Understanding (Te & Jensen, 2014). Statistics and geographic

Understanding the viral diversity of Hepatitis B virus
in Saudi Arabia using Next- Generation Sequencing (NGS)

Introduction

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Viral hepatitis is a
systemic infection affecting mainly the liver and causing its inflammation. The
condition may be acute (rapid onset, short duration) or chronic (long-term).
Viral hepatitis is caused by infection with one of the five identified hepatotropic
viruses, which are named as hepatitis A virus (HAV), hepatitis B virus (HBV),
hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV),
respectively (Gairy, 2007). These viruses differ from each other in their
genome organisation, structure, epidemiology, modes of transmission, incubation
time, clinical symptoms, diagnosis, control and prevention, and treatment
(Gairy, 2007).

HBV is a short-term
illness that causes no permanent damage for most adults, but around 2 to 6
percent of adults infected will develop a chronic infection that can possibly
lead to other complications. Around 90 percent of newborns with the virus will
develop chronic infection (Kathleen, 2017). The major complications of Chronic
Hepatitis B (CHB) infection are liver cirrhosis and hepatocellular carcinoma
(HCC) (Te & Jensen, 2014).

Statistics and
geographic distributions

CHB is a potentially
life-threatening liver disease and represents a major global health problem.
Approximately two billion people have been exposed, and 240 million people are
chronically infected with HBV. More than 780,000 people die every year due to
the consequences of hepatitis B (World Health Organisation, 2017). Hepatitis B
was recognized as a disease in ancient times, but the virus itself was
identified not until 1965 (Wolfram, 2013). The prevalence of HBV in the world
is highly variable. The highest in the Western Pacific Region and the African
Region, where 6.2% and 6.1% respectively of the adult population are infected.
The region of the Americas had the lowest number of infected individuals
representing 0.7% of the population. In the Middle East Region, South-East Asia
Region and the European Region, an estimated 3.3%, 2.0% and 1.6%% of the
general population are infected, respectively (World Health Organisation,
2017).

Classification

This virus belongs to
the genus Orthohepadnavirus of the Hepadnaviridae family and, along with the
Spumaretrovirinae subfamily of the Retroviridae family, represents the only
other animal virus with a DNA genome known to replicate by the reverse
transcription of a viral RNA intermediate (Norder et. al., 2004). The Hepatitis
B Virus is a blood-borne virus and roughly 75 – 200 times more infectious than
HIV (Bowyer & Sim, 2000).

Viral Genome and
replication cycle

HBV is a partially
double-stranded DNA virus. The HBV genome is of around 3200 nucleotides in
length (Di Bisceglie AM, 2009) and it consists of a minus-strand, which spans
the full genome, and a plus-strand of DNA spanning roughly two-thirds of the
genome. Upon infection of the liver cells, the genome is transported to the
nucleus and converted to covalently closed circular DNA (cccDNA) to produce a
double stranded genome which serves as a template for transcription by the host
cell RNA polymerase II
(Bowyer and Sim, 2000). Transcription of the template gives rise to pre-genomic
mRNA and three sub –genomic mRNAs. The 3.5 kB pre-genomic mRNA is produced with
redundant ends and is used for translation of the core protein and polymerase
enzyme. Following transport of the pre-genomic mRNA and translation in the
cytoplasm, selective encapsidation of the RNA into the nucleocapsid ensues,
along with encapsidation of RNA polymerase. In the immature capsid, the 3.5 kb
mRNA is reverse transcribed, to yield the minus strand. The RNA is degraded and
the DNA strand is replicated, producing the second, shorter DNA strand. Early in
the infection cycle, the cellular burden of viral DNA is amplified following
the transportation of mature nucleocapsid to the nucleus in order for
replication to repeat. Viral surface proteins are produced at a later stage of
the infection process, where sub-genomic mRNAs are produced and translated.
Nucleocapsids containing DNA genomes then gain their outer envelope, possibly
by budding into the endoplasmic reticulum (ER) in areas where transmembrane
HBsAg are inserted. The resulting Dane particles are then transported from the
cell by normal pathways of vesicular transport. At that point the particles are
assembled and released without cell lysis (Yen, 1993).

