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C hair of Medical biology, M icrobiology, V irology, and I mmunology. Causative agents of viral hepatites. Lecturer Prof. S.I. Klymnyuk.

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slide1

Chair of Medical biology, Microbiology, Virology, and Immunology

Causative agents of viral hepatites

Lecturer Prof. S.I. Klymnyuk

slide2

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.

slide3

Table 1

Characteristics of Human Hepatitis Viruses

slide4

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

slide5

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.

slide6

Global Prevalence of Hepatitis A Infection

HAV Prevalence

High

Intermediate

Low

Very Low

slide7

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.

slide9

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.)

slide10

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.

slide12

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.

slide13

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

slide14

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

slide15

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.

slide16

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.

slide17

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
concentration of hepatitis a virus in various body fluids
Concentration of Hepatitis A Virusin Various Body Fluids

Feces

Body Fluid

Serum

Saliva

Urine

102

104

100

106

108

1010

Infectious Doses per ml

slide19

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

slide20

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.

slide21

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

slide23

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

slide24

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

slide25

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.

slide26

>8%

2-8%

<2%

HBV - Epidemiology

Prevalence of HBsAg Carrier State

WHO

slide27

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.

slide29

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.

slide30

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).

slide33

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.

slide34

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.

slide35

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.

slide36

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.

the core particle then releases it s contents of dna and dna polymerase into the liver cell nucleus
The core particle then releases it’s contents of DNA and DNA polymerase into the liver cell nucleus.
slide40
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.

slide42

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.

slide43

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 .

slide44

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.

genome of hbv virus
Genome of HBV virus

Genome: 3.200 nucleotídes

S

P

C

X

slide46

Pré S1

Pré S2

gene S

gene C

Pré C

HBV Genome

AgHBs

DNA Polimerase

gene P

AgHBc

AgHBe

slide47

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

slide49

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.

slide50

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.

slide51

Neonatal transmission also appears to occur during childbirth. The incidence is increased significantly if the mother's blood contains HBcAg. For example, in a study from Taiwan, a 32% transmission rate was observed, and the transmission could be correlated with HBcAg-positive cord blood. The presence of HBsAg in breast milk also suggests an additional vehicle for the transmission of HBV to the newborn. The demonstration of infectious virus in semen presents the possibility that virus can be sexually transmitted. In hospitals, HBV infections are a risk for both hospital personnel and patients because of constant exposure to blood and blood products.

hbv spread mainly by parenteral route
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)
hbv spread mainly by parenteral route1
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
who is at greatest risk for hbv infection
WHO IS AT GREATEST RISK FOR HBV INFECTION?
  • DRUG ABUSERS
  • BLOOD PRODUCT RECIPIENTS
    • ACCOUNTS FOR 5-10% POSTRANSFUSION HEPATITIS
  • HEMODIALYSIS PATIENTS
  • PEOPLE FROM SOUTHEAST ASIAN COUNTRIES (70-80%)
who is at greatest risk for hbv infection1
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
slide56
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%
slide57

PATHOGENESIS OF HEPATITIS B VIRUS INFECTIONS.

Acute hepatitis caused by HBV cannot be clinically distinguished from hepatitis caused by HAV. However, several characteristics differentiate the infections caused by these viruses (see Table 2). HBV infections are characterized by a long incubation period, ranging from 50 to 180 days. Symptoms such as fever, rash, and arthritis begin insidiously, and the severity of the infection varies widely. Mild cases that do not result in jaundice are termed anicteric. In more severe cases, characterized by headache, mild fever, nausea, and loss of appetite, icterus (jaundice) occurs 3 to 5 days after the initial symptoms. The duration and severity of the disease vary from clinically inapparent to fatal fulminating hepatitis. The overall fatality rate is estimated to be 1% to 2%, with most deaths occurring in adults older than 30 years of age.

slide60

The duration of uncomplicated hepatitis rarely is more than 8 to 10 weeks, but mild symptoms can persist for more than 1 year. The mechanism of hepatic damage of HBV is not established, but considerable data support the notion that most of the liver damage that occurs during acute or chronic hepatitis is mediated by a cellular immune response directed toward the new antigens deposited in the cell membrane of the infected cell.

