Unit 4: Virology
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Lecture 14+15+16 - Viral Hepatitis
Viral hepatitis is a systemic disease primarily involving
the liver. Most cases of acute viral hepatitis in children
and adults are caused by one of the following viruses:
HAV, HBV, HCV, HDV the defective virus dependent on
co-infection with HBV, and HEV. There are additional
viruses that can cause sporadic hepatitis: yellow fever
virus, CMV, EBV, herpes virus, rubella virus and
enteroviruses.
Hepatitis viruses produce acute inflammation of the liver,
resulting in clinical illness characterized by fever, GIT
symptoms (Nausea , vomiting and jaundice).
Regardless of the virus type, identical histopathologic
lesions are observed in the liver during acute disease.
Hepatitis A virus
Disease
HAV causes hepatitis A.
Properties
HAV is a typical enterovirus classified in the picorna
virus family. It has single-stranded RNA genome and
non-enveloped icosahedral nucleocapsid and replicate in
the cytoplasm of the cell. Replicative cycle is similar to
that of other enteroviruses.
HAV is stable to treatment with 20% ether , acid (pH 1
for 2 hr),, and heat (60 c for 1 hr) and its infectivity can be
preserved for at least one month after being dried and
stored at 25 c or years at -20c .
The virus is destroyed by boiling in water for 5 min.,
autoclaving ( 121 c for 20 min.), dry heat ( 181 c for 1hr),
UV light (1 min. at 1.1 watt), treatment with formalin
(1:4000 for 3 days at 37 c) or treatment with chlorine (
10-15 ppm for 30 min.).
Heating food to above 85 c for 1 min. and disinfecting the
surfaces with sodium hypochlorite (1:100 dilution of
chlorine bleach) are necessary to inactivate HAV.
Transmission and epidemiology
1) HAV is transmitted by feco-oral route.
2) Humans are the reservoir for HAV.
3) Virus is detected in the stool from about 2 weeks before
the onset of the jaundice up to 2 weeks after.
4) Children are the most frequently infected group, and
outbreaks occur in special living situations such as
summer camps, and boarding school.
5) Common source of outbreak is from fecally contaminated
water or food.
6) HAV rarely transmitted via blood, because the level of
viremia is low and chronic infection doesn't occur.
7) There is no chronic hepatitis and no chronic carrier state.
There is no predisposition to hepatocellular carcinoma.
Clinical findings
The clinical manifestations of hepatitis are virtually the
same regardless of which hepatitis virus is the cause.
Fever, anorexia, nausea, vomiting, and jaundice arte
typical. Dark urine, pale feces, and elevated transaminase
level are seen. Most cases resolve spontaneously in 2-4
weeks.
HAV incubation period is about 3-4 weeks. Most case of
HAV infections are asymptomatic, and are detected solely
and are detected by the presence of IgG antibody.
Laboratory diagnosis
1) IgM specific Anti-HAV in the acute phase. Peaking 2
weeks after elevation of liver enzyme. Anti-HAV IgM
usually decline to non-detectable level within 3-6 months
2) Anti-HAV IgG appears soon after the onset of disease and
persists for decades. Four fold increase in antibody titer
can also be used in the diagnosis.
3) Isolation of the virus from cell culture is possible but not
used for routine clinical work.
Treatment and prevention
No antiviral therapy is available.
1) Active immunization with vaccine containing inactivated
HAV is available. The vaccine is given in 2 doses: an
initial dose and a booster 6-12 months later. No further
booster dose is required.
The vaccine is indicated in travelers to highly endemic
area, and in children age 2-18 months. The vaccine is
effective in post-exposure if it is given within 2 weeks
after the exposure. There is a combination of HAV with
HBV vaccine ( Tminrix).
2) Passive immunization with immune serum globulin prior
to infection or within 14 days after exposure can prevent
or mitigate the disease.
3) Proper hygiene is very important in the preventive
measures.
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Hepatitis E virus (HEV)
HEV is a major cause of hepatitis transmitted by the feco-
oral route.
The virus is classified in the virus family, hepeviridae, in
the genus hepevirus. HEV resembles, but is distinct from
calciviruses.