HBV genome consists of
a condensed coding region that includes four overlapping genes labelled X, C,
P, and S respectively. C gene codes for the core protein (HBcAg), P gene for
the viral polymerase, and S gene for the surface antigen, and the function of
the protein coded by X gene is not yet well know (Xu et. al., 2000, Stuyver et.
al., 2008). Among these genes, S (surface antigen-encoding) gene is composed of
a long open reading frame that contains codons dividing the gene into three
separate parts; preS1, preS2, and S. Because of the several start codons, three
different sizes of polypeptides are produced, including Large (preS1 + preS2 +
S or L-HBsAg), medium (preS2 + S or M-HBsAg), and small (S or S-HBsAg)
(Al-Qudari et. al., 2016). The region of preS1 and preS2 appear to be the main
variable sequences of the viral genome. HBV variants with point mutations and
deletions in the preS sequences are commonly found in CHB (Pollicio et, al.,
2014). S-HBsAg is a main viral antigen playing a role in binding to cell
receptors and facilitating virus entry. It includes 226 amino acid sequence and
contains an antigenic structure positioned between amino acids 99 and 169,
which is called “Major Hydrophilic Region (MHR).” The amino acid configuration
of this region mainly decides the HBV subtype. Moreover, a region between
residues 124 and 147 locates within MHR, and named “a” determinant, is
described as major target for neutralizing antibodies (Al-Qudari et. al., 2016).

There are ten different
genotypes of HBV. The classification of these genotypes is based on genetic
distance. These genotypes (A-J) differ by? 4 percent genomic sequence diversity) (Pourkarim et. al., 2014).

Virologist had
encounter difficulties due to misclassifications and lack of standardization of
genotyping methods and whether the comparable sequences represent whole genome
sequence or only S gene sequence.

Principles for
assigning a new subgenotype include: i) comparison of whole genome sequences,
ii) the genetic distance with inter-genotypic pairwise distance >7.5% and
intra-genotypic pairwise distance >4.5%, iii) well-supported phylogenetic
tree with bootstrap values of more than 75%, iv) Identification of any
recombination events, v) identification of fingerprints in the genome sequence
including amino acid motifs, vi) presence of three clinical isolates
representing the new strain, and vii) validation of all new subgenotype strains
by the evolutionary and phylogeny analysis (Pourkarim et. al., 2014).

Transmissions

HBV can survive outside
the body for at least 7 days. The virus can still be the source of infection
during this time if it enters the body of a person who is not vaccinated. The period
of incubation of the virus is about 75 days but can differ from 30 to 180 days.
It may be identified within 30 to 60 days after infection and can persist and
develop into CHB (World Health Organisation 2017). HBV is transmitted through
contact with infected body fluids and human is the only natural host. Blood is
the main mode of transmission, but other body fluids have also been involved,
including saliva and semen. At this time, only three modes of HBV transmission
have been documented: perinatal, sexual and parenteral/percutaneous
transmission. There is no reliable indication that airborne infections arise
(Jinlin, Zhihua, and Fan, 2005).

Treatments

Up to now, seven drugs
have been affected as a treatment for CHB. These agents are injectable
interferon alpha (INF- ?), pegylated interferon (PEG-INF-?), and the oral
lamivudine, entecavir, adefovir dipivoxil, telbivudine and tenofovir (Ayoub
& Keeffe, 2011).

Prevention

Hepatitis B vaccination
is routinely available; it consists of a yeast-derived recombinant HBsAg
protein. It is effective at producing protection in up to 95 percent of
immunocompetent recipients. The hepatitis B vaccine is recommended for all
infants at birth and for children up to 18 years. The vaccine is also
recommended for adults living with diabetes and those at high risk for
infection due to their jobs and lifestyle. Three doses are generally required
to complete the hepatitis B vaccine series. Up till now more than one billion
doses of the hepatitis B vaccine have been given worldwide (Hepatitis B
Foundation, 2017).

Current status of HBV
infections in Saudi Arabia and Problem Statements

Chronic HBV infection
in Saudi Arabia is a significant problem, which justifies a concerted multi-disciplinary
research effort to address key questions relating to the epidemiology,
pathogenesis, natural history, and treatment of the disease.

In Saudi Arabia, the seroprevalence of HBsAg among
Saudi children was reportedly 7%, and greater than 70% prevalence of at least
one HBV marker of the screened children in a published epidemiological study in
1988 (Abdo & Sanai, 2015). This study activated a nationwide response
resulting in the introduction of a national universal HBV immunization in
1989.  A catch-up vaccination program
followed for children at school entry, healthcare workers, and other high-risk
groups. The national efforts resulted in high vaccination uptake percentage
with almost all Saudis aged 27 years or younger had been vaccinated either at
birth or school.

In the last two
decades, there has been a significant decline in the frequency of HBV infection
in Saudi Arabia (Bashawri et. al., 2004). In this study, it was found that the
incidence of HBV decreased from 4.7% to 1.6% from 2002-2005 to 2012-2015, respectively,
thus suggesting that the vaccine program was successful.

The epidemiology and
molecular characterization of HBV is understudied in the Middle East. There is
a lack of studies characterizing the circulating variants in Saudi Arabia and
occurrence of antiviral resistance-associated mutations. The available data on
the dominant genotype in the Kingdom is based on sequencing of S gene rather
than whole genome sequences. However, data covering the prevalence of
resistance-associated mutations in treatment-naïve patients are limited.

