Understanding the viral diversity of Hepatitis B virusin Saudi Arabia using Next- Generation Sequencing (NGS)Introduction Viral hepatitis is asystemic infection affecting mainly the liver and causing its inflammation.

Thecondition may be acute (rapid onset, short duration) or chronic (long-term).Viral hepatitis is caused by infection with one of the five identified hepatotropicviruses, 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 theirgenome organisation, structure, epidemiology, modes of transmission, incubationtime, clinical symptoms, diagnosis, control and prevention, and treatment(Gairy, 2007).HBV is a short-termillness that causes no permanent damage for most adults, but around 2 to 6percent of adults infected will develop a chronic infection that can possiblylead to other complications. Around 90 percent of newborns with the virus willdevelop chronic infection (Kathleen, 2017). The major complications of ChronicHepatitis B (CHB) infection are liver cirrhosis and hepatocellular carcinoma(HCC) (Te & Jensen, 2014).Statistics andgeographic distributionsCHB is a potentiallylife-threatening liver disease and represents a major global health problem.

Approximately two billion people have been exposed, and 240 million people arechronically infected with HBV. More than 780,000 people die every year due tothe consequences of hepatitis B (World Health Organisation, 2017). Hepatitis Bwas recognized as a disease in ancient times, but the virus itself wasidentified not until 1965 (Wolfram, 2013).

The prevalence of HBV in the worldis highly variable. The highest in the Western Pacific Region and the AfricanRegion, where 6.2% and 6.1% respectively of the adult population are infected.

The region of the Americas had the lowest number of infected individualsrepresenting 0.7% of the population. In the Middle East Region, South-East AsiaRegion and the European Region, an estimated 3.3%, 2.0% and 1.

6%% of thegeneral population are infected, respectively (World Health Organisation,2017).ClassificationThis virus belongs tothe genus Orthohepadnavirus of the Hepadnaviridae family and, along with theSpumaretrovirinae subfamily of the Retroviridae family, represents the onlyother animal virus with a DNA genome known to replicate by the reversetranscription of a viral RNA intermediate (Norder et. al., 2004). The HepatitisB Virus is a blood-borne virus and roughly 75 – 200 times more infectious thanHIV (Bowyer & Sim, 2000).Viral Genome andreplication cycleHBV is a partiallydouble-stranded DNA virus. The HBV genome is of around 3200 nucleotides inlength (Di Bisceglie AM, 2009) and it consists of a minus-strand, which spansthe full genome, and a plus-strand of DNA spanning roughly two-thirds of thegenome. Upon infection of the liver cells, the genome is transported to thenucleus and converted to covalently closed circular DNA (cccDNA) to produce adouble stranded genome which serves as a template for transcription by the hostcell RNA polymerase II(Bowyer and Sim, 2000).

Transcription of the template gives rise to pre-genomicmRNA and three sub –genomic mRNAs. The 3.5 kB pre-genomic mRNA is produced withredundant ends and is used for translation of the core protein and polymeraseenzyme. Following transport of the pre-genomic mRNA and translation in thecytoplasm, selective encapsidation of the RNA into the nucleocapsid ensues,along with encapsidation of RNA polymerase. In the immature capsid, the 3.5 kbmRNA is reverse transcribed, to yield the minus strand. The RNA is degraded andthe DNA strand is replicated, producing the second, shorter DNA strand.

Early inthe infection cycle, the cellular burden of viral DNA is amplified followingthe transportation of mature nucleocapsid to the nucleus in order forreplication to repeat. Viral surface proteins are produced at a later stage ofthe infection process, where sub-genomic mRNAs are produced and translated.Nucleocapsids containing DNA genomes then gain their outer envelope, possiblyby budding into the endoplasmic reticulum (ER) in areas where transmembraneHBsAg are inserted. The resulting Dane particles are then transported from thecell by normal pathways of vesicular transport. At that point the particles areassembled and released without cell lysis (Yen, 1993).HBV genome consists ofa 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 forthe viral polymerase, and S gene for the surface antigen, and the function ofthe 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 ofa long open reading frame that contains codons dividing the gene into threeseparate parts; preS1, preS2, and S. Because of the several start codons, threedifferent 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 mainvariable sequences of the viral genome.

HBV variants with point mutations anddeletions 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 cellreceptors and facilitating virus entry. It includes 226 amino acid sequence andcontains an antigenic structure positioned between amino acids 99 and 169,which is called “Major Hydrophilic Region (MHR).” The amino acid configurationof this region mainly decides the HBV subtype.

