Download Acute Infection

Medical Virology
Lecture 05/06
Hepatitis Viruses
Youjun Feng
Center for Infection & Immunity, Zhejiang University School of Medicine
[email protected]
• Hepatitis: Inflammation of the liver,
destruction of hepatocytes
• Acute Infection-Icteric ( 黄疸的) phase
• Chronic Infection-May progress to:
Hepatic fibrosis,
Cirrhosis,
Liver failure,
Increased risk of hepatocellular carcinoma
• Focus on the viruses
– Details of hepatic disease and treatment
will be demonstrated in other courses
• Understand similarities and differences
Cryo-EM
– Viral structure, biology
– Modes of transmission
– Pathogenesis
– Diagnosis
– Prevention
• HAV
• HBV
• HCV
• HDV
• HEV
Hepatitis A Virus (HAV)
• “Epidemic jaundice”
• “Infectious hepatitis” (1912)
Biological properties
• Picornavirus classified as Enterovirus 72 in 1980s, later classified
Heparnavirus
• 27 nm naked (non-enveloped) icosahedral capsid (二十面体)
• Extremely stable capsid
Immature particle
Mature particle
http //www.ncbi.nlm.nih.gov/books
Biological properties
• Picornavirus classified as Enterovirus 72 in 1980s, later classified
Heparnavirus
• 27 nm naked (non-enveloped) icosahedral capsid
• Positive-sense, single-stranded RNA genome, 7500 nt
NCR:
IRES:
3B-VPg/primer protein
Biological properties
• Picornavirus classified as Enterovirus 72 in 1980s, later classified
Heparnavirus
• 27 nm naked (non-enveloped) icosahedral capsid
• Positive-sense, single-stranded RNA genome
• Resistance: Stronger than enterovirus, resistant to detergents,
acid (pH 1.0 for 2h), 60 ℃ for 1h survive for months in fresh
water and salt water
• one serotype and 7 genotypes
Transmission of HAV
Water/food-borne virus
Fecal-Oral spread
Contaminated water or food ( shellfish, green onions)
Risk factors:
Poor sanitation and hygiene, overcrowding, daycare
Pathogenesis of HAV
Enters bloodstream through gastrointestinal epithelium
Replicates in hepatocytes and Kupffer cells
Released by exocytosis, not cell lysis
Goes into bile, intestine, excreted in feces
Shedding of virus for 10 days prior to any symptoms
Acute Hepatitis A Infection
May be mild to asymptomatic in children
Abrupt onset of disease in adults
“Self-limited”-controlled by immune system
Low overall mortality from fulminant ( 爆发性的 ) hepaptitis
Higher risk with simultaneous liver disease such as
cirrhosis due to alcohol or chronic Hepatitis B or C
Diagnosis of HAV
Clinical syndrome
EILSA
Detection of anti-HAV specific
antibodies
IgM titer in acute infection, positive
for 4-6 months
IgG titer present for decades
Research testing
-Virus feces by electron microscopy
(no cell culture available)
RNA PCR
Prevention of HAV
Sanitation
Avoidance of questionable food and water in endemic regions
Passive immunization
Polyclonal anti-HAV antibodies that persist for 6 months
Only effective for 2 weeks prior to exposure
Expensive, painful, IM injection site reactions
Active immunization
Inactivated HAV vaccine
Live attenuated HAV vaccine
Hepatitis E virus (HEV)
Used to be called “Enteric” or “Epidemic”
or “Water-borne”
Non-A Non B Hepatitis
Identified in India in 1955
Biological properties
Non-enveloped virus
Calcivirus
Single Strand (+) RNA
7.2-7.6 kb
Mature
particle
Immature
particle
Biological properties
Cryo-EM
Pathogenesis
• Hepatic damage by host immune response
• No chronic carrier state
• Acute infection clinical syndrome very similar
to HAV, except higher rates in pregnancy
• Mortality 1-2%, higher than HAV
– 10~20% in pregnant women
– Mechanism unknown
• Diagnosed by HEV-RNA; anti-HEV Ab, IgG
and IgM
Transmission and Epidemiology
•Fecal-Oral transmission, especially from fecallycontaminated water
•Person-to-person transmission
•Highest incidence in Asia, Africa, Middle East and
Central America
•High incidence among pregnant women with 10-20%
mortality
Prevention
Sanitation
No vaccine (Phase III clinical trial in China)
Little known about pre- or post-exposure efficacy of immune
globulin
No efficacy of immunoglobulin obtained from western
populations
Hepatitis B virus (HBV)
• Baruch Blumberg, 1963: “Australian antigen – Au”
1968: Au was a viral antigen = HBsAg (surface antigen)HAA
Dane, 1970:
• Discovered 42 nm 'Dane particles‘
• HBcAg (core antigen).
