Chair of Microbiology, Virology, and Immunology Hepatitis Viruses

Characteristics of Human Hepatitis Viruses Mechanism of transmission Family/ Genus Size/ Genome Length of Incubation Mechanism of transmission Vaccine HAV Picornaviridae / Hepatovirus 72 27-30 nm, single-stranged RNA 15-40 days Mostly oral-fecal Yes HBV Hepadnaviridae / hepadnavirus 142 nm, circular double-stranged DNA 50-180 days Parenteral Recom-binant subunit vaccine HCV Flaviviridae 30-50 nm single-stranged RNA 14-28 days Parenteral, likely other sources No HDV Unclassified 35-40 nm single-stranged RNA 50 180 days* Parenteral transmission HEV Caliciviridae 27 34 nm single-stranged RNA 6 weeks Oral-fecal

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

HEPATITIS A VIRUS Virus Hepatitis A Family Picornaviridae Genus Hepatovirus Virion 27 nm icosahedral Envelope No Genome ssRNA Genome size 7,5kb Stability Heat- and acid-stable Transmission Fecal-oral Prevalence High Fulminant disease Rare Chronic disease Never Oncogenic

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.)

Genome organisation of HAV This figure shows the genome organisation of HAV The genome consist of a long ORF divided into 3 regions; The structural proteins P1 And the non-structural proteins P2 and P3. The C-terminal part of VP3, the N-terminal part of VP1, as well as the VP1-2PA junction are the most variable regions and has therefore been used in molecular epidemiological studies. Based upon sequencing 168 bp within the VP1-2PA Betty Roberston and workers mange to distinguish geographical distributed strains and classified them into genotypes.

Hepatitis A Virus Life Cycle

Hepatitis A Transmission Fecal-oral contamination of food or water Food handlers Raw shellfish Travel to endemic areas Close personal contact Household or sexual contact Daycare centers Blood-borne (rare) Injecting drug users Hepatitis A Transmission For example. An epidemic of HAV that occurred in Shanghai, China, in 1988 in which 300,000 people were infected with the virus resulted from eating Anadara subcrenata obtained from a polluted river. Slide 45 Routes of Hepatitis A Transmission Hepatitis A is transmitted primarily via the fecal-oral route by either ingestion of contaminated food or water or person-to-person contact with an individual with acute HAV infection. Examples of fecal-oral transmission from ingestion of contaminated food or water include travel of susceptible individuals to endemic areas of infection, epidemics of infection resulting from ingestion of contaminated shellfish, and epidemics of infection from ingestion of food contaminated by infected food handlers. Person-to-person transmission occurs most commonly within families and among children in day care centers. Sexual transmission occurs most frequently between men who have sex with men. Blood-borne transmission, although unusual, does occur, primarily in hemophiliacs and injection drug users. Shapiro CN, Hadler SC. Significance of hepatitis A in children in day care. Semin Pediatr Infect Dis 1990; 1:270-279. Soucie JM, Robertson BH, Bell BP, McCaustland KA, Evatt BL. Hepatitis A virus infections associated with clotting factor concentrate in the United States. Transfusion 1998; 38:573-579. Centers for Disease Control and Prevention. Prevention of hepatitis A through active or passive immunization: recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR 1999; 48(RR-12):1-38.

Pathogenesis

Pathogenesis of HAV HAV replicates slowly in the liver without producing apparent cytopathological effects (CEPs). In the absence of cytolysis, the virus readily establishes a persistent infection. Jaundice, resulting from damage to the liver Antibody is detected and cell-mediated immune responses to the virus

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 7

Concentration of Hepatitis A Virus in Various Body Fluids Feces Body Fluid Serum Saliva Urine 100 102 104 106 108 1010 Infectious Doses per ml 10

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% Clinical Variants of Hepatitis A Infection Slide 47 Clinical Variants of Hepatitis A Infection Hepatitis A infection can result in either an asymptomatic or symptomatic clinical illness. The illness typically has an abrupt onset with symptoms that can include transient fever, malaise, anorexia, nausea, vomiting, abdominal pain, and jaundice. The likelihood of symptoms and jaundice are related to the patient’s age. Fewer than 10% of children under 6 years of age have jaundice. In contrast, over two-thirds of older children and adults have symptomatic disease with jaundice. The duration of illness is < 2 months in most cases. Complete recovery and the development of natural immunity to reinfection ultimately occur in all infected individuals. Hadler SC, Webster HM, Erben JJ, Swanson JE, Maynard JE. Hepatitis A in day-care centers: a community-wide assessment. N Engl J Med 1980; 302:1222-1227. Lednar WM, Lemon SM, Kikpatrick JW, Redfield RR, Fields ML, Kelley PW. Frequency of illness associated with epidemic hepatitis A virus infection in adults. Am J Epidemiol 1985; 122:226-233.

