Chapter 50 Hepatitis Viruses.pptx education

Learning objectives
Essentials of Medical Microbiology by Apurba S Sastry
© 2018, Jaypee Brothers Medical Publishers

Hepatitis viruses
• Heterogeneous group of viruses that are hepatotropic
• Cause acute inflammation of the liver producing identical
histopathologic lesions
• Similar clinical illness such as fever, nausea, vomiting, and
jaundice.

Properties HAV HBV HCV HDV HEV
Common
name
Infectious
hepatitis
Serum hepatitis Non A non B or
post-transfusion
hepatitis
Delta agent Non A non B
enteric transmitted
hepatitis
Family Enterovirus-72
(Picornaviridae)
Hepadnaviridae Flaviviridae Unclassified
viroid-like
Unclassified
Caliciviridae-like
Genus Hepatovirus Orthohepadnaviru
s
Hepacivirus Deltavirus Hepevirus
Virion 27 nm,
icosahedral
42 nm, spherical 60 nm, spherical 35 nm,
spherical
30–32 nm,
icosahedral
Classification

Classification (cont..)
Envelope No Yes (HBsAg) Yes Yes (HBsAg) No
Genome ssRNA dsDNA ssRNA ssRNA ssRNA
Stability Heat and acid-
stable
Acid-sensitive Ether-sensitive,
acid-sensitive
Acid-sensitive Heat-stable
Onset Abrupt Insidious Insidious Insidious Abrupt
Age Children,
Young adults
Young adults
babies, toddlers
Any age, but
more common in
adults
Any age
(similar to
HBV)
Young adults
(20–40 years)
Route Feco-oral Blood (MC)
Sexual,
Vertical
Blood (MC)
Sexual(+/-)
Vertical(+/-)
Blood (MC)
Sexual(++)
Vertical(+)
Feco-oral

I.P (days) 15–45,
(Average 30)
30–180,
(Average 60–90)
15–160,
(Average 50)
30–180,
(Average60–90)
14–60,
(Average 40)
Fulminant
disease
Rare (0.1%) Rare (0.1–1%) Rare (0.1%) Frequent (5–
20%)
Usually rare(1-2% )
Pregnancy- 20-40%
Carrier None Yes (0.1–30%) Yes (1.5–3.2%) Variable None
Chronicity None Occasional (1–
10%)
Common (85%) Common None
Oncogenic No Yes (neonate) Yes +/- No

Prevalence High High Moderate Low, regional Regional
Associated
other
feature
Secondary
attack rate 10-
20%
HCC, cirrhosis,
Autoimmune
disorder like
AGN, arthritis,
PAN
HCC, cirrhosis,
Autoimmune-
AGN, arthritis,
cryoglobulinemia
HCC, cirrhosis,
fulminant
hepatitis
Secondary attack
rate (1-2%)
Rarely seen in
western countries
Prognosis Excellent Worse with age Moderate Acute-good
Chronic- poor
Good
Prophylaxis Immunoglobuli
n,
Inactivated
vaccine
HBIG,
Recombinant
vaccine
None HBV vaccine (no
vaccine for HBV
carriers)
Vaccine (HEV239)
(only in China)
Therapy None Pegylatedinterfero
n
Lamivudine
Pegylated
interferon plus
ribavirin
Interferon ± None

HEPATITIS A VIRUS
• Originally designated as ‘enterovirus 72’.
• Morphology
o 27 to 32nm
o Spherical particle with icosahedral symmetry
o Linear ssRNA
o Only one serotype, does not cross-react with other hepatitis
viruses
o Typed into seven genotypes based on gene sequences.

Resistance
• Relatively resistant to disinfectants.
• Stable to acid, heat (60°C for 1 hour), and ether (20%)
• Preserved in dried state for 1 month and stored for years
at -20°C.
• Destroyed by autoclaving, by boiling in water for 5
minutes, by hot air oven, by UV rays and by treatment
with formalin or chlorine (10–15 ppm for 30 minutes).

Mode of transmission
• Feco-oral route
• Infection is by ingestion.
• Rarely, sexual (homosexuals through oro-genital contact) and
parenteral routes (infected blood products or needle pricks)

Epidemiology
• Hosts- Humans
• Age:
o Children and adolescents (5- 14 years of age) are most commonly
affected
o Majority remain subclinical (80–95%),but excrete virus in feces
for longer time.
o Adults are more icteric (75–90%) than with higher mortality rate
• Anicteric to icteric cases ratio is about-
o In children – 12:1
o In adults – 1:3

• Outbreaks are common in summer camps, day care centers, families and
institutions, neonatal ICUs, and among military troops.
• Recurrent epidemics and sudden, explosive epidemics are common and
usually result from fecal contamination of a single source (e.g, drinking
water, milk or food such as raw vegetables, salad,frozen strawberries,
green onions and shell fish). The largest epidemic was reported from
Shanghai (1988, >3 lakh cases)
• Seasonal incidence– Though HAV infection is widespread throughout the
year, it tends to peak in late rainfall and in early winter.
• Virus excretion-Viral excretion in feces may be 2 weeks before to 2 weeks
after the appearance of jaundice (however, viremia occurs from -2 weeks
to +1 week of jaundice).

Risk factors
• Poor personal hygiene and overcrowding
• Adults have protective antibodies and are mostly immune.
• However, in developed countries with improved hygiene, the
incidence is decreasing and there is trend of shift of infection
towards the older age.

