 510% of the adult population is
chronically infected.
High rates of chronic infections are
also found in the Amazon and the southern
parts of eastern and central Europe. In the
Middle East and the Indian subcontinent, an
estimated 25% of the general population is
chronically infected. Less than 1% of the
population in Western Europe and North
America is chronically infected. [2]
V. Symptoms
During acute infection phases, most of
symptoms are not visible. However, some
people have acute illness with symptoms that
last several weeks, including yellowing of the
skin and eyes (jaundice), dark urine,
extreme fatigue, nausea, vomiting and
abdominal pain. A small subset of persons
with acute hepatitis can develop acute liver
failure which can lead to death. [3]
HBV can also cause a chronic liver
infection that can later develop into cirrhosis
of the liver or liver cancer. More than 90% of
healthy infected adults of HBV will recover
naturally from the virus within the first year.
VI. Who is at risk for chronic disease?
The likelihood that infection with the
virus becomes chronic depends upon the age
at which a person becomes infected. Children
less than 6 years of age who become infected

2
with the hepatitis B virus are the most likely to
develop chronic infections.
In infants and children [4]:
8090% of infants infected during the
first year of life develop chronic
infections.
3050% of children infected before
the age of 6 years develop chronic
infections.
<5% of otherwise healthy persons
who are infected as adults will
develop chronic infection.
2030% of adults who are chronically
infected will develop cirrhosis and/or
liver cancer.
VII. Diagnosis
Laboratory diagnosis focuses on the
detection of the hepatitis B surface antigen
HBsAg [5]. Blood donations are tested for
hepatitis B to ensure blood safety and avoid
accidental transmission to people who receive
blood products. [6]
Acute HBV infection is described by
the presence of HBsAg and
immunoglobulin M (IgM) [7] antibody
to the core antigen, HBcAg. During the
initial phase of infection, patients are
also seropositive for hepatitis B e
antigen (HBeAg). HBeAg is usually a
marker of high levels of replication of
the virus. The presence of HBeAg
indicates that the blood and body
fluids of the infected individual are
highly contagious.
Chronic infection is described by the
persistence of HBsAg for at least 6
months (with or without concurrent
HBeAg). Persistence of HBsAg is the
principal marker of risk for developing
chronic liver.
VIII. Treatment
Care is aimed at maintaining comfort
and adequate nutritional balance, including
replacement of fluids lost from vomiting and
diarrhoea [8]. Chronic hepatitis B infection
can be treated with drugs, including oral
antiviral agents.Treatment can slow the
progression of cirrhosis, reduce extent of liver
cancer and improve long term survival.
Oral treatments tenofovir [9] or
entecavir [10] are recommended, because
these are the most potent drugs to suppress
hepatitis B virus.
They rarely lead to drug resistance as
compared with other drugs, are simple to take

(1 pill a day), and have few side effects so

require only limited monitoring.
However, in most people, the
treatment does not cure hepatitis B infection,
but only suppresses the replication of the
virus. Therefore, most people who start
hepatitis B treatment must continue it for life.
[11].
Treatment using interferon (an
antiviral protein produced by cells that have
been invaded by a virus; inhibits replication of
the virus) injections may be considered in
some people in certain high income settings,
but its use is less feasible in lower source
settings due to high cost and significant
adverse effects requiring careful monitoring.
There is still limited access to
diagnosis and treatment in many resource
constrained settings, and many people are
diagnosed only when they already have
advanced liver disease. Liver cancer
progresses rapidly, and since treatment
options are limited, the outcome is in general
poor. In low income settings, most people with
liver cancer die within months of diagnosis. In
high income countries, surgery and
chemotherapy
(Chemotherapy
(also
called chemo) is a type of cancer
treatment
that
uses
drugs
to
destroy cancer cells) can prolong life for up to
a few years. In high income countries, liver
transplantation is sometimes used in people
with cirrhosis, with varying success.