Based on the ultimate pattern of the disease, this disease can be divided into two categories: self- limiting acute infections and chronic infections.

characteristics of hbv infection
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
slide62

Table 2

Differential Characteristics of Hepatitis A and Hepatitis B

slide63

Table 2

Differential Characteristics of Hepatitis A and Hepatitis B

slide64

Self-Limiting Hepatitis B Virus Infections.

Self limiting infections can be inapparent or can result in a clinical hepatitis with jaundice lasting 4 to 5 weeks. HBsAg may or may not be present in the blood, but, if present, it usually disappears as the symptoms of hepatitis subside and the jaundice clears. Antibodies to HBcAg, HBeAg, and HBsAg arise at different periods during the infection and can remain detectable for years after recovery. There seems to be a good immune response to groupspecific determinants, because recover)' appears to provide immunity to different subtypes of the virus.

slide65

Chronic Hepatitis B Virus Infections. Between 6% and 10% of clinically diagnosed patients with hepatitis B become chronically infected and continue to have HBsAg in their blood for at least 6 months, and sometimes for life. Chronic infections can be subdivided into two general categories: chronic persistent hepatitis and chronic active hepatitis. The latter is the most severe and often eventually leads to cirrhosis or the development of primary hepatocellular carcinoma. Worldwide, it has been estimated that there are more than 200 million permanently infected carriers of HBV, of which about 1million reside in the United States. The prevalence of chronic carriers varies widely in different parts of the world, from 0.1% to 0.5% in the United States to up to 20% in China, Southeast Asia, and some African countries.

slide66

The perinatal infection of newborn infants born to chronically infected mothers results in a high incidence of chronic infection (90%), which often is lifelong. This is particularly disquieting in the developing countries of Asia and Africa, where carrier rates are high. It has been estimated that HBV is the most common single cause of liver disease in the world.

All carriers have antibodies to HBcAg, and some have antibodies to HBeAg. Those who do not possess antiHBe may have circulating HBeAg. Carriers with high concentrations of Dane particles and circulating HBeAg appear to be more likely to suffer liver damage than those in whom only HBsAg can be detected, but the validity of this proposal is yet to be established. However, such persons are much more likely to be transmitters of the disease than are those who have solely HBsAg in their blood.

slide67

Several cases of membranous glomeulonephritis have been described in HBsAg-positive children, and it has been reported that the glomerulonephritis results from the deposition of immune complexes consisting of anti-HBe IgG and HBeAg.

The mechanism by which carriers can remain persistently infected and yet be asymptomatic is unknown. However, prolonged carrier status is seen in association with chronic hepatitis in patients with lowered immunity and in those infected during the neonatal period or early childhood.

slide68

Virus-Host Immune Reactions. Currently there is evidence for at least 3 hepatitis viruses—type A (short incubation hepatitis virus), type B (long incubation hepatitis virus), and the agent or agents of non-A, non-B hepatitis. A single infection with any confers homologous but not heterologous protection against reinfection. Infection with HBV of a specific subtype, eg, HBsAg/adw, appears to confer immunity to other HBsAg subtypes, probably because of their common group a specificity.

Most cases of hepatitis type A presumably occur without jaundice during childhood, and by late adult­hood there is a widespread resistance to reinfection. However, serologic studies in this country indicate that the incidence of infection among certain populations may be declining as a result of improvements in sanitation commensurate with arise in the standard of living. It has been estimated that as many as 50-75% of young middle to upper income adults in the USA may be susceptible to type A hepatitis. Younger people who live in poorer circumstances or crowded institutions (eg, the armed forces) are at increased risk.

slide69

The immunopathogenetic mechanisms that result in viral persistence and hepatocellular injury in type B hepatitis remain to be elucidated. An imbalance be­tween suppressive and cytopathic immune responses of the host has been hypothesized to account for the various pathologic manifestations of this disease. It is postulated that antibody-dependent, complement-mediated cytolysis or cellular effector mechanisms are responsible for the hepatic injury observed, whereas noncytopathic synthesis of viral components, surface expression of viral antigens or liver-specific neoantigens, and shedding of virus are primarily modulated by the humoral immune response.

