C hair of Medical biology, M icrobiology, V irology, and I mmunology

1. Chair of Medical biology, Microbiology, Virology, and Immunology Causative agents of viral hepatites Lecturer Prof. S.I. Klymnyuk

2. Although hepatitis (inflammation of the liver) was first described in the fifth century BC, it is only recently (1940 to 1950) that the viral etiology of man cases of this disease has been established. More than 50,000 cases of viral hepatitis are reported annually in the United States. Human hepatitis is caused by at least six genetically and structurally distinct viruses (Table 1). The diseases caused by each of these viruses are distinguished in part by the length of their incubation periods and the epidemiology of the infection.

3. Table 1 Characteristics of Human Hepatitis Viruses

4. Table 1 Characteristics of Human Hepatitis Viruses * Length of incubation will vary depending on the HBV status of the infected individual. HDV infection requires either coinfection with HBV or prior infection with HBV

5. HEPATITIS A VIRUS Much of the initial information about HAV resulted from the use of human volunteers to determine the epidemiology of the disease, its incubation period, and the role of the immune response in controlling it. HAV also can be transmitted to several species of marmoset monkeys and chimpanzees, and can be grown in cell cultures of some primate and human cells.

6. Global Prevalence of Hepatitis A Infection HAV Prevalence High Intermediate Low Very Low

7. STRUCTURE AND REPLICATION OF HEPATITIS A VIRUS. Hepatitis A virus, now classified as a member of the Picornaviridae, is a spherical, RNA containing particle, 27 to 32 nm in diameter. Biochemical analysis has shown that the virus possesses a single stranded RNA of about 7500 nucleotides. The mature virus particle contains three major polypeptides (VP1, VP2, and VP3) with molecular weights ranging from 14,000 to 33,000 daltons. The particle also can contain a small VP4 protein.

8. HEPATITIS A VIRUS

9. Hepatitis A virus particles found in fecal extracts by immunoelectron microscopy. Both full and empty particles are present. The virus is 27 to 29 nm in diameter. (X 125,000.)

10. HAV is one of most stable viruses infecting humans. It is resistant to treatment with diethyl ether, can withstand heating at 56 C for 30 minutes, and is remarkably resistant to many disinfectants. Electron microscopy of fecal extracts mixed with antibody to HAV has revealed clumps of virus particles about 27 nm in diameter with icosahedral symmetry. Although minor biochemical differences have been reported among HAV strains isolated in different studies, there appears to be no evidence for major antigenic differences among HAV strains isolated in various parts or the world.

11. Genome organisation of HAV

12. EPIDEMIOLOGY OF HEPATITIS A VIRUS INFECTIONS. The spread of hepatitis A is most often from person to person by a fecal-oral route, hence, the older term for the disease was infectious hepatitis. An average of 25,000 cases of hepatitis A are reported each year in the United States. However, these cases represent only a small percentage or actual infections, because many HAV infections remain undiagnosed. This is particularly true for children, in whom infections frequently are subclinical and the characteristic jaundice rarely is seen. As public health standards increase, the overall prevalence of HAV usually decreases. However, especially in developing countries, this can lead paradoxically to more disease, because it often postpones exposure to the virus until an age at which infection is more likely to produce clinical symptoms. The most common source of infection is close person to person contact.

13. Outbreaks of hepatitis A have been reported in day-care centres and institutions for the mentally retarded. In some cities in the United States, 9% to 12% of reported cases of hepatitis A occur in children in day-care centres, their parents, or staff members. Epidemics also have resulted from drinking fecally contaminated water; however, such water borne epidemics are rare. Eating food prepared by an infected person or ingesting raw oysters, clams, or mussels harvested from fecally contaminated water is the source of many HAV infections. Because there is no persistent infection with continuous viremia (as in HBV infections), HAV transmission by blood products is rare. Although the incidence of HAV infections in intravenous drug abusers is high, it has not been proven that this is due to blood borne transmission

14. Hepatitis A Transmission • Close personal contact Household or sexual contact Daycare centers • Fecal-oral contamination of food or water Food handlers Raw shellfish Travel to endemic areas • Blood-borne (rare) Injecting drug users