IX. Prevention
Vaccine is the mainstay of prevention.
All infants should receive the hepatitis B
vaccine as soon as possible after birth,
preferably within 24 hours. The birth dose
should be followed by 2 or 3 doses to complete
the primary series. In most cases, one of the
following two options is considered
appropriate:
A 3 dose schedule of hepatitis B
vaccine, with the first dose
(monovalent) being given at birth and
the second and third (monovalent or
combined vaccine) given at the same
time as the first and third doses of
diphtheria,
pertussis
(whooping
cough), and tetanus (DTP) vaccine.
[12]
4 doses, where a monovalent birth
dose is followed by three monovalent
or combined vaccine doses, usually

3
given with other
vaccines. [13]

routine

infant

The complete vaccine series induces

protective antibody levels in more than 95% of
infants, children and young adults. All children
and adolescents younger than 18 years old and
not previously vaccinated should receive the
vaccine if they live in countries where there is
low or intermediate endemicity. In those
settings it is possible that more people in high
risk groups may acquire the infection and they
should also be vaccinated. They include [14]:
People who frequently require blood
or blood products, dialysis patients,
recipients
of
solid
organ
transplantations.
People interned in prisons.
Persons who inject drugs.
Household and sexual contacts of
people with chronic HBV infection.
People with multiple sexual partners.
Health-care workers and others who
may be exposed to blood and blood
products through their work.
Travelers who have not completed
their hepatitis B vaccination series,
who should be offered the vaccine
before leaving for endemic areas.
In addition, implementing of blood
safety strategies, including quality assured
screening of all donated blood and blood
components used for transfusion, can prevent
transmission of HBV. Safe injection practices,
eliminating unnecessary and unsafe injections,
can be effective strategies to protect against
HBV transmission. Furthermore, safer sex
practices, including minimizing the number of
partners and using barrier protective
measures (condoms), also protect against
transmission.
X. Types and Phases of Hepatitis B
Infection
Chronic Hepatitis B:

Fig. 2 Photomicrograph of the liver with

chronic hepatitis B.
Adopted from [15]

This refers to active infection with

elevated liver enzymes and/ or inflammation
shown on ones liver biopsy. Individuals with
active hepatitis B are at risk for progressive
liver damage resulting in cirrhosis and are at
highest risk for developing liver cancer. Please
refer to Fig. 2.
Immune Tolerance:
Historically,
individuals
were
identified as silent carriers [16] or healthy
carriers if they had persistently normal liver
enzymes and a normal liver biopsy. Currently,
the term used is an immunotolerant patient
[17] or carrier if one is asymptomatic, has
persistently normal liver and healthy
enzymes, elevated HBV DNA and a normal liver
biopsy.
Inactive Carrier:
These individuals have undetectable
virus by PCR [18] and normal (healthy) liver
enzyme levels (an ALT less than 20 for women
and less than 30 for men at most testing sites).
These patients have a lower risk of liver
disease progression.

ioinformatics Tools

I. Introduction
Bioinformatics is the application of
computer science, statistics and information
theory to the field of biology and medicine. As
with most biosciences, bioinformatics has
become an integral part of virology research in
recent years. Numerous bioinformatics tools
are available for general analyses of viral
genomes, such as multiple nucleotide/amino
acid sequence alignment, motif identification,
recombination, genome annotation and
phylogenetic relationships. [19]
II. Bioinformatics tools and public
databases for HBV
The HBV genome is a partially doublestranded circular DNA molecule that is
approximately 3,200 nucleotides in length.
The genome has a compact gene layout, and is
composed
of
4
overlapping
ORFs
(polymerase, envelope, core and X) so that the
total coding capacity is approximately 1.5 the
length of the genome. [20]
CDS-Plotcon [21] is a tool developed
for the detection and viewing of conserved
functional elements within coding sequences,