Typical Serologic Course of Acute Hepatitis A Virus Infection Fecal HAV ALT IgM anti-HAV Months after exposure Symptoms 1 2 3 4 5 6 12 24 Total anti-HAV Slide 46 Serological Course of Acute Hepatitis A HAV replicates in the liver, is excreted in bile, and is shed in the stool. Most patients with hepatitis A become symptomatic after an average incubation period of 28 days (range 15-50 days). Peak infectivity occurs during the two-week period before the onset of elevated aminotransferases and jaundice. Fecal shedding of the virus usually disappears by the time patients become symptomatic. Specific antibodies develop rapidly in patients with HAV infection. IgM antibodies against HAV are usually detectable 5-10 days before the onset of symptoms and can persist for up to 6 months after infection. IgG antibodies, which appear early in the infection, persist indefinitely and confer lifetime protection against reinfection. The diagnosis of acute hepatitis A is made by detecting IgM anti-HAV in the serum. Skinhøj P, Mathiesen LR, Kryger P, Møller AM. Faecal excretion of hepatitis A virus in patients with symptomatic hepatitis A infection. Scand J Gastroenterol 1981; 16:1057-1059. Liaw YF, Yang CY, Chu CM, Huang MJ. Appearance and persistence of hepatitis A IgM antibody in acute clinical hepatitis A observed in an outbreak. Infection 1986; 14:156-158. Stapelton JT. Host immune response to hepatitis A virus. J Infect Dis 1995; 171(suppl 1):S9-S14.

Hepatitis A Vaccine Efficacy Studies Site/Age Efficacy Vaccine Group (95% CI) HAVRIX Ò Thailand 94% (SKB) 1-16 yrs (79%-99%) 2 doses 360 EL.U. VAQTA ä New York 100% (Merck) 2-16 yrs (85%-100%) 1 dose 25 units 17

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).

Prevalence of HBsAg Carrier State >8% 2-8% <2% Slide 61 Epidemiology of Hepatitis B Hepatitis B infection is a global health problem. Approximately 350 million people worldwide are chronic carriers of HBV. Chronic hepatitis B is endemic in most parts of Africa, Asia and Alaska, with carrier rate higher than 20%. In United States, the latest population survey showed prevalence of chronic hepatitis B to be 0.42%. However, in that study, people at high risks, such as those imprisoned or incarcerated, were not surveyed. Hence, the actual prevalence may be even higher. Kao JH, Chen DS. Global control of hepatitis B virus infection. Lancet Infec Dis 2002;2:395-403. McWuillan GM, et al. Prevalence of hepatitis B virus infection in the United States: the National Health and Nutrition Examination Surveys, 1976 through 1994. Am J Public Health. 1999 Jan;89(1):14-8. WHO

HEPATITIS B VIRUS Virus Hepatitis B Family Hepadnaviridae Genus Orthohepadnavirus Virion 42 nm, spherical Envelope Yes (HBsAg) Genome dsDNA Genome size 3,2kb Stability Acid-sensitive Transmission Parenteral Prevalence High Fulminant disease Rare Chronic disease Often Oncogenic Yes

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).

HEPATITIS B VIRUS:

HOW THE VIRUS REPRODUCES ?? 1. First the virus attached to a liver cell membrane. 2. The virus is then transported into the liver cell

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

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

7. 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. 6. 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.

HBV: Replication Reverse transcription: one of the mRNAs is replicated with a reverse transcriptase making the DNA that will eventually be the core of the progeny virion RNA intermediate: HBV replicates through an RNA intermediate and produces and release antigenic decoy particles. Integration: Some DNA integrates into host genome causing carrier state

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

Open Reading Frames There are 4 open reading frames derived from the same strand (the incomplete + strand) S - the 3 polypeptides of the surface antigen (preS1, preS2 and S - produced from alternative translation start sites. C - the core protein P - the polymerase X - a transactivator of viral transcription (and cellular genes?). HBx is conserved in all mammalian (but not avian) hepadnaviruses. Though not essential in transfected cells, it is required for infection in vivo.

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

ANTIGEN OF HEPATITIS B VIRUS: HBsAg = surface (coat) protein ( 4 phenotypes : adw, adr, ayw and ayr) HBcAg = inner core protein (a single serotype) HBeAg = secreted protein; function unknown

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 four main subtypes: “adw”, “ayw”, “adr”, “ayr”. Other determinants - f, g, j, n, t, x.