• 1973: HBeAg discovered (endogenous antigen = a truncated
• version of HBcAg).
• 1983: members of Hepadnaviridae
Biological properties
tubular particle
Dane particle
(complete virion )
spherical particle
(HBsAg)
Electron microscopy of hepatitis B virus-positive serum reveals 3
morphologically distinct forms of particles
Hepatitis B virus. Dane particle and incomplete particles that are
found in patient's serum
Dane particle
• complete 42 nm virion
• nucleocapsid
– partly double stranded DNA virus, the + strand not complete
– DNA polymerase
– HBcAg
– HBeAg
• is also found in the soluble forms in virus-positive sera
• envelope
– lipid bilayer membrane
– protein
• HBsAg, preS1, preS2
Dane particle-antigen
HBsAg
• surface (coat) protein
• “ a ” antigenic determinant 124147aa
• 4 phenotypes adw, adr, ayw, ayr
HBcAg
• inner core protein
• a single serotype
HBeAg
• secreted protein
• function unknown
Genetic structure
dsDNA
(-) 3200 nt
(+) 50-90% of (-)
LEADING SEQUENCE
DNA POLYMERASE
DR 1: direct repeat (+) 5’- TTCACCTCTGC
DR 2: direct repeat (-)
Genetic structure(L-)
• P region: DNA polymerase
(RDDP,DDDP, RNase H)
Genetic structure(L-)
• X region: HBxAg
0.8 kb mRNA
trans-activation factor
Genetic structure(L-)
•
4 open reading frames
•
S region: capsid protein
– S gene: HBsAg
– preS1 gene: preS1Ag
– preS2 gene: preS2Ag
•
C region
– C gene: HBcAg
– preC gene + C gene : HBeAg
•
P region: DNA polymerase
(RDDP,DDDP, RNase H)
•
X region: HBxAg
(trans-activation factor)
Replication
1. absorption, uncoating
2. L-DNA → dsDNA
Pre S1, Pre S2
DDDP
3. dsDNA(L-)→ mRNA
DDRP
3.5kb, 2.4kb, 2.1kb, 0.7-0.9kb
3.5kb mRNA as template for DNA replication (pre-genome)
4. mRNA→ protein
3.5kb mRNA→ inner capsid proteins, DNA polymerase
2.4kb mRNA, 2.1kb mRNA→ outer capsid proteins
0.8kb mRNA→HBxAg
5. packaging of pre-genome and inner caspid
& mRNA(pre-genome) →DNA(-)
6. DNA(-) → DNA(+) RNase H
7. virion packaging and release
budding/exocytosis
Variation
HBV DNA polymerase: no proof-reading PreS/S gene
Prec/C gene
“a” eiptope mutation (nt in S gene encode for 145aa, 126aa )
e minus (A-G at1896nt in PreC gene )
e supression(1762/1764 nt muationin promoter of PreCgene)
Isolation and culture
•Animal models:
-Chimpanzee
-Duck
•Cell culture: not available
•In vitro transfection
Resistance
Resistant to low temperature, dry, UV, 70% ethanol,
ethyl ether, chloroform, phenol
Dis-infected by 100℃ 10min, pH 2.4 6h
Sensitive to detergent
Pathogenesis
Transmission Routes
Concentration of Hepatitis B Virus
in Various Body Fluids
High
Moderate
blood
semen
serum
vaginal fluid
wound exudates
saliva
Low/Not
Detectable
urine
feces
sweat
tears
Breast-milk
Epidemiology
• Estimated 300 million HBV carriers worldwide
• High prevalence areas 10-20%
– China, southeast Asia, sub-Saharan Africa
• Intermediate prevalence areas 2-5%
– Mediterranean, Middle East, Japan, Central and S.