HEV is SS – RNA non-enveloped positive sense virus.
Clinically, the disease resembles HAV with exception of a
high mortality rate in pregnant women (20%).
Chronic liver disease doesn’t occur, and there is no
prolonged carrier state.
In acute infection, IgM antibodies to HEV are positive.
There is no antiviral therapy.
In 2007, a recombinant vaccine against HEV was shown
to be safe and effective and it is available in endemic area.
Hepatitis B virus
Disease
HBV causes hepatitis B.
Properties
HBV is a member of the hepadnavirus family. It is a 42-
nm enveloped virion (known as Dane particle (named for
the scientist who first published electron micrographs of
the virion)), with an icosahedral nucleocapsid core
containing a partially double-stranded circular DNA
genome. The envelope contains a protein called the
surface antigen (HBsAg), which is important for
laboratory diagnosis and immunization (HBsAg was
known as Australia antigen because it was first found in
the serum of an Australian aborigine).
Within the core is a DNA-dependent DNA polymerase.
The genome contains 4 genes that encode five proteins,
the S gene encodes the surface antigen, the C gene
encodes the core Ag and the e Ag, the P gene encodes the
polymerase, and the X gene encodes the X protein. The X
protein is an activator of viral RNA transcription.
The DNA polymerase has both RNA-dependent (reverse
transcriptase) and DNA-dependent activity.
Electron microscopy of a patient’s serum reveals 3
different types of particles:
A few 42-nm virions and many 22-nm spheres and long
filaments 22nm wide, which are composed of surface Ag.
In addition to HBsAg, there are two other important
antigens: the core antigen (HBcAg) forms the
nucleocapsid core of the virion and the "e" antigen
(HBeAg) is secreted from infected cells into the blood.
The e antigen is an important indicator of transmissibility.
For vaccine purposes, HBV has one serotype based on
HBsAg. However, for epidemiologic purposes, there are
four serologic subtypes of HBsAg based on a group
specific antigen, "a" and two sets of manually exclusive
epitopes, d or y and w or r. this lead to four serotypes:
adw,adr,ayw,and ayr.
The specificity of HBV for liver cells is based on two
properties: virus- specific receptors located on
hepatocyte cell membrane (facilitate entry) and
transcription factors found only in the hepatocyte that
enhance viral mRNA synthesis (act postentry).
The stability of HBsAg doesn’t always coincide with that of
the infectious agent. However, both are stable at -20c for
more than 20 years and stable to repeated freezing and
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thawing. The virus is stable at 37 c for 60 min. & remains
viable after being dried and stored at 25 c for at least 1 week
HBV (but not HBsAg) is sensitive to higher temperature
(100c for 1 minute) or to longer incubation periods (60 c
for 10 hours).
HBsAg is stable at pH of 2.4 for up to 6 hours, but HBV
infectivity is lost. Sodium hypochlorite, 0.5% (e.g, 1:10
chlorine bleach), destroys antigenicity within 3 minutes at
low protein concentrations, but undiluted serum
specimens require higher concentrations (5%). HBsAg is
not destroyed by UV irradiation of plasma or other blood
products, and viral infectivity may also resist such
treatment.
Replicative cycle
1) After entry of the virion into the cell and its uncoating.
2) Virion DNA polymerase synthesizes the missing portion
of DNA, and a double stranded closed-circular DNA is
formed in the nucleus. This DNA serves as template for
mRNA synthesis by cellular RNA polymerase.
3) After mRNAs are made, a full length positive- strand
transcript is made, which is a template for the minus
strand of the progeny DNA. The minus strand then serves
as the template for the plus strand of the genome DNA.
This RNA-dependent DNA synthesis catalyzed by reverse
transcriptase encoded by HBV takes place within the
newly assembled virion core in the cytoplasm.
The RNA-dependent DNA synthesis that produces the
genome and the DNA-dependent DNA synthesis that fills
in the missing portion of DNA soon after infection of the
next cell are carried out by the same enzyme (i.e., the
HBV genome encodes only one polymerase).
Hepadnaviruses are the only viruses that produce genome
DNA by reverse transcription with mRNA as the
template. (This type of RNA –dependent DNA synthesis
is similar to but different from the process in retroviruses,
in which the genome RNA is transcribed into a DNA
intermediate).