HBV SPREAD MAINLY BY PARENTERAL ROUTE DIRECT PERCUTANEOUS INOCULATION OF INFECTED SERUM OR PLASMA INDIRECTLY THROUGH CUTS OR ABRASIONS ABSORPTION THROUGH MUCOSAL SURFACES ABSORPTION OF OTHER INFECTIOUS SECRETIONS (SALIVA OR SEMEN DURING SEX) 28

HBV SPREAD MAINLY BY PARENTERAL ROUTE POSSIBLE TRANSFER VIA INANIMATE ENVIRONMENTAL SURFACES VERTICAL TRANSMISSION SOON AFTER CHILDBIRTH (TRANSPLACENTAL TRANSFER RARE) CLOSE, INTIMATE CONTACT WITH AN INFECTED PERSON 29

Pathogenesis and Immunity Virus enters hepatocytes via blood Immune response (cytotoxic T cell) to viral antigens expressed on hepatocyte cell surface responsible for clinical syndrome 5 % become chronic carriers (HBsAg> 6 months) Higher rate of hepatocellular carcinoma in chronic carriers, especially those who are “e” antigen positive Hepatitis B surface antibody likely confers lifelong immunity (IgG anti-HBs) Hepatitis B e Ab indicates low transmissibility

Determinants or acute and chronic HBV infection From Murray et. al., Medical Microbiology 5th edition, 2005, Chapter 66, published by Mosby Philadelphia,,

WHO IS AT GREATEST RISK FOR HBV INFECTION? LAB PERSONNEL WORKING WITH BLOOD PRODUCTS SEXUALLY ACTIVE HOMOSEXUALS PERSONS WITH MULTIPLE AND FREQUENT SEX CONTACTS MEDICAL/DENTAL PERSONNEL DRUG ABUSERS BLOOD PRODUCT RECIPIENTS ACCOUNTS FOR 5-10% POSTRANSFUSION HEPATITIS HEMODIALYSIS PATIENTS PEOPLE FROM SOUTHEAST ASIAN COUNTRIES (70-80%) 30

HBV 300,000 NEW CASES IN U.S. PER YEAR LIFETIME RISK FOR AVERAGE PERSON IS 5% SEXUAL PROMISCUITY > RISK LIFETIME RISK FOR DENTIST IS 13-28% 32

Jaundice

CHARACTERISTICS OF HBV INFECTION INFECTION IS USUALLY SELF LIMITING, COMPLETE RESOLUTION IN 6 MONTHS HOWEVER, WHEN INFECTED 5% ADULTS CHRONIC CARRIERS 20% CHILDREN CHRONIC CARRIERS 80-90% NEONATES AND INFANTS BECOME CHRONIC CARRIERS 37

Acute Infection HBsAg Anti-HBs Anti-HBc Anti-HBc IgM HBeAg HBV DNA 0 2 4 6 HBsAg Anti-HBs Anti-HBc Anti-HBc IgM Months Years HBeAg HBV DNA Anti-HBe HBV - Diagnosis Slide 80 Serological Markers of Acute HBV Infection Incubation period of HBV infection ranges from 60 to 180 days. HBsAg is the first serological marker of acute HBV infection. Early in the course of acute HBV infection, markers of active viral replication (HBeAg and HBV DNA), are also detectable. As the patients recover, serum HBV DNA level markedly decrease but may remain detectable by PCR assay for up to several decades, HBeAg to anti-HBe seroconversion occurs and finally HBsAg becomes undetectable. Persistence of HBsAg for more than 6 months indicates progression to chronic HBV infection. Anti-HBc IgM is the first antibody to be detected and usually persists for several months. It may be the only marker of acute HBV infection during the ‘window’ period after HBsAg is cleared and before anti-HBs is detected. Recovery from acute HBV infection is indicated by the presence of anti-HBc IgG and anti-HBs.

Chronic Infection HBV - Diagnosis HBV DNA HBeAg Months Years Anti-HBe Anti-HBc IgM Anti-HBc IgG Anti-HBe HBsAg Slide 81 Serological Markers of Chronic HBV Infection Chronic HBV infection is indicated by the presence of HBsAg persisting for more than 6 months and detection of anti-HBc IgG. During the early phase of chronic HBV infection, markers of HBV replication: HBeAg and high serum HBV DNA levels are also present. Over time, patients seroconvert from HBeAg to anti-HBe, accompanied by decrease in serum HBV DNA levels.

PRACTICE HBsAG N. HBcAB (TOTAL) P. HBsAB P. HAV-IGM N. HCV N. PAST INFECTION.

PRACTICE HBsAg N. HBcAB (total) N. HBsAB P. HAV-IGM N. HCV N. IMMUNIZATION.

PRACTICE HBsAg P. HBcAB (Total) P. HBsAB N. HAV-IGM N. HCV N. MAY BE ACUTE OR CHRONIC. Order Hep. B Core IgM to clarify. The IgM will be positive , If Acute.