Clinical manifestations
• Incubation period - 15–45 days (mean 30 days).
• Onset is relatively abrupt (sub-acute).
o Pre-icteric phase (predominant GI symptoms such as nausea
and vomiting) followed by
o Icteric phase or jaundice (dark urine, yellowish sclera and
mucus membrane)
• Complete recovery occurs in most (98%) cases.
• No chronic or carriers state

Complications
• Fulminant hepatitis
• Relapsing hepatitis
• Cholestatic hepatitis

Laboratory Diagnosis
• Anti HAV Antibody detection
o IgM antibodies appear during the acute phase, peak about 2
weeks after the elevation of liver enzymes, and disappear
within 3–6 months
o IgG appears a week after the appearance of IgM and persists for
decades.
o Interpretation-
Anti-HAV IgM positive- Indicates acute infection
Anti-HAV IgG antibody detection in the absence of IgM indicates past
infection or recovery.
o ELISA is the method of choice

Course of HAV markers with symptoms

• Detection of HAV particles by immune electron microscopy
o HAV appears in stool from -2 to +2weeks of jaundice.
o Also detected from liver, bile, & blood.
• Detection of HAV antigens in stool by ELISA-from -2 to +2weeks of
jaundice
• Isolation- HAV is the only hepatitis virus where isolation has been
attempted.
• Non-specific- elevated liver enzymes and serum bilirubin level
Laboratory Diagnosis

Treatment
• No specific antiviral drug available
• Prevention and containment of infection
o Hand washing before and after use of toilet
o Sanitary disposal of infected fecal material- by disinfection with
0.5% hypochlorite
o Purification of drinking water by effective filtration and
adequate chlorination (with at least 1mg/L of residual chlorine).
o Use of boiled water (boiling for at least 5 min) during outbreaks.

Vaccines
• Formaldehyde Inactivated vaccine:
o Prepared from human fetal lung fibroblast cell lines - MRC-5 and WI 38
o Given to children after 12 months of age
o Single dose is administered by IM route (deltoid) followed by booster at 6-
12months gap.
o Protective efficacy - 94%.
• Live attenuated vaccine is given as single dose, subcutaneously.
o Uses H2 and L-A-1 strains, prepared in human diploid cell line (China).
• Both vaccines are highly immunogenic, produce long lasting immunity, possibly
life-long.

Human Immunoglobulin(HAV-Ig)
• For post-exposure prophylaxis of intimate contacts (household,
day care centres)
• Dose - 0.02 mL/kg (protection – 1 to 2months).
• Administered as early as possible after exposure; (within 2
weeks).
• Not necessary for:
o Already vaccinated
o Casual contacts (office, factory, school, or hospital)
o Elderly persons (likely to be immune).

HEPATITIS B
• Discovered by Blumberg in 1963.
• Morphology: Electron microscopy of serum of the patients infected with
HBV reveals three morphologic forms:
o Spherical forms (made up of HBsAg) -most numerous, small forms
measuring 22 nm in diameter.
o Tubular or filamentous forms (made up of HBsAg) - also have the
same diameter but 200 nm long.
o Complete form or Dane particles (less frequent). They are larger, 42-
nm size spherical virions; made up of-
 Outer surface envelope - HBsAg (Hepatitis B surface antigen)
 Inner 27 nm size nucleocapsid- consists of core antigen (HBcAg) and pre-core
antigen(HBeAg) and partially double stranded DNA

Viral antigens
Hepatitis B surface antigen (HBsAg)
• Previously called as 'Australia antigen‘
• Antigenically complex and contains two components:
o Common group reactive antigen ‘a’ epitope
o Two pairs of type specific antigens d/y and w/r; only
one member of each pair being present at a time.
• Four subtypes of HBsAg - adw, ayw, adr, and ayr.

Viral antigens
Hepatitis B core antigen (HBcAg)
• Forms the intracellular core protein
• Not secreted
• Does not circulate in blood
• Can be demonstrated in hepatocytes by immunofluorescence.

Viral antigens
Hepatitis B pre core antigen (HBeAg)
• Non-particulate soluble antigen possessing a signal
protein which enables it to be secreted.
• Present in circulation.

Typing of HBV -
Serotypes
• HBV is divided into four major serotypes (adr, adw, ayr, ayw) - Based on
antigenic epitopes present on its envelope protein HBsAg.
• Serotypes - not important in immunity as the dominant ‘a’ antigen is
shared by all, but they are useful for epidemiologic investigations, as all
the cases during an epidemic would likely to have the same subtype.
• Serotypes exhibit distinct geographical distribution:
o adw - predominant sub type in Europe, Australia and America.
o adr - South and East India
o ayw - Western and Northern India

Typing of HBV-
Genotypes
• HBV can also be divided into eight genotypes (A–H)
• According to overall nucleotide sequence variation of the
genome.
• Genotypes A and D are prevalent in India.

Viral genome
• Partially circular dsDNA, of 3200 bp in length.
• minus strand of DNA (L or long strand)is complete
and full-length and is identical in all HBV isolates
• The positive strand (S or short strand) is incomplete
and of variable length (50-80%).
• HBV genome is compact and consists of four
overlapping genes:
o S gene
o C gene
o X gene
o P gene

Viral genome-
S gene
• Three regions- S, pre-S1 and pre-S2
• Code for surface antigen (HBsAg)
• S region codes for the major protein (S).
• Product of S region plus the adjacent Pre-
S2region is the middle (M) protein
• Pre-S1, Pre-S2combines with S to code for
Large (L) protein.
• The L protein is present only in the virion,
while the M and S proteins are found in
the circulating HBsAg particles also.

Viral genome-
C gene
• Consists of pre-C and C-regions, which
code for two nucleocapsid proteins
o Pre-C region codes for HBeAg
o C-region codes for HBcAg

Viral genome-
X gene
• Codes for HBxAg, which can activate the
transcription of cellular and viral genes.
• Contribute to carcinogenesis by binding to
p53.
• HBxAg and its antibody are elevated in
patients with severe chronic hepatitis and
hepatocellular carcinoma.