slide70

Various host responses, immunologic and genetic, have been proposed to account for the higher frequency of HBsAg persistence observed in infants or children compared to adults and in certain disease states, eg, Down's syndrome, leukemia (acute and chronic lymphocytic), leprosy, thalassemia, and chronic renal insufficiency. Patients with Down's syn­drome are particularly prone to persistent antigenemia (but low antibody frequency) and inapparent infec­tions, and they show a significantly greater prevalence of these disorders than is found in other mentally retarded patients. This does not imply that these patients have an increased susceptibility to HBV. On the contrary, among other equally exposed patients who are residents within the same institution, the total serologic evidence of HBV infection is similar except that the antigen carrier rate is low whereas the antibody prevalence is high. An immunologic difference in the host response to the virus is apparently responsible for this serologic dichotomy.

slide71

Persistent antigenemia and mild or subclinical infections are more frequently observed in individuals who have been infected with low doses of virus. Corre­spondingly, a direct relationship between virus dose and time of appearance of HBsAg or an abnormal ALT value has been reported; i.e., the incubation period be­comes longer as the dose of virus diminishes.

The frequency of the chronic HBsAg carrier state following acute icteric type B hepatitis is not known but is probably under 10%. More than half of these patients continue to exhibit biochemical and histologic evidence of chronic liver disease, i.e., chronic persistent or chronic active hepatitis.

slide72

Primary Hepatocellular Carcinoma. A considerable amount of evidence has documented the close association between HBV infection and the development of primary hepatocellular carcinoma. Hepatocellular carcinoma is the most common cancer in the world, with at least 250,000 new cases reported annually. Patients with hepatocellular carcinoma often have high levels of HBsAg, and the carcinoma cells often contain integrated HBV DNA. Further evidence for the link between persistent HBV infections and hepatocellular carcinoma comes from epidemiologic data showing that the risk of developing primary hepatocellular carcinoma is more than 200 times higher in HBV carriers than in noncarriers. Within some populations, the risk of developing primary hepatocellular carcinoma is as high as 50% in male chronic carriers.

slide73

However, HBV infection is not solely responsible for tumor development, because the carrier state often exists for a lengthy period (often 40 years or more) before the onset of liver cancer In addition, the predominance of hepatocellular carcinoma in men indicates that other factors, including sex related factors, contribute to the development of this cancer. Nonetheless, an important component of chronic liver disease is the continual regeneration of damaged or destroyed hepatocytes, which, coupled with HBV replication and exposure to environmental carcinogens, likely contributes in a significant fashion to tumor development and progression. The relationships between oncogene activation, loss of tumor suppressor genes, and the HBV are under active investigation.

Because of the close egidemiologic link between chronic HBV infection and hepatocellular carcinoma, it is hoped that mass vaccination of susceptible individuals in such countries as China and Taiwan will reduce the overall incidence of HBV infection, and that this eventually will reduce dramatically the incidence of hepatocarcinoma.

slide74

DIAGNOSIS OF HEPATITIS B VIRUS INFECTIONS. As in all cases of viral hepatitis, abnormal liver function is indicated by increased levels of liver enzymes such as serum glutamic oxaloacetic transaminase and alanine aminotransferase (ALT). The presence of HBsAg confirms a diagnosis of hepatitis B, and its serologic detection is routinely carried out in diagnostic laboratories and blood banks using radioimmunoassays or enzyme-linked immunosorbent assay's.

slide75

Acute Infection

HBV DNA

HBeAg

Anti-HBe

Anti-HBs

Anti-HBc

HBsAg

Anti-HBc IgM

0 2 4 6

Months Years

HBV - Diagnosis

slide76

HBV DNA

HBeAg

Anti-HBe

HBsAg

Anti-HBc IgG

Anti-HBc IgM

Months

Years

HBV - Diagnosis

Chronic Infection

practice
PRACTICE
  • HBsAg N.
  • HBcAB (TOTAL) N.
  • HBsAB N.
  • HAV-IGM N.
  • HCV N.
  • NO evidence of viral hepatitis viruses.
practice1
PRACTICE
  • HBsAG N.
  • HBcAB (TOTAL) P.
  • HBsAB P.
  • HAV-IGM N.
  • HCV N.
  • PAST INFECTION.
practice2
PRACTICE
  • HBsAg N.
  • HBcAB (total) N.
  • HBsAB P.
  • HAV-IGM N.
  • HCV N.
  • IMMUNIZATION.
practice3
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.
practice4
PRACTICE
  • HBsAg P.
  • HBcAB (TOTAL) P.
  • HBsAB N.
  • HAV-IGM P.
  • HCV P.
  • Co infection with HBV, HAV, and HCV
practice5
PRACTICE
  • HBsAG P.
  • HBcAB (total) P.
  • HBsAB P.
  • HAV-IGM N.
  • HCV N.
  • Past infection with recovery, and then re-infection that has become chronic, this is very rare but does happen.
slide84

CONTROL OF HEPATITIS B VIRUS INFECTIONS. The examination of all donor blood for the presence of HBsAg is now routine, and this practice has done much to control the occurrence of posttransfusion hepatitis B infections.