Vaccine Age Group Dose Volume # Doses HBV - Vaccine Vaccine Age Group Dose Volume # Doses (μg) (ml) Engerix-B 0-19 yr 10 0.5 3 (mo 0,1,6)  20 yr 20 1.0 3 (mo 0,1,6) Adults on hemodialysis 40 2.0 4 (mo 0,1,2,6) Recombivax HB 0-19 yr 5 0.5 3 (mo 0,1,6)  20 yr 10 1.0 3 (mo 0,1,6) (Optional 2-dose) 11-15 yr 10 1.0 2 (mo 0, 4-6) hemodialysis 40 1.0* 3 (mo 0,1,6) Slide 95 Dose schedule of HBV vaccine Two recombinant HBV vaccines are currently available in the United States: Engerix-B (GlaxoSmithKline Biologicals) and Recombivax HB (Merck & Co., Inc.). The two vaccines can be used inter-changeably. Conventional 3-dose schedules include injections at 0, 1, and 6 months for immunocompetent people. The dose recommended for children and adolescents is lower while that for immunocompromised adults such as dialysis patients is higher. In adolescents aged 11 to 15 years, Recombivax HB has been approved for use in a 2-dose, 10ug regimen, with the second dose given 4 to 6 months after the first. This 2-dose regimen should increase compliance and reduce costs. CDC. Hepatitis B virus: a comprehensive strategy for eliminating transmission in the United States through universal childhood vaccination: recommendations of the immunization practices advisory committee (ACIP). MMWR 1991;40 (RR-13):1-19. Koff RS. Hepatitis A, hepatitis B, and combination hepatitis vaccines for immunoprophylaxis. An update. Dig Dis Sci 2002;47:1183-94.

Combined HAV and HBV - Vaccine Bivalent HAV and HBV vaccine 1ml contains 720 ELISA Units of inactivated HAV and 20 ug of recombinant HBsAg protein Dosage: 1 ml at 0, 1, 6 months Recommended for all susceptible persons  18 years at risk of exposure to both HAV and HBV, including travelers to areas of high/intermediate endemicity for both viruses Slide 97 Combined hepatitis A and B vaccines Many individuals are at risk of acquiring hepatitis A and B. A vaccine that provides protection against both hepatitis A and B reduces the number of injections, offers greater convenience to the vaccinees, and improves compliance. Twinrix is a bivalent vaccine containing the antigenic components used in producing Havrix A and Engerix-B. A 1.0 mL dose of vaccine contains not less than 720 ELISA Units of inactivated hepatitis A virus and 20 ug of recombinant HBsAg protein. For those aged 18 or older, a dose of 1ml is given at 0, 1, and 6 months. Twinrix is recommended for individuals who are, or will be, at risk of exposure to both HAV and HBV infection, including travelers to areas of high or intermediate endemicity for both viruses, and patients with chronic liver disease. Knoll A, Hottentrager B, Kainz J, et al. Immunogenicity of a combined hepatitis A and B vaccine in healthy young adults. Vaccine 2000;18:2029-32.

Therapeutic Agents Immune Modulators Nucleo(s)tide analog Interferon Lamivudine Thymosin Adefovir dipivoxil Therapeutic vaccines Emtricitabine Entecavir L-dT/ L-dC Clevudine Famciclovir Slide 113 Therapeutic agents for chronic hepatitis B Antiviral therapies can be divided into immune modulators and nucleotide or nucleoside analogues. Currently, only interferon, lamivudine, and adefovir dipivoxil are approved for the treatment of chronic hepatitis B.

Characteristics of hepatitis C viruses Family Flaviviridae Genus Hepacivirus Virion 60 nm spherical Envelope Yes Genome ssRNA Genome size 9,4 kb Stability Ether-sensitive, acid-sensitive Transmission Parenteral Prevalence Moderate Fulminant disease Rare Chronic disease Often Oncogenic

Model of Human Hepatitis C Virus Lipid Envelope Capsid Protein Nucleic Acid Envelope Glycoprotein E2 HCV was first recognized as a separate entity in 1975 after it was determined that the majority of cases of transfusion-related hepatitis were not caused by the 2 types of hepatitis recognized at the time (hepatitis A and B). The disease was called “non-A non-B” hepatitis until the virus was cloned and sequenced in 1989. At that time the virus was renamed hepatitis C virus. HCV is a linear single-strand RNA virus 40-50 nanometers in size that belongs to the Flaviviridae family and the Hepacivirus genus. The virus is covered with a lipid envelope and is encased with glycoprotein peplomers or “spikes.” www.hephet.com/nih/purcell.html www.clevelandclinicmeded.com/diseasemanagement/gastro/hepatitis_c/hepatitis_c.htm www.hepcprimer.com Envelope Glycoprotein E1