15. PATHOGENESIS OF HEPATITIS A VIRUS INFECTIONS. Hepatitis A is an acute, usually self limiting disease with an asymptomatic incubation period of 15 to 40 days. During this time, the liver is infected and large amounts of virus can be shed in the feces. Symptoms usually begin abruptly with fever, nausea, and vomiting.The major area of cell necrosis occurs in the liver, and the resulting enlargement of the liver frequently causes blockage of the biliary excretions, resulting in jaundice, dark urine, and clay colored stool. A fulminant form of hepatitis A occurs in only 1% to 4% of patients. Complete recovery can require 8 to 12 weeks, especially in adults.

16. During convalescence, patients frequently remain weak and occasionally mentally depressed. In humans, the severity of the disease varies considerably with age, most cases occurring in young children are mild and undiagnosed, resolving without sequelae. In contrast to HBV, HAV infections result in no extrahepatic manifestations of acute infection and no long term carrier state, and they are not associated with either cirrhosis or primary hepatocellular carcinoma.

17. Hepatitis A - Clinical Features • Incubation period: Average 30 days • Range 15-50 days • Jaundice by <6 yrs, <10%age group: 6-14 yrs, 40%-50% >14 yrs, 70%-80% • Complications: Fulminant hepatitis Cholestatic hepatitis Relapsing hepatitis • Chronic sequelae: None

18. Concentration of Hepatitis A Virusin Various Body Fluids Feces Body Fluid Serum Saliva Urine 102 104 100 106 108 1010 Infectious Doses per ml

19. Clinical Variants of Hepatitis A Infection • Asymptomatic (anicteric) disease Children under 6 years of age, > 90% Children from 6-14 years old, 40-50% • Symptomatic (icteric) disease Adults and children over 14, 70-80% HAV

20. DIAGNOSIS OF HEPATITIS A VIRUS INFECTIONS. The diagnosis of individual cases of hepatitis A usually is not possible without supporting laboratory findings. However, a tentative diagnosis of hepatitis A is appropriate if there is the simultaneous occurrence of several cases in which the epidemiology and incubation period are consistent with that of HAV disease Such situations have been known to arise in day-care centres, summer camps, and military installations. Virus particles frequently can be detected in fecal extracts by use of immune electron microscopy, in which the fecal extract is mixed with antibodies to HAV. Standard radioimmunoassays also can be used to detect the presence of HAV antigens in fecal extracts. An enzymelinked immunosorbent assay using anti HAV linked to either horseradish peroxidase or alkaline phosphatase also is used to detect fecal HAV. In addition, a specific diagnosis of hepatitis A can be made by demonstrating at least a four fold rise in anti-HAV antibody levels in serum.

21. Symptoms ALT Total anti-HAV Fecal HAV IgM anti-HAV 4 5 6 12 24 0 1 2 3 Months after exposure Typical Serologic Course of Acute Hepatitis A Virus Infection

22. Nomenclature and definition of hepatitis A viruses, antigens, and antibodies

23. CONTROL OF HEPATITIS A VIRUS INFECTIONS. Proper sanitation to prevent fecal contamination of water and food is the most effective way to interrupt the fecal-oral transmission of hepatitis A. Pooled immune serum globulin from a large number of individuals can be used to treat potentially exposed poisons, and its effectiveness has been well established. Immune serum globulin normally contains a substantial titer of neutralizing antibodies to HAV. Studies indicate that large amounts of immune serum globulin can effectively prevent hepatitis A infection, whereas smaller amounts (0 01 mg/kg) modify the severity of the disease, resulting in a mild or asymptomatic infection Such infections can produce a long-lasting active immunity. Formalin inactivated HAV vaccines have been developed and some have been licensed. Additional approaches using recombinant DNA techniques also are being used to generate subunit vaccines or novel recombinant vaccine strains

24. Hepatitis A Vaccine Efficacy Studies Vaccine Site/Age Efficacy Vaccine Group N (95% CI) Ò HAVRIX Thailand 38,157 94% (SKB) 1-16 yrs (79%-99%) 2 doses 360 EL.U. ä VAQTA New York 1,037 100% (Merck) 2-16 yrs (85%-100%) 1 dose 25 units