America
• Low prevalence areas 0.1-2%
– N. America, Europe, Australia, New Zealand
Natural History of Chronic HBV Infection
Acute HBV Infection
• Longer incubation period prior to symptoms
• Insidious onset of symptoms rather than abrupt
• Only 25% of infected people will manifest the full
clinical syndrome of hepatitis
• Immune complex disease related to HBsAg
– Seen in ~15%
– Rash, arthritis, fever, necrotizing vasculitis (polyarteritis
nodosum), glomerulonephritis
Chronic HBV Infection
• Occurs in 5-10% of acute infection
– 90% of perinatal
– 20-50% of early childhood
– 5% of adult
• Usually after mild or asymptomatic infection
• Source of ongoing transmission
• 10% may develop cirrhosis or liver failure, mostly
due to chronic active hepatitis
结节性多动脉炎
肾小球性肾炎
HBV infection in infants and
young children
– Immature responses
– mild symptoms, chronic infection (90%)
Balance between virus clearance
and liver injury
Balance between virus clearance
and liver injury
Immunopathogenesis
• Virions released by exocytosis, not cytolysis
• Ab-mediated immune responses
– Type II hypersensitivity
– Type III hypersensitivity
• Cell-mediated immune responses
– Type IV hypersensitivity
Immune responses
CTL mediated anti-virus immune
responses vs. liver injury
Antigen/Antibody Responses
HBsAg
•surface (coat) protein
•4 phenotypes adw, adr, ayw, ayr
HBcAg
•inner core protein
•a single serotype
HBeAg
•secreted protein
•function unknown
HBsAg & anti-HBs
•HBsAg
–major sign of HBV infection
–acute infection
–chronic infection or carrier
–Hepatocellular cancer patient
•anti-HBs
–neutralization antibody
PreS1, PreS2 & anti-PreS1,
PreS2
•PreS1, PreS2
– virus replication /infectious
–newly infection
•anti-PreS1, anti-PreS2
–1st appearance antibody after infection
–neutralizing antibody, clearance of virus
–do not routinely checked for clinical
diagnosis purpose
HBcAg & anti-HBc
•HBcAg
–not detectable in the serum
•anti-HBc, IgM
– virus replication/infectious
– acute infection
– acute episode during chronic infection
– transient response
•anti-HBc, IgG
–do not protect individuals
– chronic infections
– last for a long time
HBeAg & anti-HBe
•HBeAg
–virus replication /infectious
–early stage after infection
•anti-HBc
–the sign of better prognosis
–variation: ending codon in pre-C
Laboratory Diagnosis
Laboratory Diagnosis
• Acute infection by clinical syndrome
– Minority of infections diagnosed by syndrome
• Acute and chronic infection by patterns of
serology
• HBV DNA assay (DNA hybrid or PCR)
• No cultures performed in clinical evaluation
Acute Hepatitis B Virus Infection with Recovery
Typical Serologic Course
Progression to Chronic Hepatitis B Virus Infection
Typical Serologic Course
Interpretation of Serologic Markers of HBV
infection
Prevention and Treatment
Treatment
• Interferon - for HBeAg +vs carriers with chronic active hepatitis. Response rate is
30 to 40%.