Some of the progeny DNA integrates into the host cell
genome, and this seems likely to be the DNA that
maintains the carrier state. Progeny HBV with its HBsAg
containing envelope is released from the cell by budding
through the cell membrane.
Transmission and epidemiology
Three main methods for transmission:
1) via blood
2) During sexual intercourse.
3) Perinataly from infected mother to newborn. Also by
breast feeding.
4) Needle stick injuries can transmit the virus indicating that
only very small amounts of bloods are necessary.
5) HBsAg can be detected in saliva, nasopharyngeal
washings, semen, menstrual fluid, and vaginal secretions .
6) Transmission from chronic carrier to close contacts by
oral route or by sexual or other intimate exposure occurs.
7) Transmission by feco-oral route has not been
documented.
8) Health care personnel have a higher incidence of hepatitis
and prevalence of detectable HBsAg or Anti-HBs than
those who have no occupational exposure to patients or
blood products.
9) HB infections are more common among patients and staff
of hemodialysis unit. As many as 50% of the renal
dialysis patients who contract HB may become chronic
carrier of HBsAg compared with 2% of the staff group.
Family contacts are also at increased risk.
Pathogenesis and immunity
1) After entering the blood, the virus infects hepatocytes and
the viral antigens are displayed on the surface of the cell.
2) Cytotoxic T-cells mediate an immune attack against the
viral antigens, and inflammation and necrosis occur.
3) The pathogenesis of hepatitis B is probably the result of
this cell mediated immune injury and HBV itself doesn’t
cause a cytopathic effect.
4) Antigen-antibody complexes cause some of the early
symptoms (e.g. arthralgia, arthritis and urtecaria) and
some of the complications in chronic hepatitis (e.g.,
glomerulonephritis, cryoglobulinemia, and vasculitis).
5) About 5% of patients infected with HBV become chronic
carrier and a chronic carrier is defined as patient who has
HBsAg persisting in their blood for at least 6 months. The
chronic carrier state is attributed to a persistent infection
of hepatocytes, which results in prolonged presence of
HBV and HBsAg in the blood. The main determinant for
a person to clear the infection or become a chronic carrier
is the adequacy of the cytotoxic T-cell response.
6) A high rate of hepatocellular carcinoma occurs in chronic
carriers. The HBV has no oncogene, and HCC appears to
be the result of persistent cellular regeneration that
attempts to replace the dead hepatocyte. Alternatively,
malignant transformation could be the result of insertional
mutagenesis, which could occur when the HBV genome
integrates into the hepatocyte DNA. This integration
could lead to activation of cellular oncogene leading to a
loss of growth control.
7) Chronic carrier state is more likely to occur in newborn
(about 90%) than in adults because of less competent
immune system.
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8) Lifelong immunity after the natural infection and is
mediated by an antibody against HBsAg which is a
neutralizing and protective antibody while an antibody
against core antigen is not protective.
Clinical finding
Many HBV infections are asymptomatic and are detected
only by the presence of antibody to HBsAg. The mean
incubation period is 10-12 weeks. The clinical appearance
of acute hepatitis B is similar to that of hepatitis A. but in
hepatitis B symptoms tend to be more severe and life
threatening hepatitis can occur. Extrahepatic
manifestations of viral hepatitis include a transient serum
sickness –like prodrome consisting of fever, skin rash, and
polyarthritis, necrotizing vasculitis & glomerulonephritis
Laboratory diagnosis
1) HBsAg is the most important test used for the detection of
early infection. HBsAg appears during the incubation
period and is detectable in most patients during prodrome
and acute disease. It falls to undetectable levels during
convalescence in most cases; its prolonged presence ( > 6
months ) indicates the carrier state and the risk of chronic
hepatitis and hepatic carcinoma. HBsAb is not detectable
in the chronic carrier state. HBsAb is being made but is
not detectable in the laboratory tests because itr is bound
to the large amount of HBsAg present in blood. HBsAb is
also being made during the acute disease but is similarly
undetectable because it is bound in antigen-antibody
complexes.