Viral genome-
P gene
• Largest and codes for polymerase (P)
protein which has three enzymatic
activities
o DNA polymerase activity
o Reverse transcriptase activity
o RNase H activity

Hepatitis B virus mutants
• Pre-core mutants: have defect in pre-core region of C gene which
leads to their inability to synthesize HBeAg.
o Mutation- Nonsense mutation in the pre-C gene leading to
formation of premature stop codon.
o Geographical distribution- Mediterranean countries and in
Europe.
o Patients infected with precore mutants may be diagnosed late
and they tend to have severe chronic hepatitis that progresses
more rapidly to cirrhosis.
o Markers-They lack HBeAg. Other viral markers are present as
such.

• Escape mutants: Mutations in the S gene leads to alteration of HBsAg.
o Mutation occurs in the immunodominant aantigen of HBsAg
o Escape mutants are observed in three situations-
 Infants born to HBeAg positive mothers
 Liver transplant recipients who underwent the procedure for hepatitis B
and who were treated with a high-potency human monoclonal anti-HBs
preparation.
 A small proportion of recipients of active and passive immunization, in
whom antibody pressure may favor evolutionary change in gene coding a
antigen
o Pose problems in hepatitis B vaccination strategies as well as in the
diagnosis of the disease.

• YMDD mutation:
o HBV infected patients on lamivudine therapy
o May develop resistance to the drug due to mutation in
the YMDD locus of the HBV reverse transcriptase region
of polymerase gene.

Hepatitis B replication

Transmission
• Parenteral route- Via blood & blood products transfusion and needle
prick injuries or by inoculation during surgical or dental procedures or
percutaneous inoculation via shared razors and tooth brushes.
o HBV (30%) is more infectious than HIV (0.3%) and HCV (3% ). As little
as 0.00001 ml of blood can be infectious.
• Sexual transmission
• Vertical (perinatal) transmission- Risk is maximum if the mother is
HBeAg positive
o Transmission occurs at any stage; in utero, during delivery (maximum
risk) and during breast feeding
• Direct skin contact

Transmission
• High risk groups which are more prone for acquiring
infection are:
o Surgeons(maximum risk)
o Paramedical workers
o Sex workers especially homosexual males
o Recipients of blood transfusion and organ
transplantation
o Drug addicts

Epidemiology
• Reservoir of infection-Humans are the only reservoir
o Cases may range from inapparent to symptomatic cases.
o Carries may be temporary (harbour the virus for weeks to months)
or persistent/ chronic (harbour the virus for > 6months).
• Carriers can also be grouped into-
o Simple carriers- Low infectivity, transmit the virus at a lower rate.
They possess low level of HBsAg and no HBeAg.
o Super carriers- Highly infectious and transmit the virus efficiently.
They possess higher levels of HBsAg and also have HBeAg, DNA
polymerase, and HBV DNA.

Epidemiology
• Prevalence- Based on HBsAg carrier rates,
o Type 1 pattern (low endemicity)- Carrier rate is <2%. It
is observed in SriLanka and Nepal.
o Type 2 pattern (intermediate endemicity) – Carrier rate is
between 2-8%. It is observed in India, Bhutan, Indonesia
and Maldives.
o Type 3 pattern(high endemicity)-Carrier rate is >8%. It is
observed in Bangladesh and DPR Korea.

Situation in the world
• World Hepatitis Day is celebrated on 28th July every year.
• WHO estimates that in 2015, about 257 million population
were living with chronic HBV infection with a global
prevalence of 3.5%.
• 2.7 million persons were co-infected with HBV and HIV
• The widespread use of HBV vaccine in infants has led to a
considerable reduction in the incidence of new chronic HBV
infections.

Situation in India
• Intermediate level of HBV endemicity (3.7% prevalence).
• 40 million HBV carriers, which constitutes 11% of the estimated
global burden, which is second highest to China (30%)
• Highest prevalence recorded from Andamans and Arunachal
Pradesh
• Tribal areas, the prevalence is extremely high (19%) than non-
tribal populations
• HBV accounts for 40–50% of hepatocellular carcinoma (HCC) and
10–20% of cirrhosis in India.

Epidemiology (cont..)
• HBV followed by HCV are the most common cause of:
o Chronic hepatitis
o Cirrhosis: HBV accounts for 30% of cirrhosis
o Hepatocellular carcinoma (HCC): HBV accounts for 45% of
HCC
o Post-transfusion hepatitis: HBV (1:220,000)>HCV
(1:1800,000).

• Resistance- HBV can be destroyed by hypochlorite and heat (autoclave).
• Period of infectivity:
o People infected with HBV are said to be infectious as long as the
HBsAg is present in blood i.e. during incubation period (a month
before jaundice) up to several months thereafter (occasionally years
for chronic carriers).
o Become non-infectious once HBsAg disappears and is replaced by
anti-HBs antibody.
o Maximum infectivity is observed when HBeAg is elevated in serum.

• The global HBV prevalence in HIV-infected persons is 7.4%. Conversely,
about 1% of HBV-infected persons are also infected with HIV.
• The highest burden (71%) for HIV–HBV coinfection is found in sub-
Saharan Africa
• Although HBV does not alter the progression of HIV, the presence of HIV
greatly enhances the risk of developing HBV-associated cirrhosis and
liver cancer
• Tenofovir, a drug recently included in the treatment regimen of HIV is
also active against HBV. This may have a role in controlling HIV-HBV
coinfection.