IRES = internal ribosomal entry site; UTR = untranslated region; HCV RNA Structure IRES, Translation Transcription, Replication Structural Non-Structural 5' UTR 3' UTR Structure Processing Replication C E1 E2 Nucleocapsid, Assembly Envelope Proteins, Assembly and Entry p7 Calcium Channel? NS2 NS3 NS4A Protease Serine Protease, Helicase NS3 cofactor NS4B NS5A NS5B Replication? Phosphoprotein, Replication RNA-dependent RNA polymerase HVC is composed of a single-stranded RNA genome approximately 9.6 kilobases long that encodes a single, large polyprotein of about 3,000 amino acids. This polyprotein is cleaved post-translationally into multiple structural and nonstructural (NS) peptides. The structural peptides include a nucleocapsid core (C), 2 envelope glycoproteins (E1, E2), and a calcium channel (p7). The NS proteins are labeled NS2 through NS5. Although the specific functions of the NS proteins have not been completely characterized, their presumed functions are illustrated in the slide. In particular, the highly conserved NS3 and NS5 segments of the genome contain structured RNA elements that are vital for the initiation of protein translation (both NS3 and NS5) and for RNA transcription (NS3). These structures represent potential attractive targets for anti-HCV therapy. Hoofnagle JH. Course and outcome of hepatitis C. Hepatology. 2002;36:S21-S29. Pawlotsky JM, McHutchison JG. Hepatitis C. Development of new drugs and clinical trials: promises and pitfalls. Summary of an AASLD hepatitis single topic conference, Chicago, IL, February 27-March 1, 2003. Hepatology. 2004;39:554-567. IRES = internal ribosomal entry site; UTR = untranslated region; C = nucleocapsid core; E1 = envelope protein 1; E2 = envelope protein 2; NS = non-structural

Hepatitis C: A Global Health Problem 170-200 Million (M) Carriers Worldwide Far East Asia 60 M Western Europe 5 M Eastern Europe 10 M United States 3-4 M Southeast Asia 30-35 M Africa 30-40 M Americas 12-15 M Hepatitis C: A Global Health Problem According to CDC estimates, approximately 3-4 million people in the US are currently infected with the hepatitis C virus (see map). There are, however, a few distinct geographic regions where infection is especially common. In Egypt, for example, HCV infection occurs in 10% to 30% of the general population. Likewise, the prevalence of infection is greater than 10% in certain parts of Asia and high rates of infection have been found in certain geographic regions of Japan, Taiwan, and Italy. In such areas, HCV infection is generally more prevalent among persons over the age of 40 and uncommon under the age of 20. This cohort effect suggests that transmission probably occurred through a practice that has been discontinued, such as traditional folk remedies or reuse of needles for injection. In developing countries, nosocomial transmission is most likely the principal route of HCV infection. Evidence from Egypt, Romania, and other nations, before economic transition, point to various percutaneous practices performed in the context of traditional and nontraditional medical care. The incidence of acute hepatitis C in the US has decreased during the last decade, from approximately 180,000 cases per year to approximately 30,000 to 50,000 cases per year. References Cohen J. The scientific challenge of hepatitis C. Science. 1999;285(5424):26-27. Alter MJ. Epidemiology of Hepatitis C. Hepatology. 1997;26(suppl 1):62S-65S. World Health Organization. Weekly epidemiological report. 1999;74:421-428. Available at: http://www.who.int/wer/pdf/1999/wer7449.pdf. Accessed February 19, 2003. Australia 0.2 M

Prevalence In Groups at Risk HCV - Epidemiology Prevalence In Groups at Risk Recipients of clotting factors before 1987 75 - 90% Injection drug users 70 - 85% Long-term hemodialysis patients 10% Individuals with > 50 sexual partners 10% Recipients of blood prior to 1990 5% Infants born to infected mothers 5% Long-term sexual partners of HCV positive 1 - 5% Health workers after random needlesticks 1 - 2% Slide 186 Prevalence In Groups at Risk These data are taken directly from Table 1 of the reference cited and are rounded to the nearest 5%. These numbers are estimates of the already-established prevalence in a particular group, not the prospective risk of infection. This information is useful in highlighting the relative importance of screening various groups for HCV infection.

Current Likelihood of Transmission Transfusion ~ 1 in 1,000,000 Maternal-Infant Mother HIV-negative ~ 5% Mother HIV-positive 15 - 20% Heterosexual partner ~1 in 1,000 per yr Needlestick injury HCV-positive source ~ 5% HCV status unknown ~ 1% Slide 187 Current Likelihood of Transmission A recent study of the risk of transmission by blood transfusion concluded that the risk was 1 in 250,000 to 1 in 25,000 per unit [Schreiber GB et al., New Engl J Med 1996;334:1685]. However, the current risk is likely to be lower, particularly away from high-prevalence urban areas. The risk of 1 in 1,000,000 is based on data from a major blood bank in the Midwest. See slide 185. The risk estimates for needlestick injury are averages from multiple studies.

