latrocetionIntroduction Bioinformatics is an interdisciplinary research area at the interface between computer science and biological science. It involves the technology that uses computers for storage, retrieval, manipulation and distribution of information related to biological macromolecules such as DNA, RNA and proteins. Bioinformatics is limited to sequence, structural, and functional analysis of genes and genomes and their corresponding products and is often considered computational molecular biology. It consists of two subfields: the development of computational tools and databases and the application of these tools and databases in generating biological knowledge to better understand living systems. These tools are used in three areas of genomic and molecular biological research: molecular sequence analysis, molecular structural analysis and molecalar functional analysis. The areas of sequence analysis include sequence alignment, sequence database searching, motif and pattem discovery, gene and promoter finding, reconstruction of evolutionary relationships, and genome assembly and comparison. Structural analyses include protein and nucleic acid structure analysis, comparison, Classification and prediction. The functional analysis includes gene expression profiling, protein- protein interaction prediction, protein sub cellular localization prediction, metabolic pathway reconstruction, and simulation, The three aspects of bioinformatics analysis are not isolated but often interact to produce integrated results. For example, protein structure prediction depends on sequence alignment data; clustering of gene expression profiles requires the use of phylogenetic tree construction methods derived In sequence analysis, Sequence- based prediction is related functional analysis of co expressed genes. The first major bioinformatics project was undertaken by Margaret Dayhoff in 1965, who developed a first protein sequence database called Atlas of Protein Sequence and Structure. Subsequently, in the early 1970s, the Brookhaven national laboratory established the Protein Data Bank for archiving three-dimensional protein structures. At its onset, the database stored less than a dozen protein structures, compared to more than 30,000 structures today. The first sequence alignment algorithm wasDeveloped by Needleman and Wunsch in 1970. This was a fundamental step in the development of the field of bioinformatics. which paved the way for the routine sequence comparisons and database searching practiced by modem biologists. 10 The recent advance of Bioinformatics is molecular modeling which is aimed at understanding structure-function and structure property relationship in physico-chemical processes and pharmaceuticals & thus has become increasingly important for finding and designing new drugs. In fact computers are playing an important role in new drug discovery and drug design. HEPATITIS:- Hepatitis (plural hepatitides) implies injury to liver characterized by presence of inflammatory cells in the liver tissue. Etymologically from ancient Greck hepar or hepato. meaning, ‘liver,’ and suflix -itis, denoting ‘inflammation’. The condition can be self limiting, healing on its ‘own, or can progress to scarring of the liver . Hepatitis is acute when it lasts less than 6 months and chronic when it persists longer. A group of viruses known as the hepatitis viruses cause most cases of liver damage worldwide. Hepatitis can also be due to toxins (notably alcohol), other infections or from autoimmune process. It may run a sub clinical course when the affected person may not feel ill. The patient becomes unwell and symptomatic when the disease impairs liver functions that include,among other things, screening of harmful substances, regulation of blood composition, and production of bile to help digestion. Causes Acute hepatitis + Viral Hepatitis: Hepatitis A to E (more than 95% of viral cause), Herpes simplex, Cytomegalovirus, Epstein-Barr, Yellow fever virus, Adenoviruses. + Non viral infection: Toxoplasma, Leptospira, Q fever, Rocky mountain spotted fever « Alcohol + Toxins: Amanita toxin in mushrooms, Carbon tetrachloride, Asafetida * Drugs: Paracetamol, Amoxicillin, Antituberculosis medicines, Minocycline and many others. = Ischemic hepatitis (circulatory insufficiency)(1) + Pregnancy + Auto immune conditions, e.g. Systemic Lupus Erythematosus (SLE) + Metabolic diseases, e.g. Wilson's disease Chronic hepatitis Viral hepatitis: Hepatitis B with or without hepatitis D, hepatitis C (Hepatitis A and E do not lead to chronic disease)» Autoimmune: Autoimmune hepatitis * Alcohol + Drugs: Methyl-dopa, Nitrofurantoin,lisoniazide, Ketoconazole + Non-alcoholic steatohepatitis + Heredity: Wilson's disease, alpha 1-antitrypsin deficiency + Primary biliary cirrhosis and primary sclerosing cholangitis occasionally mimic chronic hepatitis" Viral hepa’ A virus is a particle which is smaller than bacteria, and contains complex genetic information called DNA or RNA. This genetic material allows the virus to infect bacteria or living cells, set up the machinery to reproduce itself, leading to destruction of the cell in which it resides. To date, five viruses, labeled A through E, have been identified which appear to cause viral hepatitis. Viruses A and E can be contracted from contaminated water or food (by mouth), while viruses B, C and D are transmitted by direct injection into the bloodstream (through any method of injection under the skin), The term viral hepatitis describes any one of the illnesses