4
and it allows a user to select the feature
annotations to view in conjunction with the
local conserved regions identified (for
example, the overlapping region where the
regulatory element of interest is located).
The second tool is Alidot [22] from
Vienna RNA for the detection of conserved
RNA secondary structures within the four
groups of HBV genome sequences. Similar to
CDS-Plotcon, a user can select the type of
feature annotations to view in conjunction
with the local RNA secondary structures
identified, and to determine whether the
regulatory element of interest contains
conserved RNA secondary structures. Overall,
this database is a useful initial resource for
research scientists who study HBV molecular
biology and regulation of gene expression from
the HBV genome.
The HepSEQ [23] Research Database
System is a molecular, clinical and
epidemiological database for hepatitis B
infections that is useful for researchers
interested in such analyses.
HepSEQ has three sequence analysis
tools: SeqMatcher, Genotyper and Polymerase
Annotator. SeqMatcher uses a similarity
search method to identify sequences identical
or near identical to the query sequence in the
database. The resultant list of sequences is
presented in a tabular format that would
enable the user-input sequence to be linked to
potentially related cases. The Genotyper tool
was developed to determine the genotype of
an
input
sequence
containing
the
surface/polymerase genes of HBV. This tool
performs pairwise comparisons between the
input sequence, and each of the reference
sequences to identify those with >98%
sequence similarity. The HBV genotype of the
input sequence is considered equivalent to the
reference sequence with the highest similarity
scores. Finally, the Polymerase Annotator
tool was developed to predict the HBV
genotype, and to identify clinically significant
amino acid substitutions related to vaccine
escape or antiviral resistance.
Another web-accessible sequence
analysis tool dedicated for HBV drug
resistance analysis is the Geno2pheno [24] for
HBV. This tool accepts DNA sequences
containing the Pol/Rt region of HBV to
determine HBV genotype and sub genotype of
the input sequence along with confidence
level, identify the HBV Pol/Rt and HBsAg

codons covered by the input sequence, align to

the local HBV genotype D consensus sequence
or a consensus sequence of the respective HBV
genotype, identify a list of HBV Pol/Rt and
HBsAg amino acid substitutions following
comparison with the consensus reference
sequence,
and
predict
phenotypic
susceptibility to the five anti-HBV drugs.
Similar analyses can also be performed on
other regions of the HBV genome, such as the
basal core promoter and pre core regions,
large surface protein, middle surface protein
and core protein, in order to identify clinically
important mutations or amino acid
substitutions. A unique feature of this HBV
sequence analysis tool is its ability to identify
and report potential mixed HBV genotype or
subtype infections based on the single letter
ambiguous base codes present in the input
sequence.
SeqHepB [25] is an HBV sequence
analysis tool that was developed in the early
era of HBV antiviral therapy. The goals were to
provide timely and accurate drug resistance
prediction, to identify basal core promoter and
pre core mutations, and to aid in the
management of patients with chronic hepatitis
B. This sequence analysis tool is able to analyze
full-length HBV genome sequences starting
from the terminal protein domain of the
polymerase ORF, and it can automatically
identify all the overlapping genomic regions
covered by the HBV sequence submitted for
analysis.

atasets for HBV

I. B-Cell Gene Signature in HBV-Associated

Acute Liver Failure
Background: The dramatic clinical course of
acute liver failure has posed major limitations
for pathogenesis studies. Gene expression
profiling is used to establish a molecular
definition of hepatitis B virus (HBV)associated acute liver failure.
Methods: Two patients who underwent liver
transplantation for HBV-associated acute liver
failure were studied. Gene expression profiling
was performed on 8 liver specimens obtained
from the two patients with acute liver failure
(4 samples per liver) and individual liver
specimens from 8 liver donors. Statistical

5
analyses were used to identify the signature
genes of HBV-associated acute liver failure.
Results: Multivariate permutation analysis
identified 1,368 transcripts that were
differentially expressed in acute liver failure;
709 were up-regulated and 659 downregulated. The most represented up-regulated
transcripts were those involved in the immune
response, whereas the most abundant downregulated transcripts were those involved in
metabolism and hepatic synthesis. Acute liver
failure was characterized by an overriding Bcell signature comprising genes related to
mature B cells and plasma cells with abundant
polyclonal expression of immunoglobulin
genes. By contrast, there was a limited T-cell
signature and up-regulation of several
inhibitors
of
T-cell
activation.
Immunohistochemical analysis confirmed
the prominent B-cell signature showing diffuse
liver infiltration by plasma blasts and plasma
cells with strong cytoplasmic staining for IgM
and IgG, associated with a significant
deposition of complement factors.
Conclusion: The presence of a prominent
intrahepatic plasma-cell infiltrate with ectopic
immunoglobulin production and complement
deposition suggests a pivotal role of humoral
immunity in the pathogenesis of acute liver
failure associated with HBV infection. [26]