HCV ACCOUNTS FOR 90-95% OF POST TRANSFUSION HEPATITIS RISK OF SEXUAL TRANSMISSION LOWER THAN FOR HBV RISK THROUGH CASUAL CONTACT LOW 39

HCV VERTICAL TRANSMISSION POSSIBLE RISK INCREASED IF MOTHER IS POSITIVE FOR HCV RNA RISK INCREASED IF MOTHER IS HIV POSITIVE OVERALL PREVALENCE ESTIMATED AT 1.4% 40

WHO IS AT GREATEST RISK FOR HCV INFECTION? SEXUALLY ACTIVE HOMOSEXUALS PERSONS WITH MULTIPLE AND FREQUENT SEXUAL CONTACTS MEDICAL/DENTAL PERSONNEL (3-10% VIA NEEDLESTICK FROM INFECTED PATIENT) DRUG ABUSERS BLOOD PRODUCT RECIPIENTS (ANTI-HCV SCREENING HAS GREATLY REDUCED RISK) HEMODIALYSIS PATIENTS LAB PERSONNEL WORKING WITH BLOOD PRODUCTS 41

Lecture Notes Approximately 60% of cases of hepatitis C are due to injection drug use. Fifteen percent of cases may be sexually transmitted. Ten percent of cases are due to transfusion-related infections. Unknown sources account for approximately 10% of cases and nosocomial, occupational, and perinatal exposure together account for another 5%.1 Reference 1. CDC. Hepatitis C Slide set. Available at: http://www.cdc.gov/ncidod/diseases/hepatitis/slideset. September 25, 2000. Accessed: February 5, 2002.

Lecture Notes Approximately 85% of infected individuals will develop chronic hepatitis. Of these, 20% to 50% will progress to develop cirrhosis. One quarter of those with cirrhosis will progress to hepatic decompensation and 1% to 4% will progress to hepatocellular carcinoma. This progression typically takes place over the course of 20 to 30 years.

Diagnostic Tests Hepatitis C antibody tests Qualitative HCV RNA tests Quantitative HCV RNA tests Genotyping Slide 222 Diagnostic Tests A variety of tests are available for the diagnosis and management of patients with chronic hepatitis C. These include antibody tests for detection of anti-HCV, qualitative and quantitative tests for detection of HCV RNA, and various methods of determining the genotype of HCV infection. Carithers RL Jr, Marquart A, Gretch DR. Diagnostic testing for hepatitis C. Semin Liver Dis 2000; 20:159-171. Pawlotsky JM. Molecular diagnosis of viral hepatitis. Gastroenterology 2002; 122:1554-1568. Pawlosky JM. Use and interpretation of virological tests for hepatitis C. Hepatology 2002; 36(Suppl 1):S65-S73.

Acute HCV Infection Anti-HCV ALT Symptoms Time After Exposure Weeks 400 600 800 1000 ALT (IU/L) 2 4 6 8 10 12 24 1 3 5 Anti-HCV Symptoms Weeks Months HCV RNA positive 200 7 Normal ALT Slide 229 Acute hepatitis C infection The diagnosis of acute hepatitis C can be quite difficult. Jaundice is infrequent and many patients have few, if any, symptoms. These diagnostic difficulties are compounded by considerable delay between HCV infection and the detection of antibodies in the patient’s serum. This was particularly true for early antibody tests for HCV in which the average time between infection and seroconversion was 16 weeks. This window has been progressively shorted with newer generations of antibody tests. However, even with the most sensitive newer tests the “window period” between HCV infection and seroconversion is at least 6-8 weeks. In contrast, HCV RNA can usually be detected within 10-14 days after infection. Carithers RL Jr, Marquart A, Gretch DR. Diagnostic testing for hepatitis C. Semin Liver Dis 2000; 20:159-171. Pawlosky JM. Use and interpretation of virological tests for hepatitis C. Hepatology 2002; 36(Suppl 1):S65-S73.

HCV Antibody Test Indicates past or present infection Inexpensive, sensitive and specific Poor positive predictive value in low prevalence populations Low sensitivity in immunosuppressed patients Slide 227 Antibody tests for hepatitis C Antibody tests for hepatitis C are inexpensive, sensitive and specific for prior HCV infection. In patients with clinical evidence of chronic liver disease, a positive antibody test for HCV has a positive predictive value > 90%. In contrast, in low risk populations, such as blood donors, the positive predictive value for a positive test is extremely low. Antibody tests for HCV have low sensitivity for infection in immunocompromised individuals. Carithers RL Jr, Marquart A, Gretch DR. Diagnostic testing for hepatitis C. Semin Liver Dis 2000; 20:159-171. Pawlosky JM. Use and interpretation of virological tests for hepatitis C. Hepatology 2002; 36(Suppl 1):S65-S73.