Various outcomes of HBV infection

• Age- The outcome of HBV infection depends on the age. Following HBV
infection-
• Chance of developing acute hepatitis is directly related to the age-
o 1% (perinatal)
o 10% (early childhood; 1-5 years age)
o 30% (late child hood; after 5 years age)
• Chance of developing chronic hepatitis or carrier state is inversely
related to age-
o 80-90% (perinatal)
o 30% (early child hood; 1-5 years age)
o 5% (late child hood; after 5 years age)

• Age- The outcome of HBV infection depends on the age.
• Explanation-
o Pathogenesis HBV infection is immune mediated
o Hepatocytes carrying viral antigen are subjected to NK cell
mediated antibody dependent cell cytotoxicity(ADCC) or CD8 T
cell mediated cytotoxicity
o Absence of an effective immune system (infants) - leads to
carrier state

Clinical Manifestations
• Incubation period - 30–180 days.
• Onset is slow and insidious
• Patients may present with subclinical infection or acute or chronic
hepatitis
• Clinically, HBV infection is indistinguishable from other hepatitis viruses;
characterized by-
o Pre-icteric phase (predominant gastrointestinal symptoms such as
nausea and vomiting) followed by
o Icteric phase or jaundice
• Clinical outcome may be either development of carrier state or
complete recovery.

Complications
• Hepatic complications:
o Fulminant hepatitis
o Cirrhosis
o Hepatocellular carcinoma.
• Extrahepatic complications:
o Prodromal phase - Serum sickness-like syndrome (5–10%
of patients)
o This is due to immune complex deposition.

Laboratory diagnosis
• Viral markers of HBV infection:
• Definitive diagnosis of hepatitis B depends on the serological
demonstration of the viral markers which can be classified as-
o Antigen markers- HBsAg and HBeAg
o Antibody markers- Anti-HBs, Anti-HBe and Anti-HBc
o Molecular markers- HBV DNA
o Non-specific markers- elevated liver enzymes and serum bilirubin
• Detection method – ELISA, ICT
• Viral DNA can be detected by PCR; but quantified by real time PCR.
• HBV does not grow in any conventional culture system.

Serological markers of hepatitis B virus in various
stages of hepatitis B virus infection

Hepatitis B surface antigen (HBsAg)
• First marker to be elevated following infection; appears within 1–12
week (usually between 8–12 weeks of infection)
• Appears during incubation period;2–6 weeks before the biochemical and
clinical evidence of hepatitis.
• Presence of HBsAg indicates onset of infectivity (i.e. patient is capable of
transmission of HBV)
• Remains elevated in the entire duration of acute hepatitis;
o Becomes undetectable 1-2 months after the onset of jaundice or
o Rarely persists beyond 6 months during chronic hepatitis and carrier
state
• HBsAg is used as an epidemiological marker of hepatitis B infection.

Hepatitis B precoreantigen (HBeAg) and HBV DNA
• Appear concurrently with or shortly after appearance of HBsAg in serum
• They are the markers of
o Active viral replication
o High viral infectivity (i.e. they are highly infectious to others)
• Present in either acute, chronic and carrier state and it cannot
differentiate between these stages.
• Indicates that the virus is actively multiplying, which could be either-
o Acute active hepatitis
o Chronic active hepatitis
o Or a carrier in whom HBV is actively multiplying and highly infectious

Hepatitis B coreantigen (HBcAg)
• Hidden antigen due to surrounding HBsAg coat.
• Also non-secretory in nature - hence, it cannot be detected in
blood.
• HBcAg may be detected in hepatocytes by
immunofluorescence microscopy.

Anti-HBc
• Anti-HBc IgM(Hepatitis B core antibody)
o Anti-HBc IgM is the first antibody to elevate following infection.
o Appears within the first 1–2 weeks after the appearance of HBsAg and lasts
for 3-6months.
o Indicates acute hepatitis B infection,
o Only marker (sometimes anti-HBc IgG)present during the period between
appearance of anti-HBs antibody and disappearance of HBsAg.
• Anti-HBc IgG (Hepatitis B core antibody)
o Appears in late acute stage and remains positive indefinitely whether the
patient proceeds to
 Chronic stage (with persistence of HBsAg, symptomatic and elevated liver enzymes)
 Carrier state (with persistence of HBsAg but asymptomatic) or
 Recovery (appearance of Anti-HBs antibody)

Anti-HBe (Hepatitis B pre-core antibody)
• Appear after the clearance of HBeAg and remain elevated
for variable period
• Signifies diminished viral replication and decreased
infectivity.

Anti-HBs (Hepatitis B surface antibody)
• Appears after the clearance of HBsAg and remains elevated
indefinitely
• Indicates recovery , immunity and non-infectivity (i.e.
stoppage of transmission)
• Only marker of vaccination.

Interpretation of markers in various stages of
hepatitis B infection
HBsAg Anti-
HBs
Anti-
HBc
HBeAg Anti-
HBe
Symptoms Liver
enzyme
s
DNA Interpretation
+ - - - - Absent Normal + Early acute hepatitis (Incubating)
+ – IgM + – Present Elevated + Acute hepatitis B, high infectivity
+ – IgG ++ – Present Elevated ++ Chronic hepatitis B, high infectivity
(Immunoreactive chronic hepatitis)
+ – IgG – + Present Elevated + Chronic hepatitis B, low infectivity
(Chronic inactive hepatitis)
+ - IgG + - Absent Normal ++ Immuno tolerant chronic HBV infection
(previously called as super carriers)

Interpretation of markers in various stages of
hepatitis B infection
HBsAg Anti-
HBs
Anti-
HBc
HBeAg Anti-
HBe
Symptoms Liver
enzyme
s
DNA Interpretation
+ - IgG - +/- Absent Normal + Chronic inactive HBV infection
(previously called as simple carriers)
+ – IgG – - Present Elevated + Precore–mutant hepatitis B infection
- - IgG -/+ -/+ Present Elevated + Escape mutant hepatitis B infection
- + IgG - +/– Absent Normal - Recovery
- + - - - Absent Normal - Post vaccination
- - IgG - - Absent Normal + Occult hepatitis B infection

