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Globally, Hepatitis B virus infection is the 10th main reason of death. (Shakeri et al., 2013)
The Hepatitis B virus infection is the major worldwide health issue.(Eke, Eke, Okafor, Ezebialu, & Ogbuagu, 2011)The severe and persistant consequences of HBV infection is said to be a main public health issue in the US , the annual infections estimated to be 200K to 300K happening in the past two decades.(McQuillan et al., 1999). Hepatitis B virus infection is more communicable disease than HIV and HCV infection. It is 50 – 100 times more infectious than HIV and 10 times more infectious than hepatitis C virus. HBV is a silent killer disease of the liver with many carriers not realizing that they are infected with the virus . It is estimated that 400 million persons have been infected with HBV all over the world . Annually over a half million deaths results because of cirrhosis and hepatocellular carcinoma, occurred as a result of long term chronic hepatitis B infection.(Caliendo et al., 2011) Even though the secure and efficient vaccine is available in the market for the couple of decades, the HBV spreads all around the world. (Custer et al., 2004). According to Anna Ruggieri and Walter Malroni, more than 240 million people are at the threat of severe infection such that cirrhosis and hepatocellular carcinoma(Ruggieri, Malorni, & Ricciardi, 2016). HBV infection is significantly creating serious health problems and the new heath cases still being recorded every year(Eke et al., 2011).In the African and Asian continents, HBV is the main cause of illness and death. Every year, approximately 1 million of infected people die because of the effects of the illness such as HCC and liver cirrhosis(Eke et al., 2011).Pakistan carries one of the world’s highest burdens of chronic hepatitis and mortality due to liver cirrhosis and HCC.Viral Hepatitis is widespread public health issue in Pakistan(Umer ; Iqbal, 2016). In Pakistan, where a majority of the people are below the poverty line, the incidence of hepatitis is very high. It is the frequent cause of death in the country’s population. Sufferers are usually neglected by society. According to some rough estimates, the hepatitis-infected population is around 15 million 5. Health professionals and officials often make conflicting statements on the incidence rate of Hepatitis-B in Pakistan, with incidence rate ranging from 1.7 to 5.5 % 6,7 (Xu et al., 2014).
1.1. How HBV burdenize the society:
The prevalence of HBV infection is uneven throughout the world, with significant burdens in Asia and the Pacific Islands, sub?Saharan Africa, the Amazon Basin, and Eastern Europe.
The incidence (rate of new cases) of acute HBV infection has decreased dramatically in the United States since the mid?1980s. This reduction can be attributed to the availability of an effective vaccine and widespread immunization of infants and high?risk populations. However, the number of people who have chronic HBV infection remains high because of the long duration of infection and influx of immigrants who have chronic infection. It is estimated that more than 1 million U.S. residents have chronic infection, which contributes to an estimated 2000 to 4000 deaths each year. National surveys indicate that 0.3% to 0.5% of U.S. residents have chronic infection, and 47% to 70% of these persons were born outside the United States. The prevalence of HBV infection is higher among people who were born in countries with a high HBV prevalence and members of subpopulations that have behavioral risk factors for HBV transmission, including injection?drug users and men who have sex with men. More comprehensive screening for HBV is needed for public health evaluation and management of chronically infected persons and their contacts.(Sorrell et al., 2009)
1.2. Overview:
A .Identification and molecular characteristics:
In 1967, the hepatitis B virus (HBV) surface antigen (HBsAg) was first identified by Blumberg et al(Suk?Fong Lok, 2016). The particle of HBV was initially observed by the Scientists Dan et al. in 1970(Busch & Thimme, 2015). These new findings and observations of HBV help in the contribution to the development of valuable surveillance tools, vaccines and HBV treatment methods (A. W.-c. Lin & Wong, 2016). The vaccine of HBV is available in the market, which provokes protective antibody reaction in 90% to 99% vaccinees(Aggeletopoulou, Davoulou, Konstantakis, Thomopoulos, & Triantos, 2017). However, the protective antibody response is not permanent, and therefore vaccination renewal is required(Liao & Liang, 2015).
B .HBV Organization
HBV belongs toHepadnaviridae family. It contains a circular and partially double-stranded DNA. (Shakeri et al., 2013)The human HBV is an enveloped incomplete double-strand DNA virus which belongs to the family Hepadnaviridae(A. W.-c. Lin & Wong, 2016). The genome of HBV is unusual because the DNA is not fully double-stranded. One end of the total length of the DNA strand is connected to the viral DNA polymerase. The 5′- end of the two DNA strands have two direct repeats (DRs) which are DR1 and DR2. Both DRs maintain the circular configuration and the HBV viral replication (C.-L. Lin ; Kao, 2015). The HBV DNA genome is 3.2 kb in size and contains a 3020 – 3320 nucleotides long negative strand DNA (for the full-length strand) and an 1700 – 2800 nucleotides long positive stand DNA (for the short length-strand) (Araujo, 2015). HBV genome encodes four highly overlapping open reading frames (ORF) which are called C, P, S and X, respectively (Zhang ; Cao, 2011). The HBV core protein, known as HBV core antigen (HBcAg), is encoded by the C gene and produced by proteolytic processing of the precore protein. After cleavage of the N-terminal signal peptide and C-terminal arginine-rich sequence, the precore/core protein is converted to HBV e antigen (HBeAg). HBeAg can secrete into the serum which is not necessary for HBV replication but essential for chronic infection (Tong ; Revill, 2016). The gene P encodes the HBV P proteins. HBV P proteins are essential for reconstitution of synthesis of HBV DNA. Gene S contains the gene that codes for the surface antigen (HBsAg). The gene S consists of one long open reading frame but has three in-frame “start codons ” (ATG) which divide the gene into three parts, pre-S1, pre-S2, and S. Polypeptides of three diverse sizes called large, middle, and the small (L, M and S) have been produced due to the multiple start codons. Gene X, whose function is the transcriptional transactivation of host genes, encodes a nonstructural protein named as HBV X protein (HBx) (Beck ; Nassal, 2007) (Fig. 1.2B). The two HBV enhancers encode the HBV gene transcription:
a) HBV enhancer I/X promoter
b) HBV enhancer II/core.
The HBV enhancers are activated by HBx and thus enhances HBV replication (Choi, Park, ; Rho, 1999; Hodgson, Hyser, Keasler, Cang, ; Slagle, 2012).