Passive immunization of human volunteers with hepatitis B immune globulin (HBIG) has been shown to prevent disease when the volunteers were challenged with infectious material, but the use of immune globulin is not effective for the treatment of active disease. One important and effective use for HBIG, however, is the prevention of active hepatitis B infections in neonates born to mothers who are chronic carriers of HBsAg. HBIG also can be given to nonimmune individuals known to have been exposed to HBV.

Active immunization with HBsAg promises to provide a vehicle for the control of hepatitis B.

slide85

Clinical trials in high-risk populations have shown that the incidence of hepatitis B in persons actively immunized with HBsAg is decreased by about 95%. Moreover, immunization even during the long incubation period may be efficacious in preventing HBV infections. Because HBV has not been grown in cell cultures, the first vaccine consisted of highly purified, formalin-inactivated HBsAg particles obtained from the plasma of persistently infected carriers. This vaccine has now been superseded by a recombinant vaccine, in which the gene for HBsAg has been cloned in yeast, enabling the production of polypeptides carrying the antigenic determinants of HBsAg in large amounts. The yeast-produced vaccine has been licensed for use and has been given to more than 2 million people in the United States.

slide86

The vaccine is considered safe and provides effective protection. Administration of the HBV vaccine world-wide has the potential to reduce drastically the incidence of HBV infection. Early studies have shown that its use in HBV-positive pregnant women reduces the percentage of infants who become carriers from 90% to 23%. In addition, if HBIG is used in conjunction with the vaccine, the newborn carrier incidence can be reduced to less than 5%. Taking note of the fact that many chronic HBV carriers eventually die of liver disease, tills vaccine represents the first prophylactic measure to substantially reduce or prevent cirrhosis and human cancer.

slide87

Vaccine Age Group Dose Volume # Doses

(ug) (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)

Adults on

hemodialysis 40 1.0* 3 (mo 0,1,6)

HBV - Vaccine

slide88

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

slide89

HBV - Therapy

Therapeutic Agents

Immune Modulators Nucleo(s)tide analog

InterferonLamivudine

Thymosin Adefovir dipivoxil

Therapeutic vaccines Emtricitabine

Entecavir

L-dT/ L-dC

Clevudine

Famciclovir

slide90

HEPATITIS C VIRUS. When it became clear that most cases of transfusion-associated hepatitis probably were caused by a hitherto unknown virus, molecular genetic and recombinant DNA techniques were used to identify, clone, and sequence putative agents. This led to the isolation of a new RNA virus, HCV. Sequence analysis has revealed that HCV is organized in a manner similar to the flaviviruses and that it shares biologic characteristics with this family.

slide93

Model of Human Hepatitis C Virus

Lipid Envelope

Capsid Protein

Nucleic Acid

Envelope Glycoprotein E2

Envelope Glycoprotein E1

slide94

This has led to a classification of HCV as a genus within the flavivirus family. About 80% of patients with chronic, post-transfusion NANB hepatitis in Italy and Japan have been shown to have antibodies to HCV, and 58% of patients with NANB hepatitis in the United States, with no known parenteral exposure to the virus, have HCV antibodies. Based on these data, it seems likely that HCV is a major contributor to NANB hepatitis throughout the world. Most infected individuals become chronic carriers of the virus, and many develop chronic hepatitis. Studies in several urban areas have shown that as many as 80% of intravenous drug abusers have been infected with HCV. The development of commercial antibody tests to detect HCV infection has markedly reduced the number of cases of NANB hepatitis acquired from transfusions and blood products.

slide95

C

E1

E2

p7

NS2

NS3

NS4A

NS4B

NS5A

NS5B

Nucleocapsid, Assembly

Protease

NS3 cofactor

Phosphoprotein, Replication

Calcium Channel?