Treatment of Hepatitis B
• Most acute hepatitis B infection is self-limiting; do not require
any specific treatment.
• In contrast, treatment of chronic hepatitis B has perceived
many recent advances.
• With the advent of newer antiviral drugs, now it’s possible to
contain the disease.
Adapted from EACL guideline 2017 (European Association for the Study of the Liver)

Treatment of Hepatitis B
• Indications
o The indications to initiate treatment for patients with hepatitis B infection are:
o Acute hepatitis with acute liver failure (coagulopathy or encephalopathy)
o Chronic active hepatitis (immunoreactive hepatitis, HBeAg+ve)
o Chronic inactive hepatitis (HBeAg-ve)
o HBV DNA > 2,000 IU/ml plus ALT ↑ (> normal) plus moderate degree of liver ﬁbrosis
o HBV DNA > 20,000 IU/ml and ALT ↑↑(>2 times normal); regardless of the degree of
liver ﬁbrosis
o Associated cirrhosis (regardless of ALT level, viral load)
o Super carriers or immunotolerant hepatitis (if >30 years age)
o Carriers (super or simple) with family history of HCC or cirrhosis
Adapted from EACL guideline 2017 (European Association for the Study of the Liver)

Antiviral agents
• Antiviral agents (nucleoside analogues)- such as telbivudine
and tenofovir.
• Lamivudine, adefovir, entecavir were used previously; now
not in use because of high risk of developing resistance.
• Pegylated interferon alfa (used previously; now not in use
because of adverse effects)

Treatment options available for chronic hepatitis B infection
Interferon alfa Antiviral agents (Telbivudine
and tenofovir)
Route and dosage Subcutaneous, weekly Oral, daily
Mode Monotherapy Monotherapy
Duration 48 weeks Long-term until HBsAg loss
Side effects High Minimal
Decompensated disease,
immunocompromised
patients
Cannot be given Can be given
HBV DNA <2000 IU/mL Undetectable i.e. <10 IU/mL
HBsAg loss or HBeAg
seroconversion
Occurs relatively earlier (1
year)
Occurs very slowly
Preference Was preferred in past. First choice of treatment
currently.
**HBV DNA clearance from blood may occur early; however cccDNA may be present in hepatocytes quiescently; which may get activated later. Hence, HBeAg
seroconversion (for previously HBeAg positive cases) and loss of HBsAg with seroconversion are more reliable indicator of successful therapy.

Prophylaxis
Active Immunization (Hepatitis B Vaccine)
• Hepatitis B vaccine is a recombinant subunit vaccine.
• The surface antigen (HBsAg) is used as vaccine candidate which
is prepared in Baker’s yeast by DNA recombinant technology by
cloning the S gene into the yeast chromosome
• Route of administration: Intramuscular route over deltoid
region (in infant- anterolateral thigh)
• Dosage: 10–20 µg/dose (half of the dose is given to children
below 10 years)

Prophylaxis
Schedule:
• Recommended schedule for adults: Three doses are given at 0, 1
and 6 months
• Under national immunization schedule: It is given at 6, 10, 14
weeks (along with DPT vaccine). Additional dose at birth may be
given in areas with prevalence of HBV more than 8%.
• Minimum interval between the doses—4 weeks
• If there is documentation of partial vaccination (1 or 2 doses): Then
do not restart; just complete the vaccine series regardless of when
the last dose was taken.

Prophylaxis
• Marker of protection: Recipients are said to be protected if they develop
seroconversion with an anti-HBsAg antibody titer of more than 10
mIU/mL
• Re-vaccination: If titer remain <10 mIU/mL after first series of
vaccination; the HCW is subjected to second series of vaccination (3
doses at 0, 1, 6 months)
• Non/low responders: About 5–10% of individuals mount an impaired
immune response following vaccination. They may be either:
o Non-responders (do not show seroconversion after two series of
vaccination; i.e six doses of vaccination.) or;
o Low responders (seroconversion occurs slowly after the second series
of vaccination.).

Prophylaxis
• Seroconversion:
o 95% of infants, children and young adults
o Older people (>60 years), the protection is about 65–75% only
• Protection may last for about 30 years or even longer
• Booster doses are not needed: The health care workers once protected,
should not check their titer again or should not take booster vaccines.
They remain protective even if the titer falls <10 mIU/mL. This is because
the memory cells get stimulated much faster and the titer rises very soon
following an infection with HBV
• Newer vaccine containing whole HBsAg (i.e. product of Pre S1 + Pre S2 +
S genes) is under development which may provide a better
seroconversion.

Passive immunization (Hepatitis B Immunoglobulin or HBIG)
• Indications: HBIG is used in the following situations where an
immediate protection is warranted.
o Acutely exposed to HBsAg positive blood- e.g. surgeons,
nurses, laboratory workers
o Sexual contact of acute hepatitis B patients
o Neonates borne to hepatitis B carrier mothers
o Post liver transplant patients who need protection against HBV
infection
o Following accidental exposure, HBIG should be started
immediately (ideally within 6hr, but not later than 48 hrs).

Passive immunization (Hepatitis B Immunoglobulin or HBIG)
• Recommended dose - 0.05-0.07ml/kg body weight, two
doses of HBIG should be given 30 days apart
• HBIG gives short term passive protection which lasts for
about 100 days. Since the median incubation period is less
than 100 days, two doses given one month apart should
suffice.

Combined immunization
• Combined immunization with HBIG + vaccine is more
efficacious than HBIG alone.
• Recommended for neonates born to HBV infected mother,
where a single injection of 0.5 ml of HBIG is given to the
neonate immediately after the birth, followed by full course
of vaccine given at a different site (the first dose being given
within 12 hours of birth).