caused by the five viruses mentioned, and consists of an infection of liver cells which leads to damage of the liver over days in some cases, but over many years in others. Thirty years ago, none of the hepatitis viruses had been identified. In the 1960's, transfusion-related viral hepatitis was extremely common, with 30% of patients receiving blood products becoming infected. By 1970, a blood test called the Australia antigen, was developed which appeared to identify those infected with one hepatitis virus which we now call hepatitis B. The investigator who discovered the Australia antigen, the protein which makes up the coat of the virus and which is now called the hepatitis B surface antigen (HBsAg), was awarded the Nobel prize. Our understanding of viral hepatitis has grown tremendously since the discovery of the Australia amigen.Currently 11 viruses are recognized as causing hepatitis, Two are herpes viruses (cytomegalovirus virus[CMV] and Epstein- Barr virus[EBV]) and 9 are hepatotropic viruses EBV and CMV cause mild ,self-resolving forms of hepatitis with no permanent hepatic damage. Both viruses causes the typical infectious mononucleosis of fatigue nausea , and malaise. Of the nine human hepatotrofic viruses ,only five are well characterized; hepatitis G and TTV(transfusion transmitted virus) are newly discovered viruses hepatitis A (sometimes called infectious hepatitis), and hepatic E (formally called enteric —transmitted NANB hepatitis) ,are transmitted by fecal-oral contamination .The most important type include hepatitis B(sometime called serum hepatitis), hepatitis C (formally called formally non-A ,non-B hepatic), and hepatitis D (formally called delta hepatitis). Hepatitis A Incubation period 35 weeks (mean 28 days) Milder disease than Hepatitis B; asymptomatic infections are very common, especially in children Adults, especially pregnant women, may develop mor severe disease. Although convalescence may be prolonged, there is no chronic form of the disease. Fulminant hepatitis is rare: 0.1% of cases Virus enters via the gut: replicates in the alimentary tract and spreads to infect the liver, where it multiplies in hepatocytes. Viraemia is transient. Virus is excreted in the stools for two weeks preceding the onset of symptoms. World-wide distribution; endemic in most countries. The incidence in first world countries is declining. There is an especially high incidence in developing countries and rural areas. In rural areas of South Africa , the seroprevalence is 100%.Hepatitis E Incubation period 30-40 days Acute, self limiting hepatitis, no chronic carrier state Age: predominantly young adults, 15-40 years .Fulminate hepatitis in pregnant women. Mortality rate is high (up to 40%). Similar to hepatitis A; virus replicates in the gut initially, before invading the liver. and virus is shed in the stool prior to the onset of symptoms. Viraemia is transient. A large inoculum of virus is needed to establish infection.Little is known yet. The incidence of infection appears to be low in first world countries. Hepatitis C Putative Togavirus related =~ to. the_—“Flavi_~—and_—Pesti_—_—viruses. Thus probably enveloped. Has a ssRNA genome Does not grow in cell culture, but can infect Chimpanzees Incubation period 6-8 weeks Causes a milder form of acute hepatitis than does hepatitis. B But 50% individuals develop chronic infection, following exposure. 1) Chronic liver disease 2) Hepatocellular carcinoma Incidence endemic world-wide; high incidence in Japan, Italy and Spain In South Africa, 1% blood donors have antibodies Hepatitis D Defective virus which requires Hepatitis B as a helper virus in order to replicate. Infection therefore @ljlaceurs i patients| who are already infected witht Hepalitis B.increased severity of liver discase in Hepatitis B carriers. virus particle 36 nm indiameter encapsulated with HBsAg, derived from) HBV delta antigen is associated with virus particles ssRNA genome Identified in intra-venous drug abusers Hepatitis G A virus originally cloned from the serum of a surgeon with non-A, non-B, non-C hepatitis, has been called Hepatitis G virus. It was implicated as a cause of parenterally transmitted hepatitis, but is no longer believed to be a major agent of liver disease. It has been classified as a Flavivirus Hepatitis B What is the Hepatitis B is? The hepatitis B virus (HBV) is a DNA-containing virus which is capable of infecting human liver cells and other cells in the body, once it gains access to the blood stream. One of the most interesting features of the hepatitis B virus is that the virus itself does not damage the liver, the damage being caused by the individual's own immune system attacking the virus-infected cells. Since liver damage from the virus may be very little, many patients are called healthy carriers. This means that although they may transmit the disease to others, they have normal-appearing livers and normal liver function tests. While many individuals remain healthy for many years or a lifetime, others develop chronic hepatitis, cirrhosis, and occasionally liver cell cancer. These outcomes are linked to the virus and its effects, although it is unlikely that the vi s directly causes cancer. ‘Those patients who develop hepatitis (damage to liver cells with inflammation), do so on account of the body's normal inclination to attack the foreign proteins contained in viruses, and in the cells in which the viruses are found. This process, called the immune response, determines the pace and the severity of the liver