Envelope Proteins, Assembly and Entry

Serine Protease, Helicase

Replication?

RNA-dependent RNA polymerase

HCV RNA Structure

Transcription, Replication

IRES, Translation

Structural

Non-Structural

5' UTR

3' UTR

Structure

Processing

Replication

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
Hepatitis C: A Global Health Problem

170-200 Million (M) Carriers Worldwide

Far East Asia

60 M

Eastern

Europe

10 M

Western

Europe

5 M

United States

3-4 M

Southeast Asia

30-35 M

Africa

30-40 M

Americas

12-15 M

Australia

0.2 M

slide97

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%

HCV - Epidemiology

slide98

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%

slide99
HCV
  • ACCOUNTS FOR 90-95% OF POST TRANSFUSION HEPATITIS
  • RISK OF SEXUAL TRANSMISSION LOWER THAN FOR HBV
  • RISK THROUGH CASUAL CONTACT LOW
slide100
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%

who is at greatest risk for hcv infection
WHO IS AT GREATEST RISK FOR HCV INFECTION?
  • DRUG ABUSERS
  • BLOOD PRODUCT RECIPIENTS (ANTI-HCV SCREENING HAS GREATLY REDUCED RISK)
  • HEMODIALYSIS PATIENTS
  • LAB PERSONNEL WORKING WITH BLOOD PRODUCTS
who is at greatest risk for hcv infection1
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)
slide106

Diagnostic Tests

Hepatitis C antibody tests

Qualitative HCV RNA tests

Quantitative HCV RNA tests

Genotyping

HCV - Diagnosis

slide107

Acute HCV Infection

1000

HCV RNA positive

800

Anti-HCV

600

ALT

(IU/L)

Symptoms

400

200

Normal ALT

0

0

2

4

6

8

10

12

24

1

2

3

4

5

6

7

Weeks

Months

Time After Exposure

HCV - Diagnosis

Hoofnagle JH, Hepatology 1997; 26:15S

slide108

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

HCV - Diagnosis

slide109

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

HCV - Diagnosis

potential hcv therapies
Potential HCV Therapies

HCV-086

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

VX-497

Vertex

Phase II

Infergen/gamma IFN

InterMune

Civacir

NABI

E-1

Innogenetics

Omega IFN

Biomedicine

IP-501

Indevus

Viramidine

Valeant

REBIF

Ares-Serono

Amantadine

Endo Labs

Solvay

Zadaxin

SciClone

Phase III

slide111

HEPATITIS DELTA VIRUS. Hepatitis delta virus was first described in 1977 as a novel antigen-antibody complex detected by immunofluorescence in hepatocyte nuclei of patients with chronic HBV infection and chronic hepatitis. Although HDV antigen was initially observed in Italy, it has been detected world-wide, primarily in HBV carriers who have had multiple exposures to blood and blood products (Table 3).

slide115

Table 3.

Prevalence of Delta Infection in Hepatitis B Virus (HBV) Carriers and Persons With HBsAg-Positive Acute and Chronic Hepatitis in North America

slide116

Transmission experiments in chimpanzees and other studies have shown that HDV is a transmissible and pathogenic agent that requires concomitant replication of HBV to provide certain helper functions. The HDV virion is a spherical, 36-nm enveloped particle with a chimeric structure; the genome consists of a 1.7-kilobase RNA molecule specific for HDV, whereas the envelope contains HBV encoded HBsAg. The HDV genomic RNA is a circular, single stranded RNA similar in structure to certain pathogenic RNAs or plants (viroids), and its replication requires the concomitant expression of HBV gene products

slide117

Two principal modes of HDV infection have been described (1) coinfection (the simultaneous introduction of both HBV and HDV into a susceptible host), and(2) superinfection (the infection of an HBV carrier with HDV). Simultaneous exposure to HBV and HDV leads to a typical pattern of HBV disease, with the duration of HBV infection being the limiting factor to the expression of HDV. The outcome of such HBV/HDV coinfections usually is similar to that of infection with HBV alone, and chronic infections seem to be established with the same frequency.