Dataset for HBV

GSM366072
GSM366071
GSM366070
GSM366069
GSM366068
GSM366067
GSM366066
GSM366065
GSM366064
GSM366063
GSM366062
GSM366061
GSM366060
GSM366059
GSM366058
GSM366057
0

50

100

150

200

250

II. Structure Determination of Hepatitis B

virus core particles
Report: Hepatitis B is human pathogen of
major importance. There are estimated to be
300 million carriers worldwide. Chronic
infection usually leads to cirrhosis and cancer
of the liver. Hepatitis B virus is a small
enveloped DNA virus. The protein capsid is
made up of a single particle (HBcAg). When
expressed in E. coli, the protein assembles into
shells like those in native vu-ions. Cryoelectron
microscopy has been used to study these viral
capsids, and has shown that there are two sizes
of particle, one with 180 copies of HBcAg
arranged with T=3 icosahedral symmetry and
the other with 240 copies and T=4 symmetry
(Crowther et al.). We wish to determine the
atomic structure of one or both of these viral
shells, as part of a more general program
aimed at understanding the molecular biology
of the whole virus. This in turn may provide a
basis for the design of therapeutic agents that
block viral assembly. [27]
Methods: Crystals of the T=4 protein capsids
using material expressed in E. coli were grown.
Depending on the exact crystallization
conditions, some crystals are triclinic (cell
285x337x396& a=91, p=90, F93) while others
are monoclinic (P21, cell 293x341x38781,
p=95), or when grown in the presence of
cryoprotectants the space group is C2, cell
538x353x370& p=132.30. Diffraction to 7.5A
resolution is observed using a laboratory
source.
Results: Diffraction from the unfrozen crystals
at BL4 was significantly worse than the
diffraction observed at the SRS, Daresbury.
Most crystals only diffracted to 8A resolution,
while the best diffracted to 5A. In addition, the
crystal lifetime was much shorter, allowing
only a single (10 second) exposure per crystal
while several exposures per crystal had been
possible at the SRS. It is possible that
temperature or pressure changes during
transport to Grenoble (by air in hanging drop
trays) may have produced the deterioration in
crystal quality.
However crystals grown in the
presence of the cryoprotectants butane diol
and flash frozen at 1OOK in the N2 gas stream
diffracted to between 3.6A and 3.9A resolution.
It was possible to collect a complete dataset to
a nominal resolution of 3.6A from four crystals,

6
using up to 3 positions per crystal. The overall
Rmerge was 16.2% (60% at 3.6A) and the
mean F/o(F) was 11.6 (4 at 3.6A). There was
significant radiation damage, and translating
the crystals did not restore the original quality
of the diffraction, even if the two positions
were separated by several times the size of the
incident beam. However this level of radiation
damage was tolerated in order to be able to
collect a complete dataset within the time
available. A low resolution (8A) dataset was
subsequently collected in house in order to
improve the completeness of the synchrotron
dataset at low resolution. The final dataset was
96% complete to 3.6A.
The 7.5A resolution structure
determined by cryo electron-microscopy
(Bottcher et al) was used as an initial phasing
model, after determining the particle
orientation from a self-rotation function. This
model gave an excellent fit (R-factor 22%) to
the 7.581 resolution X-ray data. There is one
half of the virus capsid in the crystallographic
asymmetric subunit, and the 30-fold noncrystallographic symmetry was used to extend
the phasing to 3.6A. An atomic model for the
four icosahedrally independent subunits has
been built into the resulting electron density
map. This has largely confirmed the protein
fold proposed on the basis of the 7.5A cryo
electron-microscopy. The path of the
polypeptide backbone is unambiguous,
although there are a few regions where the
exact main chain conformation is not clear.
[28]