cell injury in this condition, and will be described in more detail below. Since the identification of the hepatitis B virus, several other viruses which are nearly identical, have been identified in Eastern woodchucks, ground squirrels and Pekingducks. The members of this virus family, termed the 'Hepadna’ viruses, have similar life cycles to that observed in man and can serve as animal models, allowing further study of these unique disease-causing agents. Classification and general features: Family : hepadnaviridae Genera : orthohepadnavirus(e.g hepatitis B [HBV] of human ) ,Avihepadnavirus (e.g. Duck hepatitis B virus) Size 42nm Virions (also known as "Dane particles") contain a circular dsDNA genome. Fig.hepatitis B virus structure HBV Antigens HBsAg = surface (coat) protein produced in excess as small spheres and tubulesHBeAg = inner core protein HBeAg = secreted protein; function unknown. Clinical Features Incubation period 2 - § months Insidious onset of symptoms. Tends to cause a more severe disease than Hepatitis A. Asymptomatic infections occur frequently. Pathogenesis Infection is parenterally transmitted. The virus replicates in the liver and virus Particles, as well as excess viral surface protein, are shed in large amounts into the blood. Viraemia is prolonged and the blood of infected individuals is highly infectious. Complications 1) Persistant infection:~ Following acute infection, approximately 5% of infected individuals fail to eliminate the virus completely and become persistantly infected. ‘Those who are at particular risk include: babies, young children immunocompromised patients males > females ‘The virus persists in the hepatocytes and on-going liver damage occurs because of the host immune response against the infected liver cells. Chronic infection may take one of two forms: Chronic persistent Hepatitis - the virus persists, but there is minimal liver damageChronic Active Hepatitis - There is aggressive destruction of liver tissue and rapid progression to cirrhosis or liver failure, Patients who become persistently infected are at risk of developing hepatocellular carcinoma (HCC). HBV is thought to play a role in the development of this malignancy because: a) 80% of patients with HCC are carriers of hepatitis B. b) Virus DNA can be identified in hepatocellular carcinoma cells, ©) Virus DNA can integrate into the host chromosome. 3) Fulminant Hepatitis Rare; accounts for 1% of infections. Epidemiology Prevalence of disease in Africa World-wide there are 450 million persistant carriers of hepatitis B, 50 million of which are in Africa. Carriage rates vary markedly in different areas. In South Africa, infection is much more common in rural communities than in the cities. Hepatitis B is paremerally transmitted 1) Blood: * Blood transfusior ., serum products, * sharing of needles, razors + Tattooing, acupuncture * Renal dialysis * Organ donation 2) Sexual intercourse 3) Horizontal transmission in children, fami ‘close personal contact’ This is the major mode of transmission in South Africa where the majority of individualsbecome infected at between three and nine years of age. Horizontal transmission also occurs in children’s institutions and mental homes. 4) Vertical transmission - perinatal transmission from a carrier mother to her baby * Tran placental (rare) * during delivery * Post natal , ?? breast feeding , ??close contact (This is the major mode of transmission in South East Asia) Diagnosis: Serology Acute infection with resolution Viral antigens: 1) Surface antigen (HBsAg) is secreted in excess into the blood as 22 nm spheres and tubules. Its presence in serum indicates that virus replication is occurring in the liver 2) 'e" antigen (HBeAg) secreted protein is shed in small amounts into the blood. Its presence in serum indicates that a high level of viral replication is occurring in the liver 3) core antigen (HBeAg) core protein is not found in blood Antibody response: 1) Surface antibody (anti-HBs) becomes detectable late in convalescence, and indicates immunity following infection. It remains detectable for life and is not found in chronic carriers (see below), 2) ¢ antibody (anti-HBc) becomes detectable as viral replication falls. It indicates low infectivity in a carrier. 3) Core IgM rises early in infection and indicates recent infection 4) Core IgG rises soon after 1gM., and remains present for life in both chronic carriers as well as those who clear the infection. Its presence indicates exposure to HBV.of the chronic carrier 12Fig.Hepatitis B virus in serum. Prevention 1) Active Immunization Two types of vaccine are available: Serum derived - prepared from HBsAg purified from the serum of HBV carriers Recombinant HBsAg - made by genetic engineering in yeasts Both vaccines are equally safe and effective. The administration of three doses induces protective levels of antibodies in 95% of vaccine _ recipients. Universal immunization of infants was introduced in April 1995. Infants receive 3 doses at 6, 10 and 14 weeks of age. Vaccine should be administered to people at high risk of infection with HBV: 1) Health care workers 2) Sexual partners of chronic carriers 3) Infants of HBV carrier mothers 2) Passive Antibody Hepatitis B immune globulin should be administered to non immune individualsfollowing single episode exposure to HBV-infected blood. For example: needlestick injuries. ‘What is Hepatitis B Infection Like? When most individuals become infected with the hepatitis B virus, they are not aware of the infection for several