2) There is a period which is called Window phase in which
the HBsAg has disappeared and the HBsAb is not yet
detectable and at this time, the HBcAb is always positive
and can be used to make the diagnosis. HBcAb is present
in the acute and chronic infection and in patients
recovered from acute infection. Therefore it cant be used
to distinguish between acute and chronic infection. The
IgM form of HBcAb is present during acute infection and
disappears approximately 6 months after infection. The
test for HBcAg is not readily available.
3) HBeAg arises during the incubation period and is present
during the prodrome and early acute disease and in certain
chronic carrier. Its presence indicates a high likelihood of
transmissibility, and , conversely the finding of HBeAb
indicates a lower likelihood, but transmission can still
occur.
4) The detection of viral DNA in the serum is strong
evidence that infectious virions are present.
5) DNA polymerase activity is detectable during the
incubation period and early in the disease, but the assay is
not available in most clinical laboratories.
Treatment
1) No antiviral therapy is typically used in acute hepatitis B.
2) For chronic hepatitis B, pegINF alfa -2a or PegINF alfa-
2b and / or nucleoside analogues can be used.
These drugs reduce hepatic inflammation and lower the
viral load of HBV in patients with chronic active
hepatitis. Neither INF nor the nucleoside analogues cure
the HBV infection. In most patient, when the drug is
stopped, HBV replication resumes.
Prevention
1) The vaccine (Recombivax) contains HBsAg produced in
yeasts by recombinant DNA techniques. The seroconversion
rate is 95% in healthy adults.It is indicated in:
a) Certain health personnel ( medical students, surgeons
and dentists)
b) Patient receiving multiple transfusion or dialysis.
c) Drug abusers
d) Travelers to an endemic area.
The vaccine is given in three doses, and there is no need
for booster dose after the completion of the regime.
2) Hepatitis B immune globulin (HBIG) contains a high titer
of HBsAb. It is used to provide immediate passive
protection to individuals known to be exposed to HBsAg
positive blood. HBIG with HB vaccine can be given to
infant whose mother is HBsAg positive.
Unit 4: Virology
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Hepatitis C virus (HCV)
Properties
HCV is a member of flavivirus family. It is an enveloped
virion containing a genome of single stranded positive
polarity RNA. It has no virion polymerase.
HCV has at least six genotypes and multiple subgenotype
based on the differences n the genes that encode one of its
two envelope glycoprotein. The genetic variability is due
to high mutation rate in the envelope gene coupled with
the absence of proofreading function in the virion
encoded RNA polymerase. As a result, multiple
subspecies often occur in the blood of an infected
individual at the same time.
Replicative cycle:
The replication of HCV is uncertain because it has not
been grown in cell culture. It follows the replicative cycle
of other flaviviruses, as these viruses replicate in the
cytoplasm and translate their genome RNA into large
polyproteins, from which functional viral proteins are
cleaved by a virion-encoded protease. This protease is the
target of potent Anti-HCV therapy. The replication of
HCV in the liver is enhanced by liver –specific micro-
RNA. This micro-RNA acts by increasing the synthesis of
HCV mRNA.
Transmission and epidemiology
Humans are reservoir of HCV.
It is transmitted primarily via blood.
10-15% of HC cases, source of HCV can’t be detected.
At present injection drug use accounts for almost all new
HCV infections.
Transmission via blood transfusion rarely occurs because
donated blood containing Anti-HCV antibody is
discarded.
Transmission via needle stick injury occurs, but the risk is
lower than that of HBV.
Sexual transmission and transmission from mother to
child occur but are inefficient mode. No risk of
transmission has been associated with breast feeding.
Commercially prepared immuneglobulin preparations are
generally very safe, but several instances of transmission
of HCV have occurred.
WHO in 1997 estimate that about 3% of world population
has been infected, with a prevalence of 10% in African
population and other high prevalence area in Asia and
South America.
Transmission of HCV has been linked to an attempt (from
1950s to 1980s) to treat parasitic disease shistosomiasis
by therapy that involved multiple injections, often with
improperly sterilized or reused needles.
HCV infection has been associated with tattooing.
There was a case in 2009 in which HCV was transmitted
to an organ transplant recipient by the use of a blood
vessel conduit from a HCV-positive donor.