In Saudi Arabia, the
predominant HBV genotypes are genotype D and E. The awareness of hepatitis B
virus serologic and genotypic patterns would improve the disease management
plans and a plan for eradication of HBV infection at the national level (Asaad
et. al., 2015).

Phyloepidemiology of
HBV is an essential step toward the eradication of HBV in Saudi Arabia as the
genotype and subgenotype of HBV are related to the clinical outcome,
transmission network, and treatment outcome (Bui et. al., 2017). HBV genotyping
is not a routine diagnostic test in the microbiology laboratories due to
limited sequencing facilities and inaccuracy of available technologies. There
is a recent drive globally to identify circulating variants in the population
to improve clinical decision making, including treatment guidelines, prevention
strategies via disease modelling, and health resource allocation for the
management of chronic HBV patients (Bui et al, 2017).

The diagnosis of HBV infection is based mainly on the
detection of HBsAg. However, the presence of a mutation in the S gene had a
profound effect on the diagnostic sensitivity of the current serological tests,
mainly in the “a” determinant, basically, this might lead to false-negative
results. The antigenic alterations may also help the virus
escape the host immune system (Bernard, 2005). This is another indication for studies to
understand the circulating variants in Saudi Arabia, as there is a reported genetic diversity in the short frame of
“a” determinant (24 amino acids) of S-HBsAg even in different geographical
regions within the same country (Al-Qudari et. al., 2016).

Despite the reported
diversity of the “a” determinant, a conserved 17 amino acid residues were
identified, in a Spanish study, in which, the gene fragment encoding the region
between amino acids 112–212 of S-HBsAg was sequenced from sera collected from
chronic HBV carriers (Avellon, 2006).

The implication of the
genetic variability in the S gene is well described at both diagnostic and
clinical levels. As the mutations lead to the emergence of immune escape
variants that are missed by the current diagnostic tests based on the detection
of HBsAg, which increases the number of false negatives in the laboratories.

Report from a Spanish
study in 2005 mentioned that substitution mutations in S region in HBV variants
causes diagnostic failure in 12.5% of cases, the mutant variant is also
responsible for 6.6% of cases of invalid vaccination, and 9.2% escape from
immunoglobulin therapy (Avellon, 2006). The mutations in the S gene cause changes in the conformational structure
of HBsAg leading to diagnostic failure by the clinically-applied detection
assays. Diagnosis failure can result in an increased risk of post-transfusion
infection, consequently leading to a rise in HBV incidence (Allain, 2012).

HBV circulates as a
quasispecies in the infected patient. Therefore, there are limitations in
detecting resistance-associated variants using the most commonly utilized
methods for identifying HBV resistance, namely Sanger sequencing that has poor
sensitivity and only detects consensus sequence while missing populations of
less than 20% frequency. The other used method is line-probe hybridization
assays, which are susceptible to hybridization errors.

Next-generation
sequencing (NGS) is superior to the routine diagnostic methods in understanding
the viral population with an enhanced ability to detect minority variants with
a frequency as low as one percent (Abdelrahman et. al., 2015)

NGS assays have been introduced
in clinical laboratories in the last decade due to their ability to provide
comprehensive and detailed information. The main application in clinical
laboratories has been molecular genetics to identify rare diseases and
malignancies.

Outline of the Project

The introduction of NGS
technologies to the infectious diseases field had shed light on the intrahost
viral population and enabled researchers to address several questions related
to viral genomics and natural history of the disease. This study will provide
one of the initial efforts worldwide to offer a better understanding of the
dynamics of HBV circulating variants and link it to the epidemiology of the
infection in Saudi Arabia.

The aim of the project
is to understand the viral diversity of HBV in Saudi Arabia and development of
antiviral efficacy in vitro.

In this study, we will
develop protocols for HBV full genome sequencing using NGS technologies and use
this protocol to identify the circulating variants in Saudi Arabia and
determine their antiviral resistance profile.

The identified
sequences will be used to generate a cell culture-based HBV infectious system
that will be a base for downstream projects such as testing antiviral efficacy
of clinically approved drugs against circulating variants. Furthermore, the in
vitro infection model will be used to test the antiviral efficacy of natural
peptides and synthetic products to identify potential new treatment of HBV.
Such a model will also be used to test a panel of novel anti-preS1 monoclonal
antibodies (previously isolated in our laboratory) for their ability to
potentially neutralize virus infection.

Expected Outcome

The expected
phyloepidemiology map will clarify the circulating HBV variants and enable
understanding the network of transmission in the Kingdom. It will also enable
monitoring the evolution of HBV. The antiviral resistance profile of the
circulating variants will inform any planned national guidelines for the
management of HBV. The molecular characterization will introduce a genetic
model for any future development of antiviral against HBV in Saudi Arabia, as
these variants could be introduced to any in vitro model for antiviral
screening studies. The study will enable us to establish a national HBV
sequence database linked with relevant clinical data.

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