General Prophylactic Measures
• Screening of blood bags, semen and organ donors
• Following safe sex practices (e.g. using condoms, avoiding
multiple sex partners)
• Following safe injection practices - use of the disposable
syringes and needles
• Following safe aseptic surgical practices
• Health education

HEPATITIS C VIRUS –
Morphology
• Family flaviviridae, genus Hepacivirus.
• Spherical, 60 nm size
• Enveloped virus
• Nucleic acid- It contains positive sense ssRNA

HEPATITIS C VIRUS –
Morphology
• Proteins- HCV possesses
• Four structural proteins, the nucleocapsid core protein C; two
envelope glycoproteins (E1 and E2) and a membrane protein
p7 (functions as an ion channel)
• Six non-structural (NS) proteins-NS2, NS3, NS4A, NS4B, NS5A,
and NS5B

Genetic diversity of HCV
• Similar to HIV, Hepatitis C virus displays diversity in the RNA genome
that occurs because of high rates of mutations seen in the virus
which in-turn is due to (i) the high replicative activity and (ii) the lack
of proofreading activity of the RNA polymerase.
• Genotypes: HCV is divided into six major genotypes or clades,
which differ from each other by 31–33% in their RNA sequence
• Subtypes: Genotypes are further divided into more than 100
subtypes, which differ from each other by 20–25% in their RNA
nucleotide sequence.

• E2 envelope protein is the most variable region of the entire HCV
genome followed by the non-structural proteins (especially, NS5B
encoded RNA polymerase);
• More prone to undergo mutations
• Unfortunately, E2 protein happens to be the target against which
most of the neutralizing (protective) antibodies are produced
• Diversity in the gene coding E2 protein enables the emergent
mutant virus strains to escape from host’s humoral immunity, which
in turn can result in:
o Establishment of chronic infection
o Failure of development of effective vaccine.

• HCV genotypes vary in their epidemiological distribution:
o Genotype 1 and 2 - Western countries
o Genotype 3 - South and East Asia with subtype 3a common
among IV drug users from Europe
o Genotype 4 - North Africa and Middle-East
o Genotype 5 - South Africa
o Genotype 6 - Asia
o In India, genotypes 1 and 3 are more prevalent.

Transmission
• Various modes of transmission are-
o Parenteral- HCV is most commonly transmitted through exposure to
infectious blood-
 Recipients of contaminated blood transfusions, blood products or organ
transplantations
 Contaminated needles and sharps pricks
 Injection drug users
o Vertical transmission from infected mother to fetus may occur but at
much lower rate(6%) than that of HBV(20%).
o Sexual transmission (rare)
o HCV doesn’t spread through breast milk, food or casual contacts
including hugging or kissing

Clinical manifestations
• Incubation period - 15–160 days (average 50 days).
• Following an infection with HCV-
o About 20% of people develop acute hepatitis; characterized by symptoms similar to
that of other hepatitis viruses described earlier.
o About 75-80% directly develop chronic disease; out of which-
o 60-70% develop chronic hepatitis
o 5-20% develop cirrhosis
o 1-5% develop hepatocellular carcinoma. (HCV accounts for 25% of total liver cancer
patients)
• Extrahepatic manifestations- Due to deposition of circulating immune complexes in
various extra hepatic sites, various manifestations can set in such as-
o Mixed cryoglobulinemia
o Glomerulonephritis
o Arthritis and joint pain

Epidemiology
• Hepatitis C virus infection occurs worldwide.
• Every year, 3–4 million people are infected with HCV with more
than 3.5 lakhs deaths.
• Population prevalence rate:
o About 3% of the world population has been infected with HCV
worldwide with more than 170 million chronic carriers
o Higher prevalence rates have been documented from Africa
(up to 10%) followed by South America and Asia
o In India, the prevalence of HCV is about 1%.

Laboratory Diagnosis-
HCV antibody detection assay (ELISA)
• ELISA is the most common platform used; followed by rapid test
and chemiluminescence formats.
o First and second generation ELISA (used previously) - These
assays were using limited HCV antigens (core, NS3 and NS4) for
detection of antibodies. These assays are now obsolete as they
are less sensitive (not able to detect few genotypes) and used
to become positive only after 10 weeks of infection.
o Third generation ELISA: Standard method for HCV serology. It
employs antigens from NS5 region in addition to core, NS3 and
NS4 regions.

HCV antibody detection assay (ELISA)
Advantages Disadvantages
Increased sensitivity and specificity
(> 99%);
These assays detect IgG antibodies to HCV, hence they
do not discriminate between active or past infection;
for which HCV RNA test is required
Not influenced by HCV genotype False positive result- seen in with autoimmune
diseases, mononucleosis, and pregnancy
Becomes positive in 5 weeks of
infection.
False negative result- seen in with immunosuppression
such as HIV infection, transplant recipients etc.

Recombinant immunoblot antibody assay (RIBA)
• Used in past as a supplementary test to confirm the ELISA
result; not in use currently.
• Works based on the principle of western blot.
• Detects antibody against core, E2 hypervariable region and
NS3, NS4A, NS4B and NS5A regions.

HCV core antigen assay
• Automated quantitative HCV antigen test detecting core antigen has
been available recently (e.g. Abbott ARCHITECT HCV antigen assay).
• This test is less expensive and less time consuming than HCV RNA PCR.
• Advantages: Antigen detection assays can be used as an alternative to
HCV RNA testing for: (i) diagnosis of active/current infection, (ii)
monitoring response to treatment.
• Disadvantage: They are less sensitive than RT-PCR; hence not
recommended for blood screening purpose.
• An ELISA format has recently been available that detects
simultaneously antigen (core Ag) and antibody (to NS3 and S4 Ag).

Molecular methods
• Real time reverse transcriptase PCR detecting HCV RNA has
been the gold standard method for:
o Confirmation of active HCV infection.
o Quantification of HCV RNA for monitoring the response to
treatment.
o For determining HCV genotype and subtype (also done by
sequence based method).