– α-interferon 2b (original)
– α-interferon 2a (newer, claims to be more efficacious and efficient)
• Lamivudine (拉米夫定) – a nucleoside analogue reverse transcriptase inhibitor.
Well tolerated, most patients will respond favorably. However, tendency to relapse on
cessation of treatment. Another problem is the rapid emergence of drug resistance.
• Adefovir (阿德福韦) – less likely to develop resistance than Lamivudine and may be used to
treat Lamivudine resistance HBV. However more expensive and toxic
• Entecavir (恩替卡韦) – most powerful antiviral known, similar to Adefovir
• Successful response to treatment will result in the disappearance of HBsAg, HBVDNA, and seroconversion to HBeAg.
Prevention
• Vaccination – highly effective recombinant vaccines are now
available. Vaccine can be given to those who are at increased
risk of HBV infection such as health care workers. It is also given
routinely to neonates as universal vaccination in many countries.
• Hepatitis B Immunoglobulin – HBIG may be used to protect
persons who are exposed to hepatitis B. It is particular
efficacious within 48 hours of the incident. It may also be given
to neonates who are at increased risk of contracting hepatitis B
i.e. whose mothers are HBsAg and HBeAg positive.
• Other measures - screening of blood donors, blood and body fluid
precautions
Hepatitis B can be prevented!
If you have never had hepatitis B,
you can get 3 shots . . .
1
2
3
. . . and get long lasting protection.
Baby Shots
for Hepatitis B
if the mother has Hepatitis B
1 - 2 months old
Birth
Hepatitis B
Vaccine
+
Hepatitis B
Vaccine
H-BIG
6 months old
Hepatitis B
Vaccine
Hepatitis D virus (HDV)
Hepatitis D
• “Delta agent”
• Defective virus similar to plant viruses
– Small single-stranded circular RNA genome
– Single HDV antigen
– Lipid envelope from HBV, HBsAg needed for packaging
• Depends on HBV for life cycle
– Co-infection with acute HBV
– Superinfection in chronic HBV
• Replicates very efficiently in hepatocytes
Gene structure
Hepatitis delta agent. Three RNA forms. Adapted from Wagner and Hewitt.: Basic Virology.
Blackwell Publishing
Transmission and Epidemiology
• Parenteral
– Injected drug use
– Less efficient sexual transmission than HBV
• Up to 5% of chronic HBV carriers may also carry HDV
• Varies greatly by region
– Endemic in Mediterranean (地中海)
– Rare in the West
Pathogenesis of HDV
• Viral replication causes hepatocyte cell death
• Additive to HBV-induced host inflammatory
response
• Antibodies to HDVAg not likely to be
protective
Clinical Consequences of Infection
• Increases risk of fulminant hepatitis greatly
upon co-infection
– Estimated 2-20% fulminant cases
• Increases risk of cirrhosis in chronic infection
with HBV
– More rapid progression
– More likely progression
Diagnosis
• Detection of HDV antigen or antibody
• Clinical setting
– Acute fulminant disease
– Chronic co-infection
Hepatitis C virus (HCV)
•“Non-A Non-B Hepatitis”
•Identified in 1989 by molecular methods
Biological Properties
• Related to flaviviruses (黄病毒）
and pestivirus (瘟病毒)
•
•
•
•
•
40-60 nm particle, spherical
an enveloped virion
Genome: (+)ss RNA
Six genotypes, regional prevalence
Great heterogeneity, many “quasispecies”
Prevention
• No vaccine
• Prevention of HBV
• Prevention of further exposure risks in HBV
chronic infection
Biological Properties
HCV binds to either the CD81 antigen or low density lipoprotein (LDL) receptor on
hepatocytes via its E2 glycoprotein.
There is also some evidence that it may bind to glycosaminoglycans.