The clinical outcome from HDV superinfection of an HBV carrier is markedly different In this case, the persistent HBV infection promotes the efficient replication of the defective HDV and leads to a fulminant HBsAg-positive hepatitis with a significant mortality rate (5% to 15%). In addition, the chronic infection with HBV potentiates the continued replication of HDV, establishing a chronic HDV infection. There are few data to support a role for HDV in the development of primary hepatocellular carcinoma.

slide118

HDV transmission is linked closely to that of its helper, HBV. Parenteral inoculation accounts for the world-wide distribution of HDV among drug addicts. In parts of the world with a low incidence of HBV, HDV infections are found mostly in drug addicts and other individuals at risk for being HBV carriers HDV infection of newborns occurs only in babies born to HBcAg-positive, HDV infected mothers. Although HDV is found worldwide, an interesting anomaly exists in that HDV infection is endemic in South America, resulting in severe outbreaks of fulminant hepatitis. In contrast, HDV infections are rare in Asia, although the prevalence of HBsAg carriers is similar to that in South America. Overall, it has been estimated that about 5% of chronic HBV carriers also are infected with HDV.

Because no HDV vaccine is available, controlling the transmission of HBV is the only approach to controlling the spread of HDV. Unfortunately for the estimated 200 million HBsAg carriers in the world, there is no effective measure to prevent HDV infection per se

hdv infection patterns
HDV INFECTION PATTERNS
  • COINFECTION
    • ACUTE SIMULTANEOUS INFECTION WITH HBV AND HDV
    • OFTEN RESULTS IN FULMINANT INFECTION (70% CIRRHOSIS)
    • SURVIVORS RARELY DEVELOP CHRONIC INFECTION (< 5%)
hdv infection patterns1
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
slide121

ALT

HDV RNA

IgG anti-HDV

IgM anti-HDV

HDAg

IgM anti-HBc

IgG anti-HBc

HBsAg

anti-HBs

Months

HDV

HDV - Coinfection

slide122

ALT

HDV RNA

IgG anti-HDV

IgM anti-HDV

HDAg

HBV DNA

HBsAg, IgG anti-HBc

Years

HDV

HDV - Superinfection

slide124
HDV

Transmission

Oral No

Percutaneous Common

Sexual Yes, rare

Perinatal No

Incubation period 21 - 45 (days)

Clinical Illness at 10%, higher with

presentation superinfection

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%

slide125

HEPATITIS E VIRUS. Many cases of acute viral hepatitis in Asia and Africa are caused by a virus that is transmitted through the fecal-oral route but is unrelated to HAV. Outbreaks of this disease also have been confirmed in other parts of the world, including the Middle East and Mexico. The disease usually is caused by the ingestion of fecally contaminated water. The virus causing this kind of hepatitis has been named HEV. The first verified hepatitis E outbreak was documented in New Delhi, India, in 1955 In this epidemic, 29,000 cases of icteric hepatitis were reported after fecal contamination of the city's drinking water. Several other outbreaks have been linked to HEV since then HEV is a small, nonenveloped RNA virus. Recent information about the genomic organization and other properties of the virus strongly suggests that it is a calicivirus and should be placed in a new genus within this family.

slide128

Epidemiology

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

Hepatitis E

slide129

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

Hepatitis E

slide130

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)

Hepatitis E

slide131

Course of Acute Infection

Viral Replication

IgM Antibody

IgG Antibody

ALT

Viremia

Symptoms

Virus in Stool

0

10

20

30

40

50

60

1

2

Time After Infection (days)

(years)

Hepatitis E

slide132

Prevention

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

Hepatitis E

slide133

HEV

Transmission

Oral Common

Percutaneous Unknown

Sexual No

Perinatal Yes, unknown frequency

Incubation period 15 - 60

(days)

Clinical Illness at 70 – 80% in adults

presentation

Jaundice Common

Fulminant <1%, in pregnancy up to 30%

Diagnostic tests

Acute infection IgG anti-HEV (sero-

conversion)

Chronic infection Not applicable

Immunity Not applicable

Case-fatality rate 0.5 – 4%

1.5 – 21% in pregnant women

Chronic infection None

hgv and gvb c
HGV AND GVB-C
  • SHARE 95% AMINO ACID IDENTITY
  • THUS REPRESENT DIFFERENT ISOLATES OF THE SAME HUMAN VIRUS
slide136
HGV
  • “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
hgv epidemiology
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%

hgv clinical significance
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