Pathogenesis and immunity
HCV infects hepatocytes primarily, but there is no
evidence for a virus-induced cytopathic effect on the liver
cells. Rather, death of the hepatocytes is probably caused
by immune attack by cytotoxic T cells. HCV infection
strongly predisposes to hepatocellular carcinoma, but
there is no evidence for an oncogene in the viral genome
for insertion of a copy of the viral genome into the DNA
of the cancer cells.
Alcoholism greatly enhances the rate of hepatocellular
carcinoma in HCV-infected individuals.
Antibodies against HCV are made, but approximately
75% of patients are chronically infected and continue to
produce virus for at least 1 year.
Chronic active hepatitis and cirrhosis occur approximately
10% of these patients.
For patients who clear the infection, it is not known
whether re-infection can occur or whether there is life
long immunity.
Clinical findings
HCV acute infection is milder than that of HBV infection.
The average incubation period for HCV is 6-7 weeks. The
average time from exposure to seroconversion is 8-9
weeks, and about 90% of patients are Anti-HCV positive
within 5 months. Many infections with HCV including
both acute and chronic infections are asymptomatic and
are detected by the presence of antibody. HCV can also
lead to significant autoimmune reactions, including,
vasculitis, arthralgia, purpura, and membranoproliferative
glomerulonephritis. HCV is the main cause of essential
mixed cryoglobulinemia.
Laboratory diagnosis
1) Antibody detection to HCV by ELISA. The test doesn’t
distinguish between acute, chronic, or resolved infection.
2) Positive result by ELISA should be confirmed by RIBA
(recombinant immunoblot assay), because of false
positive results of ELISA.
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3) If RIBA result is positive, a PCR based test that detects
the presence of viral RNA (viral load) in the serum should
be performed to determine whether active disease exist.
4) A chronic infection is characterized by elevated
transaminase levels, a positive RIBA, and detectable viral
RNA for at least 6 months.
Treatment and prevention
1) Treatment of acute hepatitis C with alpha INF
significantly decreases the number of patients who
become chronic carrier.
2) Treatment of choice for chronic patients is by pegINF and
ribavirin.
3) Genotype 1 is less responsive to the above regimen
(treatment should continue for 1 year) than genotype 2
and 3 (treatment should be for 6 months).
4) Pooled immune serum globulins are not useful for post
exposure prophylaxis.
5) There is no effective regimen for prophylaxis following
needle-stick injury; only monitoring is recommended.
6) Patient with chronic HCV and are alcoholics, should be
advised to eliminate drinking.
7) Patient with chronic HCV should be followed by alpha-
fetoprotein and liver US to detect carcinoma at early
stage.
8) Patients with liver failure due to HCV infection can
receive a liver transplant, but infection of the graft with
HCV typically occurs.
Hepatitis G virus (HGV)
In 1996, HGV was isolated from patients with
posttransfusion hepatitis. HGV is a member of the
flavivirus family. But unlike HCV, HGV don’t cause
acute or chronic hepatitis and don’t predispose to
hepatocellular carcinoma. The role of HGV in the
causation of liver disease has yet to be established, but it
can cause chronic infection lasting for decades.
Approximately 60-70% of those infected clear the virus
and develop antibodies. HGV transmitted via sexual
intercourse and blood. It is carried in the blood of millions
of people worldwide.
Hepatitis D virus (Delta virus)
HDV is a defective virus can replicate only on the cells
also infected with HBV because HDV uses the surface
antigen of HBV as its envelope protein.
HDV is an enveloped SS RNA negative polarity,
covalently closed circle. The RNA genome encodes only
one protein called Delta protein.
HDV has one serotype & there is no animal reservoir for
the virus.
It is transmitted by the same method ad that of HBV.
The patient is either previously infected with hepatitis BV
and then infected with HDV or the infection with both
viruses is acquired at the same time. Hepatitis in patients
co infected with HBV and HDV is more.
The diagnosis can be done by detecting either delta Ag or
IgM antibody to delta antigen in the patient's serum.
There is no specific antiviral therapy but alpha interferon
can mitigate some of the effects of the chronic hepatitis