Diagnosis of acute HCV infection
• Most cases of HCV infections remain asymptomatic in early stage and are
directly presented with chronic infection (i.e. after 6 months). Acute HCV
infection (i.e. onset of symptoms within 6 months of exposure) occurs very
rarely.
• Both IgM and IgG antibodies appear during acute stage; IgM may persist
during chronic stage for variable time; whereas IgG persists for long
duration both in chronic stage and during recovery.
• Most assays available detect IgG antibodies. There is no IgM based assay
available currently.
• The acute infection can be diagnosed by: (i) anti-HCV IgG sero-conversion,
(ii) HCV RNA positivity or (iii) IgG avidity assay (low avidity indicates acute
infection, strong avidity- may indicate chronic/past infection).

Diagnosis of acute HCV infection
• Most cases remain asymptomatic in early stage and are
directly presented with chronic infection (i.e. after 6 months).
• Acute HCV infection (i.e. onset of symptoms within 6 months
of exposure) occurs very rarely.
• Both IgM and IgG antibodies appear during acute stage:
o IgM may persist during chronic stage for variable time
o IgG persists for long duration both in chronic stage and
during recovery.

Algorithm of HCV diagnosis (Adapted from CDC
guideline, 2013)
Test outcome Interpretation Further actions to be taken
HCV antibody
non-reactive
No HCV antibody
detected
 Reported as non-reactive. No further action required.
 If history of recent exposure within 6 months, then :
o Do follow-up testing for HCV antibody (for immunocompetent
patient)
o Test for HCV RNA (for immunocompromised patient)
HCV antibody
Reactive
Presumptive HCV
infection
 A repeatedly reactive result indicates either:
(i)current HCV infection, or (ii) past HCV infection that has resolved, or
(iii) biologic false positivity for HCV antibody.
 HCV RNA PCR should be done - to identify current infection.

Algorithm of HCV diagnosis (Adapted from CDC
guideline, 2013)
Test outcome Interpretation Further actions to be taken
HCV antibody
reactive,
HCV RNA
detected
Current HCV infection  Provide appropriate counselling to the patient and refer to
HCV clinic.
 Repeat HCV RNA PCR should be done before initiating
treatment.
HCV antibody
reactive,
HCV RNA not
detected
No current HCV
infection
 No further action required in most cases.
 If clinically suspected or history of exposure within 6 months: then
follow up with HCV RNA testing and appropriate counselling.

Treatment Hepatitis C
• The goal of treatment in HCV infection is:
o To prevent development of complications such as cirrhosis and liver cancer.
o To achieve sustainable viral response (SVR) which defined as undetectable HCV RNA
in blood (≤15 IU/ml) at the end of treatment.
• Interferon alfa plus ribavirin
o Earlier, pegylated-interferon (PEG-IFN) alpha plus ribavirin (RBV) was used for
treatment of HCV infection.
o This was associated with intolerable adverse effects with a moderate degree of viral
clearance (SVR 40- 50%).
• Direct-acting antiviral agents (DAAs)
o After the discovery of direct-acting antiviral agents (DAAs), the treatment of HCV
infection has been revolutionized.
o DAAs are the now the treatment of choice for HCV infection as they can produce SVR
rate up to 90% with minimal side effects.

• Three classes of DAAs are available: NS3/4A (proteases)
inhibitors, NS5B (polymerases) inhibitors and NS5A inhibitors.
Class Agents
Directly acting antiviral agents (DAAs)
NS3/4A inhibitors (proteases) Ends with suffix ‘previr’
Grazoprevir Paritaprevir , Simeprevir
NS5B inhibitors (polymerases) Ends with suffix ‘buvir’
Dasabuvir, Sofosbuvir
NS5A inhibitors Ends with suffix ‘asvir’
Daclatasvir, Ledipasvir, Velpatasvir
Other classes
Interferons Pegylated interferon alfa-2a & 2b
Nucleoside inhibitor Ribavirin

• Combination therapy: The treatment regimen for HCV
includes combinations of different DAAs with or without PEG-
IFN and ribavirin.
• Genotype specific: The drugs to be included in the regimen
depends upon the genotype detected. Most DAAs are
genotype specific; except sofosbuvir/ velpatasvir which are
active against all genotypes.
• Duration: Either 12 or 24 weeks; depending up on the HCV
genotype and associated liver cirrhosis.

Predictors of Treatment Response
• Genotypes: HCV genotype 1 and 4 are associated with poor prognosis than other
genotypes
• Stage of disease and associated co-infections such as HIV and HBV: associated with poor
prognosis
• Adherence: Poor compliance to treatment, is associated with worse prognosis
• Viral RNA load: Higher the viral load (> 800,000 IU/mL), worse is the prognosis
• Interleukin 28 B is a strong inducer of interferon-a.
• People possessing a subtype of IL 28B (called CC genotype), produce a stronger immune
response to HCV infection by inducing IFN-a release; hence have a better outcome
• Race: Caucasians and African Americans lack CC genotype - poor treatment response
than Asians
• Others: Metabolic disorders such as insulin resistance, obesity metabolic syndrome,
steatosis, lack of vitamin D supplement, older age, alcohol consumption—all reduce the
chance of responding to HCV therapy.

Prevention
• No effective vaccine available for HCV.
• General prophylactic measures are essentially same as that for
HBV.

HEPATITIS D
• Hepatitis D is a defective virus; cannot replicate by itself; depends on Hepatitis B
for its survival.
• Morphology:
o Hepatitis D is taxonomically unclassified though resembles viroids. It is small
in size(35nm), consisting of-
o Circular, negative-sense ssRNA
o Protein coat made up of single protein called as- hepatitis D antigen (HDAg)
o Surrounded by envelope protein derived from HBsAg from hepatitis B; hence
it is called as defective virus.
• Transmission:
o Transmission is similar to that of HBV and HCV.