25. HEPATITIS B VIRUS About 300 million people world-wide are thought to be carriers of HBV, and many carriers eventually die of resultant liver disease. HBV causes acute hepatitis that can vary from a mild and self limiting form to an aggressive and destructive disease leading to postnecrotic cirrhosis. Many HBV infections are asymptomatic (especially in children). However, many infections become persistent, leading to a chronic carrier state. This can lead to chronic active hepatitis and cirrhosis later in life. The HBV carrier state also is strongly associated with one of the most common visceral malignancies world-wide, primary hepatocellular carcinoma. Much of our early knowledge concerning HBV infections stems from studies with human volunteers, because the virus does not readily infect cell cultures More recently, the application of molecular biologic techniques, especially recombinant DNA technology, has yielded significant insights into the structure and replication of HBV.

26. >8% 2-8% <2% HBV - Epidemiology Prevalence of HBsAg Carrier State WHO

27. STRUCTURE OF THE HEPATITIS B VIRION In spite of our inability to grow HBV in cell cultures, several details have been learned about the structure of the hepatitis B virion through studies of new antigens appearing in the blood of infected persons Such information indicates that HBV is unlike any known group of human viruses. Interestingly, similar viruses have been identified in other species Woodchucks, Beechy ground squirrels, and Peking ducks all harbour viruses that are similar in structure and in biologic properties to human HBV. In 1964, it was discovered that numerous virus-like particles were present in the blood of both patients with HBV hepatitis and asymptomatic carriers of HBV. These virus-like particles, first discovered in the serum of an Australian aborigine, originally were referred to as Australia antigen or hepatitis-associated antigen.

28. HEPATITIS B VIRUS

29. The particles are uniformly 22 nm in diameter, existing as both spherical particles and filaments (Fig. 1). Treatment with ether removes a 2-nm envelope, leaving a 20-nm particle. However, these particles do not contain nucleic acid and since have been shown to represent incomplete virus particles containing HRV envelope protein but lacking nucleocapsids. The standard terminology for these particles is HBsAg to designate that they contain the surface antigens of HBV. In 1970, another particle, 42 nm in diameter, was found in the serum of patients with hepatitis B. These larger particles (named Dane particles after their discoverer) occurred in much lower concentrations than did the HBsAg particles. Dane particles were shown to contain the double-stranded, circular viral DNA genome. It has now been demonstrated that the 42-nm Dane particle represents the intact, infectious HBV particle.

30. FIGURE 1. Fraction of the blood scrum from a patient with a severe ease of hepatitis. The larger spherical particles, or Dane particles, are 42 nm in diameter and are the complete hepatitis B virus. Also evident are filaments of capsid protein (HBsAg).

32. HEPATITIS B VIRUS:

33. Treatment of the Dane particles with a non-ionic detergent dissociates the HBsAg and liberates a 27-nm inner core. This inner core contains a core protein, defined serologically as the HBcAg, as well as viral DNA. It also contains two virally encoded enzymes (a DNA polymerase and a protein kinase). Another HBV antigen, designated HBcAg, is often found in the serum of patients during the early stages of infection and in patients with chronic active hepatitis. HBcAg is structurally related to the HBV core protein and is encoded by the C gene. It can be detected in preparations of Dane particles and, therefore, appears to be an integral part of the infectious virion. Its presence in serum is believed to reflect active replication of HBV and is a marker for active disease. The appearance of anti-HBc antibodies generally correlates with a good prognosis and a decline in virus replication.

34. REPLICATION OF HEPATITIS B VIRUS. Studies on the replication of HBV and HBV-related viruses (ie, woodchuck, ground squirrel, and duck hepatitis viruses) have suggested a unique mode of replication for HBV. This replication involves reverse transcription, indicating that HBV is phylogenetically related to the retrovirus family. The viral genome of HBV is about 3000 to3300 nucleotides in length, and molecular cloning and DNA sequencing experiments have established the relative organization of the genes for the various structural proteins (Fig. 2). In addition, an open reading frame encoding a putative DNA polymerase has been identified.