weeks, until they develop symptoms of acute hepatitis, such as nausea, fatigue and jaundice (yellowing of the eyes). The acute hepatitis phase may last for several weeks and occasionally leads to hospitalization, but acute hepatitis B resolves completely in 95% of those infected. Others who do not develop significant symptoms following exposure may not be aware of the infection. These individuals may also overcome the infection completely and develop immunity, but frequently become chronic carriers. The outcome of hepatitis B infection depends to a great extent on the status of the person's immune system at the time of exposure. Most chronic carriers or those with chronic hepatitis B are not aware of their on-going infection, although some have persistent fatigue. Molecular virology Genome : circular and 3.2kb in size, double stranded.It has compact 14is through the used of multiple i ‘frame start codons. The HBV genome also contains parts that regulate transcription, determine the site of polyadenylation and a specific transcript for encapsidation into the nucleocapsid. Life cycle In order to reproduce, the hepatitis B virus, must first attach onto a cell which is capable of supporting its replication. Although hepatocytes are known to be the most effective cell type for replicating HBV, other types of cells in the human body have be found to be able to support replication to a lesser degree. The initial steps following HBV entry are not clearly defined although it is known that the virion initially attaches to a susceptible hepatocyte through recognition of cell surface receptor that has yet to be indified (Garces. HBVP). The DNA is then enters into the nucleus, where it is known to form a convalently close circular form called cccDNA The (-) strand of eceDNA is the template for transcription by RNA polll of a longer than genome length RNA called the pregenome and shorter subgenomic transcripts, all of which serve as mRNAs. The shorter viral mRNAs are translated by ribosomes attached to the cell's endoplasmic reticulum and the proteins that are destined to become HBV surface antigens in the viral envelope are assembled. The pregenome RNA is translated to produce a polymerase protein, P, which then binds to a specific site at the 3' end of its own transcript, where viral DNA synthesis eventually ‘occurs. Occuring at the same time as capsid formation, the RNA-P protein complex is packaged and reverse transcription begins. Atearly times after the infection, the DNA is recirculated to the nucleus, where the process is repeated, resulting in the the accumulation of 10 to 30 molecules of CCC DNA andan increase in viral mRNA concentrations (Flint et al.. 765). 16The hepatitis B virion, also known as the Dane particle, is the one infectious particle found within the body of an infected patient, This virion has a diameter of 42nm and its outer envelope contains a high quantity of hepatitis b surface proteins. The envelope surrounds the inner nucleocapsid which is made up of 180 hepatitis B core proteins arranged in an icosahedral arrangement. The nucleocapsid also contains at least one hepatitis b ploymerase protein (P) along with the HBV genome. In infected people, virions actually compose a small minority of HBV-derived particles. Large numbers of smaller subviral particles are also present,that usually outnumber the virions in the ratio of 100:1.These two subviral particles the hepatitis B filament and a hepatitis B sphereare often referred to as a group named surface antigen particles. The sphere contains both middle and small surface proteins whereas the filament also includes large hepatitis B surface protein Iso includes large hepatitis B surface protein. The absence of the hepatitis B core, polymerase, and genome causes these particles to have a non-infectious nature. High levels of these non-infectious particles can be found during the acute phase of the infection. Since the non-infectious particles present the same sites as the virion, they induce a significant immune response and are thought to be non-advantagous for the virus. However, it is also believed that the presence of high levels of non-infectious particles may allow the infectious viral particles to travel undetected by antibodies through the blood stream (Garces. HBVP Hepatitis B Antigens: ‘There are three different types of hepatitis b antigens encoded by the HBV genome- Hepatitis B Surface antigen (HBsAg)- There are three different types of hepatitis B surface antigens; small hepatitis B surface antigen (HBsAg or SHBsAg), middle hepatitis, itis B surface Antigen (LHBsAg). HBsAg is the smallest protein of the hepatitis B surface proteins and has historically been known B surface antigen (MHBsAg), and large hep: as the Australia antigen (Au antigen). It is very hydrophobic, containing four- transmembrane spanning regions. This protein is the prime constituent of all hepatitis b particle forms and appears to be manufactured by the virus in high quantities. It also contains a highly antigenic epitope which may be responsible for triggering immuneresponse. Regardless of the high Antigenicity and prevalence of these particles,the immune system appears basically oblivious to their presence. Hepatitis B Core Antigen (HBcAg)- The only HBV antigen that can not be detected directly by blood test, this antigen can only be isolated by analyzing an infected hepatocyte. A 185 amino acid protein is expressed in the cytoplasm of infected cells, they are highly associated with nucleocapsid assembly (Strauss 2002). 19