Association of HDV with HBV
• Co-infection occurs when a person is exposed simultaneously to serum containing both
HDV and HBV.
o Hepatitis B infection sets in first so that HBsAg becomes available for HDV
o Transient and self-limited condition (indistinguishable from acute hepatitis B)
o Rarely progresses to chronic stage; at a rate similar to that of HBV.
o Vaccination against hepatitis B can prevent against HDV.
• Super-infection occurs when a chronic carrier of HBV is exposed to serum containing
HDV. This results in disease 30–50 days later which may have two phases:
o Acute phase - HDV replicates actively with high transaminase levels with
suppression of HBV.
o Chronic phase - HDV replication decreases, HBV replication increases, transaminase
levels fluctuate, and the disease progresses to cirrhosis and hepatocellular
carcinoma (HCC). Mortality rate is much higher (>20%).

Differences between HDV-HBV co-infection and
super-infection
Co-infection Super-infection
Definition HBV and HDV infection occurs
simultaneously
HDV infection occurs to a
carrier of HBV
Patient status Healthy HBV carrier
Risk of development of-
Fulminant >HBV alone >Co-infection
Chronic Rare Much greater
Cirrhosis Rare More
HCC Rare More
Mortality Rare >20%
Diagnosis 1.HBsAg
2.Anti-HBc (IgM)
3.Anti-HDV (IgM)
4. HDV RNA
1.HBsAg
2.HBeAg
3.Anti-HBc (IgG)
4.Anti-HDV -
In acutephase- IgM
In Chronic phase- IgG and IgM
5. HDV RNA

Epidemiology
• Globally, about 15 million people are infected with HDV (about 5% of 350
million of HBV infected persons).
• Two epidemiologic patterns have been identified-
o In endemic areas such as Mediterranean countries - HDV is endemic
among persons with hepatitis B and is transmitted predominantly by
non-percutaneous means, especially by close personal contact.
o In non-endemic areas (USA and Northern Europe):
 Confined to persons exposed frequently exposed to blood and blood
products, primarily injection drug users and hemophiliacs who are infected
with HBV.
 Introduced into a non-endemic population through drug users or by
migration of persons from endemic to non-endemic areas.

Prevention
• Vaccination for HBV can also prevent HDV infection.
• General prophylactic measures are essentially same as that
for HBV.

HEPATITIS E VIRUS
• Hepatitis E virus (HEV) causes an enterically transmitted hepatitis.
• Morphology:
o HEV is small (30–32 nm size),non-enveloped with icosahedral
symmetry.
o Positive-sense, ssRNA, and a specific antigen (HEV-Ag).
• Genotypes- HEV has single serotype; however, five genotypes
exist in nature, which differ up to 25% in their RNA sequence.
Only four genotypes have been detected in humans
o Genotypes 1 & 2 appear to be more virulent
o Genotypes 3 & 4 are more attenuated and account for
subclinical infections.

Clinical manifestation
• Incubation period is about14–60 days (average 40days).
• Most of the patients present as self-limiting acute hepatitis
lasting for several weeks followed by complete recovery.
• Fulminant hepatitis may occur rarely in 1-2% of cases
• Pregnant women who are particularly at higher risk (20%) of
developing fulminant hepatitis.
• There is no chronic infection or carrier state.

Epidemiology
• Zoonotic disease - monkeys, cats, pigs, and dogs
• Transmission- It is feco-orally transmitted via sewage contamination of drinking water or
food.
• Epidemics have been reported primarily from India, Asia, Africa, and Central America; in
those geographic areas, HEV is the most common cause of acute hepatitis in this zone.
• The first major epidemic cof HEV was reported from New Delhi (1995) where 30,000
people were affected due to sewage contamination of the city's drinking water supply
following flood in Yamuna river.
• In India, HEV infection accounts for maximum (30-60%) cases of sporadic acute hepatitis.
• Though it resembles to HAV, the striking features that differentiate HEV from that of HAV
are:
o Secondary attack rate is rare (1-2%) in HEV, compared to 10-20% in HAV.
o Age- Young adults (20-40 years age)

Laboratory diagnosis
• HEV RNA (by reverse transcriptase PCR) and HEV virions ( by
electron microscopy) can be detected in stool and serum
even before the onset of clinical illness
• Serum antibody detection by ELISA-
o IgM anti-HEV appears in serum at the same time with the
appearance of liver enzymes and indicates acute infection.
o IgG anti-HEV replaces IgM in 2 to 4 weeks (once the
symptoms resolve)and persists for years; indicates
recovery or past infection.

Treatment & Prevention
• Treatment - No specific antiviral drug available.
• Prevention-
o General measures for prevention and containment of
infection are the same as described for HAV.
o China has produced and licensed the first HEV vaccine
called ‘HEV239’ using recombinant proteins.
o However, it is not yet available globally.

HEPATITIS G VIRUS
• Hepatitis G virus(HGV, also referred to as GB virus C) was
discovered in 1995.
• It is related to Hepatitis C virus, belongs to family Flaviviridae,
under the genus Pegivirus.
• Transmitted by contaminated blood or blood products, or via
sexual contact.
• Wrongly named as it is not hepatotropic and does not cause
hepatitis.
• Replicates in the bone marrow and spleen
• Not associated with any known human disease so far.

HEPATITIS G VIRUS
• Classified into six genotypes, each has its own geographical
distribution
• HIV co-infection- HGV commonly co-infects people infected
with HIV (prevalence 35%)
• Surprisingly this dual infection is protective against HIV and
patients survive longer.

Chapter 50 Hepatitis Viruses.pptx education

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