Moreover, a region betweenresidues 124 and 147 locates within MHR, and named “a” determinant, isdescribed as major target for neutralizing antibodies (Al-Qudari et. al., 2016).There are ten differentgenotypes of HBV. The classification of these genotypes is based on geneticdistance. These genotypes (A-J) differ by? <7.

5 percent genomic sequencediversity. HBV is further classified into 40 different subtypes (separated by>4 percent genomic sequence diversity) (Pourkarim et. al., 2014).Virologist hadencounter difficulties due to misclassifications and lack of standardization ofgenotyping methods and whether the comparable sequences represent whole genomesequence or only S gene sequence. Principles forassigning a new subgenotype include: i) comparison of whole genome sequences,ii) the genetic distance with inter-genotypic pairwise distance >7.5% andintra-genotypic pairwise distance >4.

5%, iii) well-supported phylogenetictree with bootstrap values of more than 75%, iv) Identification of anyrecombination events, v) identification of fingerprints in the genome sequenceincluding amino acid motifs, vi) presence of three clinical isolatesrepresenting the new strain, and vii) validation of all new subgenotype strainsby the evolutionary and phylogeny analysis (Pourkarim et. al., 2014).TransmissionsHBV can survive outsidethe body for at least 7 days. The virus can still be the source of infectionduring this time if it enters the body of a person who is not vaccinated. The periodof 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 anddevelop into CHB (World Health Organisation 2017). HBV is transmitted throughcontact with infected body fluids and human is the only natural host. Blood isthe main mode of transmission, but other body fluids have also been involved,including saliva and semen. At this time, only three modes of HBV transmissionhave been documented: perinatal, sexual and parenteral/percutaneoustransmission.

There is no reliable indication that airborne infections arise(Jinlin, Zhihua, and Fan, 2005).TreatmentsUp to now, seven drugshave been affected as a treatment for CHB. These agents are injectableinterferon alpha (INF- ?), pegylated interferon (PEG-INF-?), and the orallamivudine, entecavir, adefovir dipivoxil, telbivudine and tenofovir (Ayoub& Keeffe, 2011).PreventionHepatitis B vaccinationis routinely available; it consists of a yeast-derived recombinant HBsAgprotein. It is effective at producing protection in up to 95 percent ofimmunocompetent recipients. The hepatitis B vaccine is recommended for allinfants at birth and for children up to 18 years.

The vaccine is alsorecommended for adults living with diabetes and those at high risk forinfection due to their jobs and lifestyle. Three doses are generally requiredto complete the hepatitis B vaccine series. Up till now more than one billiondoses of the hepatitis B vaccine have been given worldwide (Hepatitis BFoundation, 2017).

Current status of HBVinfections in Saudi Arabia and Problem StatementsChronic HBV infectionin Saudi Arabia is a significant problem, which justifies a concerted multi-disciplinaryresearch effort to address key questions relating to the epidemiology,pathogenesis, natural history, and treatment of the disease.In Saudi Arabia, the seroprevalence of HBsAg amongSaudi children was reportedly 7%, and greater than 70% prevalence of at leastone HBV marker of the screened children in a published epidemiological study in1988 (Abdo & Sanai, 2015). This study activated a nationwide responseresulting in the introduction of a national universal HBV immunization in1989.  A catch-up vaccination programfollowed for children at school entry, healthcare workers, and other high-riskgroups.

The national efforts resulted in high vaccination uptake percentagewith almost all Saudis aged 27 years or younger had been vaccinated either atbirth or school.In the last twodecades, there has been a significant decline in the frequency of HBV infectionin Saudi Arabia (Bashawri et. al.

, 2004). In this study, it was found that theincidence 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 andmolecular characterization of HBV is understudied in the Middle East. There isa lack of studies characterizing the circulating variants in Saudi Arabia andoccurrence of antiviral resistance-associated mutations.

The available data onthe dominant genotype in the Kingdom is based on sequencing of S gene ratherthan whole genome sequences. However, data covering the prevalence ofresistance-associated mutations in treatment-naïve patients are limited.In Saudi Arabia, thepredominant HBV genotypes are genotype D and E. The awareness of hepatitis Bvirus serologic and genotypic patterns would improve the disease managementplans and a plan for eradication of HBV infection at the national level (Asaadet.

al., 2015).Phyloepidemiology ofHBV is an essential step toward the eradication of HBV in Saudi Arabia as thegenotype and subgenotype of HBV are related to the clinical outcome,transmission network, and treatment outcome (Bui et. al.