Fig:1.2B Organization of HBV genome

C .HBV life cycle : HBV particle entry requires host receptors hepatocyte-associated heparin sulphate proteoglycans (HSPGs) and sodium taurocholate co-transporting polypeptide (NTCP). After HBV particle uncoating, HBV releases relaxed circular DNA (rcDNA) in endosomes, rcDNA then associates with nuclear pore complex (NPC) to import into the nucleus. After the translocation, rcDNA is converted to covalently closed circular DNA (cccDNA). Then cccDNA is transcribed to pregenomic RNAs (pgRNAs) and subgenomic RNA which act as both the templates for reverse transcription to generate viral DNA and message RNAs (mRNAs) for viral protein translation after export into the cytoplasm by associating with a cellular Tip-associated protein/nuclear export factor-1 (TAP/NFX1) and HBcAg. The pgRNAs are selectively packaged inside core particles, and then followed by P protein-mediated negative strand DNA synthesis. After the degradation of pgRNAs, the positive strand DNA is synthesized to generate rcDNA. At last, HBV virions are assembled in the endoplasmic reticulum (ER)-Golgi compartment and then released from the cell. HBV rcDNA may also return to the nucleus to repeat the replication process producing more cccDNA(Datta, Chatterjee, Veer, ; Chakravarty, 2012; Ezzikouri et al., 2014) (Fig. 1.2C).
Fig:1.2C Life cycle of HBV

Hepatitis is a swelling of liver which is represented by the existence of swelled cells in the
organ tissue with confined or sometimes no sign and symptoms.If hepatitis lasts for approximately six months, it is called acute hepatitis and if it lasts longer, it is said to be chronic hepatitis(Ugbebor, Aigbirior, Osazuwa, Enabudoso, ; Zabayo, 2011).Most of the cases of hepatitis in the world are caused by hepatitis viruses but some cases are due to the toxins (such as alcohols, some kind of medications and also several plants), some kinds of infections and the autoimmune diseases might be the reason(Ugbebor et al., 2011).