References
[1] Disease and Treatment Profile: Hepatitis B by
Stewart Cooper, Timothy Davern.
[2] Advances in the Molecular Diagnosis of Hepatitis B
Infection by Julianne Bayliss
[3] Epidemiology and Prevention of VaccinePreventable Diseases by William Atkinson
[4] http://www.mayoclinic.org/diseasesconditions/hepatitis-b/basics/risk-factors/con20022210
[5] Nguyen T, Thompson AJ, Bowden S, et al. Hepatitis
B surface antigen levels during the natural history of
chronic hepatitis B: a perspective on Asia. J Hepatol
2010;52(4):508513
[6] Deguchi M, Yamashita N, Kagita M, et al.
Quantitation of hepatitis B surface antigen by an
automated
chemiluminescent
micro
particle
immunoassay. J Virol Methods 2004;115(2):217222
[7] Chan HL, Thompson A, Martinot-Peignoux M, et al.
Hepatitis B surface antigen quantification: why and

how to use it in 2011a core group report. J Hepatol

2011;55(5):11211131
[8]http://www.nhs.uk/Conditions/Diarrhoea/Pages/
Introduction.aspx
[9]https://www.nlm.nih.gov/medlineplus/druginfo/
meds/a602018.html
[10] http://www.drugs.com/cdi/entecavir.html
[11] Rijckborst V, Hansen BE, Cakaloglu Y, et al. Early
on-treatment prediction of response to peginterferon
alfa-2a for HBeAg-negative chronic hepatitis B using
HBsAg and HBV DNA levels. Hepatology
2010;52(2):454461
[12] Ascherio A, Zhang SM, Hernan MA, et al. Hepatitis
B vaccination and the risk of multiple sclerosis. N Engl
J Med 2001;344:32732
[13] Institute of Medicine. 2002. Immunization Safety
Review: Hepatitis B Vaccine and Demyelinating
Neurological Disorders. Washington D.C. The National
Academy Press.
[14] Lewis E, Shinefield HR, Woodruff BA, et al. Safety
of neonatal hepatitis B vaccine administration. Pediatr
Infect Dis J 2001;20:104954.
[15] Disease and Treatment Profile: Hepatitis B by
Stewart Cooper, Timothy Davern.
[16][17] Nishijima N, Marusawa H, Ueda Y, et al.
Dynamics of hepatitis B virus quasispecies in
association with nucleos(t)ide analogue treatment
determined by ultra-deep sequencing. PLoS ONE
2012;7(4):e35052
[18][19][20] Yan Q. Bioinformatics databases and
tools in virology research: an overview. In Silico Biol
2008; 8:7185.
[21] CDS-plotcon - a.k.a. MLRGD Andrew E. Firth
[22]http://citeseerx.ist.psu.edu/viewdoc/summary?d
oi=10.1.1.511.7510
[23] Gnaneshan S, Ijaz S, Moran J, Ramsay M, Green J.
HepSEQ: international public health repository for
hepatitis B. Nucleic Acids Res 2007; 35:D367D370.
[24] J.R. Quinlan, C4.5: Programs for Machine Learning,
Morgan Kaufmann, San Francisco, 1993.
[25] Zoulim F, Locarnini S. Hepatitis B virus resistance
to nucleos(t)ide analogues. Gastroenterology.
[26]http://biogps.org/#goto=genereport&id=1017&s
how_dataset=E-GEOD-14668
[27] B. Bottcher, S.A.Wynne and R.A. Crowther (1997)
Determination of the fold of the core protein of
hepatitis B virus by electron cryomicroscopy. Nature
386, 88-91.
[28] R.A. Crowther, N.A. Kiselev, B. Biittcher, J.A.
Berriman, G.P. Borisova, V. Ose and P. Pumpens.
(1994) Three-dimensional structure of hepatitis B
virus core particles determined by electron
microscopy. Cell, 21, 943-950.