Qualitative HCV RNA (PCR) Confirms diagnosis of HCV infection Useful in the early diagnosis of acute hepatitis C Demonstrates the presence of active infection “Gold standard” for documenting response to treatment Slide 228 Qualitative tests for HCV RNA Detection of HCV RNA in a patient’s blood confirms the presence of active infection. The most sensitive methods for doing so are the reverse transcription polymerase chain reaction (PCR) and transcription-mediated amplification (TMA). Qualitative tests for HCV RNA are extremely sensitive methods for determining the presence of hepatitis C virus. Most of these tests have a sensitivity of at least 50 IU/ml. Because of their sensitivity, these tests are useful in documenting successful clearance of virus during and after completion of antiviral therapy. To avoid false negative results from these tests, serum or plasma should be separated from whole blood within 4 hours, followed by rapid storage of specimens at –70°C. Carithers RL Jr, Marquart A, Gretch DR. Diagnostic testing for hepatitis C. Semin Liver Dis 2000; 20:159-171. Pawlotsky JM. Molecular diagnosis of viral hepatitis. Gastroenterology 2002; 122:1554-1568 Pawlosky JM. Use and interpretation of virological tests for hepatitis C. Hepatology 2002; 36(Suppl 1):S65-S73.

Potential HCV Therapies ViroPharma/ Wyeth Albuferon Human Genome Sciences VX-950 Vertex R803 Rigel HCV/MF59 Chiron Phase I JTK 003 AKROS Pharma Oral IFN alpha Amarillo Biosciences SCH-6 Schering HepX-C XTL NM283 Idenix ANA245 ANADYS Ceplene Maxim Multiferon Viragen IDN-6556 Idun Time to Market ISIS 14803 Isis Phase II VX-497 Vertex Infergen/gamma IFN InterMune Civacir NABI E-1 Innogenetics Omega IFN Biomedicine IP-501 Indevus Viramidine Valeant Amantadine Endo Labs Solvay REBIF Ares-Serono Zadaxin SciClone Phase III

Characteristics of hepatitis D viruses Family Unclassified Genus Deltavirus Virion 35 nm spherical Envelope Yes (HBsAg) Genome ssRNA Genome size 1,7 kb Stability Acid-sensitive Transmission Parenteral Prevalence Low, regional Fulminant disease Frequent Chronic disease Often Oncogenic ?

Hepatitis Delta Virus (HDV)

HDV INFECTION PATTERNS COINFECTION ACUTE SIMULTANEOUS INFECTION WITH HBV AND HDV OFTEN RESULTS IN FULMINANT INFECTION (70% CIRRHOSIS) SURVIVORS RARELY DEVELOP CHRONIC INFECTION (< 5%) 51

HDV INFECTION PATTERNS SUPERINFECTION RESULTS IN HDV SUPERINFECTION IN AN HBsAg CARRIER (CHRONIC HBV) CAN OCCUR ANYTIME DURING CHRONIC DISEASE USUALLY RESULTS IN RAPIDLY PROGRESSIVE SUBACUTE OR CHRONIC HEPATITIS 52

HDV - Coinfection Months HDV RNA IgM anti-HDV IgG anti-HDV HDAg IgG anti-HBc ALT HBsAg anti-HBs IgM anti-HBc Slide 168 HDV Co-infection Coinfection with HDV and HBV is characterized by the presence of markers of HDV and HBV during the acute phase. Coinfection is more likely to lead to a fulminant course than acute HBV infection alone.

HDV - Superinfection ALT HDV RNA IgG anti-HDV IgM anti-HDV HDAg HBV DNA Years HBsAg, IgG anti-HBc Slide 169 HDV Superinfection HDV superinfection occurs in an individual already chronically infected with HBV. Diagnosis is suggested by a negative test for anti-HBc IgM, positive HBsAg, and presence of HDV markers. Clinically, it presents like an acute exacerbation of chronic hepatitis in a HBV carrier, occasionally the course is fulminant. Since the HBsAg carrier permits continuous replication of HDV, the vast majority (over 90%) of HDV superinfection progresses to chronic infection. Hadziyannis S, et al. The role of the hepatitis delta virus in acute hepatitis and in chronic liver disease in Greece. Prog Clin Biol Res1991;364:51-62.

HDV Transmission: Oral No Percutaneous Common Sexual Yes, rare Perinatal Incubation period 21 - 45 days Jaundice Unknown Fulminant 2 – 7.5% Diagnostic tests: Acute infection IgM anti-HDV Chronic infection IgG anti-HDV, HBsAg + Immunity Not applicable Case-fatality rate 1 – 2% Chronic infection: Superinfection 80% Coinfection < 5%

HEPATITIS E VIRUS Virus Hepatitis E Family Caliciviridae Genus Unnamed Virion 30-32 nm, icosahedral Envelope No Genome ssRNA Genome size 7,6kb Stability Heat-stable Transmission Fecal-oral Prevalence Regional Fulminant disease In pregnancy Chronic disease Never Oncogenic

Suspected from study of waterborne hepatitis in India in 1980 Confirmed by transmission to chimp and human in 1983 Probably accounts for many historical outbreaks of hepatitis Endemic mainly in Asia, Middle East, North Africa Epidemiology Slide 315 Epidemiology Although HEV was discovered relatively recently, it is likely that it has accounted for most of the large-scale outbreaks of hepatitis around the world. Cases outside of endemic areas are uncommon and sporadic.