Hepatitis virus is present in the blood and some other type of body fluids. It can be transferred from one person to another by different ways such as transmission of blood, medical or dental instruments which are not sterilized properly,making tattoos and piercing of the body if not done by the sterilized equipment, from mother to newly born baby during birth, sexual relation, sharing injections,straws, notes, razors, toothbrushes or some other household equipments(Ugbebor et al., 2011).
Viral hepatitis have five main types: A, B, C, D, and E. Amongst all of these, hepatitis B is the most usual and serious infection that is caused due to the hepatitis B virus (HBV). HBV belongs to the Hepadnaviridae family and it is double-stranded DNA virus to some extent. At the hepatitis B surface antigen (HBsAg), it has four main and nine inconsequential serotypes as well as eight genotypes with number of its subgenotypes. Hepatic deterioration, cirrhosis and HCC are caused by HBV. The patients having strong immunity develop an acute disease, while the chronic disease occurs when the patients have weak immune response which will cause new health problems. Occurrence of chronic HBV infection depends on the age of the person such that the younger and newly born children are mostly infected(Gaia et al., 2011). HBV infection is a serious risk factor for the development of chronic HBV infection. The chronic HBV infection can be assessed and managed by the measured quantity of HBV DNA present in the blood. The level of chronic HBV infection is determined by the testing of HBV viral load and it is used to differentiate the acute and chronic disease. There are different antiviral drugs are commercially available in the market currently for the cure of chronic phase of HBV infection(Caliendo, 2011). HBV infection and its various effects can be effectively prevented by the vaccination of hepatitis B. Nevertheless, different antiviral drugs are available for the already infected HBV patients to minimize the effects of the chronic liver disease. (Wasley et al., 2010). In HBV infected patients, the risk of disease development which leads to HCC and cirrhosis will be high as level of HBV DNA is high in serum.(Blach et al., 2017; Kim et al., 2017).
D . Symptoms and Transmission of HBV:
Many people have no symptoms during the initial infection of HBV. Some develop a rapid onset of sickness with vomiting, yellowish skin, tiredness, dark urine and abdominal pain. Often these symptoms last a few weeks and rarely does the initial infection result in death. It may take 30 to 180 days for symptoms to begin. In those who get infected around the time of birth 90% develop chronic hepatitis B while less than 10% of those infected after the age of five do. Most of those with chronic disease have no symptoms; however, cirrhosis and liver cancer may eventually develop. These complications result in the death of 15 to 25% of those with chronic disease (Sorrell et al., 2009).Modes of transmission are the same as those for the human immunodeficiency Virus (HIV), but the hepatitis B virus is 50 to 100 times more infectious. Unlike HIV, the hepatitis B virus can survive outside the body for at least seven days (Afrin, 2017; BEN-ENUKORA).
E .TREATMENT:Vaccination is the most effective measure to reduce the global incidence of hepatitis B. Compared to other healthcare interventions, vaccination is an economically advantageous option, both in terms of cost-effectiveness and benefit-cost ratios. In 1991, the World Health Organization (WHO) recommended that all countries introduce a policy of universal hepatitis B vaccination to prevent and control HBV infection and its long term sequelae on a global scale. By the end of 2008, hepatitis B vaccine for infants was introduced nationwide in 177 countries. To date, global hepatitis B vaccine coverage is estimated at 69% (Gaia et al., 2011).
1.3. DISTRIBUTION OF GENOTYPES AND THEIR CLINICAL SIGNIFICANCE
Currently, there are eight major genotypes of HBV, named A to H, which are in circulation worldwide. Genomic differences of at least 8% are required for sequences to qualify as a new genotype (Dong et al., 2015). Following this criteria, two additional genotypes, I and J, have recently been described but they are not as well characterised(Moyo, Nicholson, ; Arbuthnot, 2016)and the classification of genotype I is controversial (Araujo, 2015). Within some of the genotypes, HBV strains which differ by at least 4% but less than 8% are classified into subgenotypes. These genotypes and their subgenotypes are distributed in specific regions of the world (Figure 1.3A) (Kramvis, Kew, ; François, 2005).

Fig 1.3A Phylogenetic tree representing the worldwide distribution of HBV genotypes (Source: Kramvis et al. (2005) Reproduced with permission.)
Within Africa, HBV strains belonging to genotypes A, D and E predominate. Genotype A is most prevalent in Southern and East Africa and subgenotype A1 has been commonly isolated in African patients including South Africa (Kramvis ; Kew, 2007) Other less common subgenotypes, A3 to A7, have been described in West and Central African countries (Kramvis ; Kew, 2007)although it is suggested that subgenotypes A3, A 4 and A5 might have been misclassified (Pourkarim, Amini-Bavil-Olyaee, Lemey, Maes, ; Van Ranst, 2010). Subgenotype A2 on the other hand is most commonly seen in Europe and Japan (Kramvis et al., 2005; Tamada et al., 2012). Genotype A does not produce pre-core mutants as typically seen with genotype D with a stop codon at position 1896 of the precore region (Chen, Crowther, Kew, ; Kramvis, 2008). This is because of the instability this mutation would cause in the folding of the pregenomic mRNA during encapsidation(Chen et al., 2008). However, mutants with a stop codon at position 1862 of the precore region have been identified and have been hypothesised to reduce the expression of HBeAg(Chen et al., 2008; Kramvis et al., 2005). Genotype D is mostlyconcentrated in the Mediterranean region (Kramvis et al., 2005) and is therefore the most common genotype in North Africa. Isolates belonging to genotype D have been described in South Africa. However, the strains found in these two different geographical areas belong to different subgenotypes(Kramvis ; Kew, 2007).
Genotype E which is an African genotype is most commonly seen in Central and West Africa (Kramvis ; Kew, 2007). These different genotypes have an impact on treatment as they show different disease progression have different susceptibilities to therapy. These are summarised in figure 1.3B

Figure 1.3B Comparison of clinical and virological differences among hepatitis B virus genotypes. (Adapted from Lin and Kao (2011) Reproduced with permission.)

1.4 . IMPORTANCE OF MY WORK/OBJECTIVES:

The Objective of my work is to diagnose the Hepatitis B virus at molecular level via fully automated real time PCR (CAP/CTM) and To determine the prevalence of hepatic infection, viz, hepatitis B (HBV) virus, in the population of Southern Punjab Province of Pakistan.

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