, 2017). HBV genotypingis not a routine diagnostic test in the microbiology laboratories due tolimited sequencing facilities and inaccuracy of available technologies. Thereis a recent drive globally to identify circulating variants in the populationto improve clinical decision making, including treatment guidelines, preventionstrategies via disease modelling, and health resource allocation for themanagement of chronic HBV patients (Bui et al, 2017).The diagnosis of HBV infection is based mainly on thedetection of HBsAg. However, the presence of a mutation in the S gene had aprofound effect on the diagnostic sensitivity of the current serological tests,mainly in the “a” determinant, basically, this might lead to false-negativeresults. The antigenic alterations may also help the virusescape the host immune system (Bernard, 2005). This is another indication for studies tounderstand 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 geographicalregions within the same country (Al-Qudari et. al.

, 2016).Despite the reporteddiversity of the “a” determinant, a conserved 17 amino acid residues wereidentified, in a Spanish study, in which, the gene fragment encoding the regionbetween amino acids 112–212 of S-HBsAg was sequenced from sera collected fromchronic HBV carriers (Avellon, 2006). The implication of thegenetic variability in the S gene is well described at both diagnostic andclinical levels. As the mutations lead to the emergence of immune escapevariants that are missed by the current diagnostic tests based on the detectionof HBsAg, which increases the number of false negatives in the laboratories. Report from a Spanishstudy in 2005 mentioned that substitution mutations in S region in HBV variantscauses diagnostic failure in 12.

5% of cases, the mutant variant is alsoresponsible for 6.6% of cases of invalid vaccination, and 9.2% escape fromimmunoglobulin therapy (Avellon, 2006). The mutations in the S gene cause changes in the conformational structureof HBsAg leading to diagnostic failure by the clinically-applied detectionassays. Diagnosis failure can result in an increased risk of post-transfusioninfection, consequently leading to a rise in HBV incidence (Allain, 2012).HBV circulates as aquasispecies in the infected patient. Therefore, there are limitations indetecting resistance-associated variants using the most commonly utilizedmethods for identifying HBV resistance, namely Sanger sequencing that has poorsensitivity and only detects consensus sequence while missing populations ofless than 20% frequency.

The other used method is line-probe hybridizationassays, which are susceptible to hybridization errors.Next-generationsequencing (NGS) is superior to the routine diagnostic methods in understandingthe viral population with an enhanced ability to detect minority variants witha frequency as low as one percent (Abdelrahman et. al., 2015)NGS assays have been introducedin clinical laboratories in the last decade due to their ability to providecomprehensive and detailed information. The main application in clinicallaboratories has been molecular genetics to identify rare diseases andmalignancies.Outline of the ProjectThe introduction of NGStechnologies to the infectious diseases field had shed light on the intrahostviral population and enabled researchers to address several questions relatedto viral genomics and natural history of the disease. This study will provideone of the initial efforts worldwide to offer a better understanding of thedynamics of HBV circulating variants and link it to the epidemiology of theinfection in Saudi Arabia.

The aim of the projectis to understand the viral diversity of HBV in Saudi Arabia and development ofantiviral efficacy in vitro.In this study, we willdevelop protocols for HBV full genome sequencing using NGS technologies and usethis protocol to identify the circulating variants in Saudi Arabia anddetermine their antiviral resistance profile.The identifiedsequences will be used to generate a cell culture-based HBV infectious systemthat will be a base for downstream projects such as testing antiviral efficacyof clinically approved drugs against circulating variants. Furthermore, the invitro infection model will be used to test the antiviral efficacy of naturalpeptides 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 monoclonalantibodies (previously isolated in our laboratory) for their ability topotentially neutralize virus infection.Expected OutcomeThe expectedphyloepidemiology map will clarify the circulating HBV variants and enableunderstanding the network of transmission in the Kingdom. It will also enablemonitoring the evolution of HBV. The antiviral resistance profile of thecirculating variants will inform any planned national guidelines for themanagement of HBV.

The molecular characterization will introduce a geneticmodel for any future development of antiviral against HBV in Saudi Arabia, asthese variants could be introduced to any in vitro model for antiviralscreening studies. The study will enable us to establish a national HBVsequence database linked with relevant clinical data.

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