Epidemiology Fecal-oral transmission (human to human) Contaminated water supplies in tropical or subtropical developing countries Mainly young adults Can infect primates, swine, sheep, rats Swine may be reservoir of infection in North America (attenuated virus) Maternal-infant transmission occurs and is often fatal Epidemiology Slide 316 Epidemiology Transmission is not usually directly from human to human, as the attack ratre within households is only about 2% [Smith]. Contaminated water supplies are usually the route of infection in large epidemics. Sporadic cases often have no clear source of infection. The rare individuals who acquire infection from swine seem to have a mild illness due to attenuation of the virus. Hepatitis E is a dangerous illness in the last trimester of pregnancy, because essentially all newborns acquire the infection from their mothers and the case fatality rate is high (next slide).

Clinical Characteristics Similar to hepatitis A Can cause severe acute hepatitis Subclinical infection is common Attenuated virus from animal reservoirs Low-dose infections often asymptomatic No chronic infection Up to 20% mortality among pregnant women (esp. third trimester) Clinical Characteristics Slide 317 Clinical Characteristics The clinical spectrum of hepatitis E is similar to that of hepatitis A. Both may cause a severe acute hepatitis, but the infection usually is associated with mild disease. Probably half of infections are subclinical. Neither hepatitis A nor hepatitis E infection becomes chronic. Both have a high case fatality rate in late pregnancy. See slide 6 for a further comparison.

Course of Acute Infection 10 20 30 40 50 60 Time After Infection (days) 1 2 Viral Replication IgM Antibody IgG Antibody Viremia ALT Virus in Stool (years) Symptoms Slide 318 Course of Acute Infection Virus can be detected in stool for some time prior to the onset of symptoms, accounting for the virus’s success during large-scale outbreaks. IgM antibody may persist for up to a year. IgG antibody can be detected long-term and confers immunity.

Prevention Does not prevent infection May ameliorate hepatitis Passive (Immune serum globulin) Does not prevent infection May ameliorate hepatitis Active (Vaccine) Anti-ORF2 prevents infection in chimps and humans Clinical trials in progress Prevention Slide 320 Prevention Worldwide prevention mainly relates to increasing the level of sanitation in developing countries. Travelers to endemic areas can protect themselves by boiling drinking water and eating only thoroughly cooked food.

HEV Transmission: Oral Common No Percutaneous Unknown Sexual No Perinatal Yes, unknown frequency Incubation period 15 - 60 days Jaundice Common Fulminant <1%, in pregnancy up to 30% Diagnostic tests: Acute infection IgG anti-HEV (seroconversion) Chronic infection Not applicable Immunity Case-fatality rate 0,5 – 4 % 1.5 – 21% in pregnant women None

HEPATITIS G VIRUS Virus Hepatitis G Family Flaviviridae Genus Unnamed Virion 60 nm, spherical Envelope Yes Genome ssRNA Genome size 9,4 kb Stability Ether-sensitive Transmission Parenteral Prevalence Moderate Fulminant disease ? Chronic disease Oncogenic

HGV AND GVB-C SHARE 95% AMINO ACID IDENTITY THUS REPRESENT DIFFERENT ISOLATES OF THE SAME HUMAN VIRUS “HEPATITIS C-LIKE VIRUS” CLASSIFIED IN THE FLAVIVIRIDAE FAMILY  SAME AS HCV GENETIC ORGANIZATION SIMILAR TO HCV GENONE CONSISTS OF SINGLE-STRANDED RNA MOLECULE OF POSITIVE POLARITY 2

HGV - EPIDEMIOLOGY TRANSMISSABLE BY BLOOD AND BLOOD PRODUCTS PRESENT IN ASYMPTOMATIC BLOOD DONORS WITH NORMAL ALT LEVELS FOUND IN: GENERAL POPULATION 1-2 % HEMOPHILIA PATIENTS 18 % IV DRUG USERS 33 % Patients with chronic Hepatitis B 10 % Patients with chronic Hepatitis C 20% 2

HGV - CLINICAL SIGNIFICANCE RECENT DATA SUGGESTS: HGV INFECTION DOES NOT CAUSE ACUTE HEPATITIS HGV MAY ESTABLISH CHRONIC INFECTIONS FREQUENTLY OCCURS WITH HBC AND HCV INFECTIONS MAY NOTQUALIFY AS A TRUE HEPATITIS VIRUS 2

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