35. Although the viral DNA is circular, both strands of the duplex are linear, and the circular conformation is maintained solely by extensive base pairing between the two gapped DNA strands. Within the virus particle, the negative strand appears to be uniform in length, about 3200 nucleotides. In contrast, the positive strand is shorter and varies in length between different virions, due to single-stranded gaps of variable size. Oninfection, the DNA polymerase in the nucleocapsid core is activated and completes the synthesis of the positive strand, using the negative strand as a template.

36. After the conversion of gapped double-stranded viral DNA to fully double-stranded DNA, a full-length positive-strand RNA (a "pre-genome") is transcribed from the HBV DNA template. This RNA serves as the mRNA for the translation of the HBcAg. Evidence suggests that this form of RNA also is packaged with viral core proteins and the viral DNA polymerase within the cell to form an "immature core". A DNA strand of negative polarity then is synthesized through reverse transcription. This step is followed by the synthesis of a partial positive strand and the full maturation of the virus particle containing a gapped DNA genome.

37. HOW THE VIRUSREPRODUCES ??First the virus attached to a liver cell membrane.

38. The virus is then transported into the liver cell

39. The core particle then releases it’s contents of DNA and DNA polymerase into the liver cell nucleus.

40. Once within the cell nucleus the hepatitis B DNA causes the liver cell to produce, via messenger RNA ; HBs protein , HBc protein , DNA polymerase, the HBe protein , and other undetected protein and enzymes. DNA polymerase causes the liver cell to make copies of hepatitis B DNA from messenger RNA.

41. The cell then assembles ’live’ copies of virus.

42. However because of the excess numbers of surface proteins produced many of these stick together to form small spheres and chains. These can give a characteristic “ ground glass” appearance to blood samples seen under a microscope.

43. The copies of the virus and excess surface antigen are released from the liver cell membrane into blood stream and from there can infect other liver cells .

44. Organization of the genes in hepatitis B virus (HBV). The dashed line represents the variable single- stranded region. The EcoRI site denotes the point of origin for the physical map. The broad arrows define the four large open reading frames of the L strand transcript. The four coding regions arc designated S(made up of pre-S and S genes), P (polymerise), X (regulatory gene), and C. The two regions encoding the S (surface antigen) and C (core antigen) proteins are represented by stippling.

45. Genome of HBV virus Genome: 3.200 nucleotídes S P C X

46. Pré S1 Pré S2 gene S gene C Pré C HBV Genome AgHBs DNA Polimerase gene P AgHBc AgHBe

47. Hepatitis B Virus Gene Products and Functions Polymerase Terminal protein (priming) Reverse transcriptase, RNAse H Surface Envelope proteins Pre - S1Receptor binding, Regulation of cccDNA, viral assembly Pre - S2Viral assembly, fusion sequence S Primary structural component, major antigenic determinants Core HBeAg Secreted, immunomodulatory function Core Nucleocapsid component HBX Pleiotropic effects

48. ANTIGEN OF HEPATITIS B VIRUS: HBe

49. In HBsAg there are some determinants, which are responsible for 10 subtypes of this antigen. “а” determinat is general. There are d and y, and two additional - w and r. So, there are foe main subtypes: adw, ayw, adr, ayr. Other determinants - f, g, j, n, t, x.

50. EPIDEMIOLOGY OF HEPATITIS B VIRUS INFECTIONS. Early volunteer studies failed to show a normal portal of exit for HBV and, for years, it was believed that a person could become infected only by the injection of blood or serum from an infected person or by the use of contaminated needles or syringes. As a result, the older name for this disease was serum hepatitis. It has now been shown that this supposition is not true. Using serologic techniques, HBsAg has been found in feces, urine, saliva, vaginal secretions, semen, and breast milk. Undoubtedly, the mechanical transmission of infected blood or blood products is one of the most efficient methods of viral transmission, and infections have been traced to tattooing, ear piercing, acupuncture, and drug abuse. About5% to 10% of intravenous drug abusers are HBV carriers, and as many as 60% show evidence of previous HBV infections.