Biological Properties
Highly varied genome of HCV
Genotypes & subtypes
Fig 1 Phylogenetic tree of HCV NS5B sequences. Nucleotide sequences for positions 7975–8196 (numbered
from the polyprotein AUG initiation codon) of NS5B were analyzed using the program Phylipas described
previously (76). Major branches are labeled with the type number, and minor branches with letters indicating the
subtype The variant “10a” can be considered as a subtype of type 3, and the variants “7a”, “7b”, “8a,” and so forth
as subtypes of type 6 (2,3).
Biological Properties
Highly varied genome of HCV
Quasispecies& strains
Quasispecies
Transmission
• Parenteral
– Injected drug use
– Blood transfusions (rare since screening in 1990)
– Nosocomial (医院的)
– Efficiency of sexual transmission is relatively low
– Perinatal (围产期) risk 5%
Epidemiology
Pathogenesis
• Not fully understood
• Prolonged cell-associated state
• Likely low level chronic cell-mediated host immune
response
• Progression to hepatic fibrosis and cirrhosis
• More severe disease progression
– Alcohol
– HIV co-infection
Acute Infection
•
•
•
•
Vast majority are asymptomatic
Few cases of symptomatic acute hepatitis
Very rare or non-existent fulminant cases
Very high rate of chronic infection
Diagnosis
• Acute infection
– HCV RNA in serum, liver biopsy
– HCV Ab negative
• Chronic Infection
– HCV Ab positive (not protective)
– HCV RNA in serum, liver biopsy
• Virus can not be cultured
Prevention
•
•
•
•
No vaccine available
No immune globulin
Behavioral interventions to reduce risk
Treatment: Recombinant IFN-α alone or with
ribavirin
Viral Hepatitis - Overview
Type of Hepatitis
A
Source of
virus
feces
B
C
D
blood/
blood/
blood/
blood-derived blood-derived blood-derived
body fluids
body fluids
body fluids
E
feces
Route of
fecal-oral percutaneous percutaneous percutaneous fecal-oral
transmission
permucosal permucosal permucosal
Chronic
infection
Prevention
no
pre/postexposure
immunization
经由皮肤的 >>粘膜的
yes
yes
yes
no
pre/post- blood donor
pre/post- ensure safe
exposure
screening;
exposure
drinking
immunization risk behavior immunization; water
risk behavior
modification
modification
Self control questions-part I
1. Each of the following statements concerning hepatitis C virus (HCV) and hepatitis D
virus (HDV) is correct EXCEPT:
(A) HCV is an important cause of post-transfusion hepatitis
(B) Delta virus is a defective virus with an RNA genome and a capsid composed of
hepatitis B surface antigen
(C) HDV is transmitted primary by the fecal-oral route
(D) People infected with HCV commonly become chronic carriers of HCV and are
predisposed to hepatocellular carcinoma
2. A 35-year-old man addicted to intravenous drugs has been a carrier of HBs antigen for
10 years. He suddenly develops acute fulminant hepatitis and dies within 10 days. Which
one of the following laboratory tests would contribute MOST to a diagnosis:
(A) Anti-HBs antibody
(B) HBe antigen
(C) Anti-HBc antibody
(D) Anti-delta virus antibody
3. The routine screening of transfused blood for HBs antigen has not eliminated the problem
of post-transfusion hepatitis. For which one of the following viruses screening has
eliminated a large number of cases of post-transfusion hepatitis?
(A) Hepatitis A virus
(B) Hepatitis C virus
(C) Cytomegalovirus
(D) Epstein-Barr virus
•Please summarize the similarities and differences of hepatitis viruses (Viral structure;
Modes of spread; Pathogenesis; Diagnosis; Prevention)
•What is the structure of a complete HBV particle (Dane particle)?
•What is the antigenic composition of a Dane particle?
•How many ORFs (open reading frame) does a HBV gene (long form) have? What are the
gene products from each ORF?
•What kinds of antigen and antibody can be detected in the sera of HBV infected patients?
What is the diagnostic value of each item?
•Term explanation: Dane particle
Thank You!