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Hepatitis Viruses Youjun Feng
Medical Virology Lecture 10/06 Hepatitis Viruses Youjun Feng Center for Infection & Immunity, Zhejiang University School of Medicine
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• 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
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• HAV • HBV • HCV • HDV • HEV
• Focus on the viruses – Details of hepatic disease and treatment will be demonstrated in other courses • Understand similarities and differences – Viral structure, biology – Modes of transmission – Pathogenesis – Diagnosis – Prevention • HAV • HBV • HCV • HDV • HEV Cryo-EM
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Hepatitis A Virus (HAV)
• “Epidemic jaundice” • “Infectious hepatitis” (1912)
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• Picornavirus classified as Enterovirus 72 in 1980s, later classified
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 //
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• Picornavirus classified as Enterovirus 72 in 1980s, later classified
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
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• Picornavirus classified as Enterovirus 72 in 1980s, later classified
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 water and salt water • one serotype and 7 genotypes ℃ for 1h survive for months in fresh
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Transmission of HAV Water/food-borne virus Ø Fecal-Oral spread
Ø Contaminated water or food ( shellfish, green onions) Ø Risk factors: Poor sanitation and hygiene, overcrowding, daycare
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Pathogenesis of HAV ØEnters bloodstream through gastrointestinal epithelium ØReplicates in hepatocytes and Kupffer cells ØReleased by exocytosis, not cell lysis ØGoes into bile, intestine, excreted in feces ØShedding of virus for 10 days prior to any symptoms
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Ø May be mild to asymptomatic in children
Acute Hepatitis A Infection Ø May be mild to asymptomatic in children Ø Abrupt onset of disease in adults Ø “Self-limited”-controlled by immune system Ø Low overall mortality from fulminant ( 爆发性的 ) hepaptitis Ø Higher risk with simultaneous liver disease such as cirrhosis due to alcohol or chronic Hepatitis B or C
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Diagnosis of HAV ELISA Clinical syndrome
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
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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
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Hepatitis E virus (HEV)
Used to be called “Enteric” or “Epidemic” or “Water-borne” Non-A Non B Hepatitis Identified in India in 1955
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Non-enveloped virus Calcivirus Single Strand (+) RNA 7.2-7.6 kb
Biological properties Non-enveloped virus Calcivirus Single Strand (+) RNA kb Mature particle Immature
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Biological properties
Cryo-EM
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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
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Transmission and Epidemiology
•Fecal-Oral transmission, especially from fecally- contaminated water •Person-to-person transmission •Highest incidence in Asia, Africa, Middle East and Central America •High incidence among pregnant women with 10-20% mortality
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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
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Baruch Blumberg, 1963: “Australian antigen – Au”
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
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Biological properties
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tubular particle Dane particle (complete virion ) spherical particle (HBsAg) Electron microscopy of hepatitis B virus-positive serum reveals 3 morphologically distinct forms of particles
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Hepatitis B virus. Dane particle and incomplete particles that are
found in patient's serum
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– partly double stranded DNA virus, the + strand not complete
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
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Dane particle-antigen HBsAg
• surface (coat) protein • “ a ” antigenic determinant 124- 147aa • phenotypes adw, adr, ayw, ayr HBcAg • inner core protein • a single serotype HBeAg • secreted protein • function unknown
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Genetic structure dsDNA (-) 3200 nt (+) 50-90% of (-) LEADING SEQUENCE
DNA POLYMERASE DR 1: direct repeat (+) 5’- TTCACCTCTGC DR 2: direct repeat (-)
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Genetic structure(L-)
• P region: DNA polymerase (RDDP,DDDP, RNase H)
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Genetic structure(L-)
• X region: HBxAg 0.8 kb mRNA trans-activation factor
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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) •
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Pre S1, Pre S2 Replication DDDP DDRP 1. absorption, uncoating
2. L-DNA → dsDNA 3. dsDNA(L-)→ mRNA 3.5kb, 2.4kb, 2.1kb, kb 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
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Variation HBV DNA polymerase: no proof-reading PreS/S gene PreC/C gene “a” epitope mutation (nt in S gene encode for 145aa, 126aa ) e minus (A-G at1896nt in PreC gene ) e suppression(1762/1764 nt muationin promoter of PreCgene)
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Isolation and culture •Animal models: -Chimpanzee -Duck
•Cell culture: not available •In vitro transfection
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Resistant to low temperature, dry, UV, 70% ethanol,
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
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Pathogenesis
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Transmission Routes
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Concentration of Hepatitis B Virus in Various Body Fluids Low/Not
High Moderate Detectable blood semen urine serum vaginal fluid feces wound exudates saliva sweat tears Breast-milk
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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
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Natural History of Chronic HBV Infection
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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
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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
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结节性多动脉炎 肾小球性肾炎
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HBV infection in infants and
young children – Immature responses – Mild symptoms, chronic infection (90%)
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Balance between virus clearance
and liver injury
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Balance between virus clearance
and liver injury
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• Virions released by exocytosis, not cytolysis
Immunopathogenesis • Virions released by exocytosis, not cytolysis • Ab-mediated immune responses – Type II hypersensitivity – Type III hypersensitivity • Cell-mediated immune responses – Type IV hypersensitivity
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Immune responses
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CTL mediated anti-virus immune responses vs. liver injury
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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
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HBsAg & anti-HBs •HBsAg –major sign of HBV infection –acute infection
–chronic infection or carrier –Hepatocellular cancer patient •anti-HBs –neutralization antibody
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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
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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
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–virus replication /infectious –early stage after infection
HBeAg & anti-HBe •HBeAg –virus replication /infectious –early stage after infection •anti-HBc –the sign of better prognosis –variation: ending codon in pre-C
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Laboratory Diagnosis
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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
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Acute Hepatitis B Virus Infection with Recovery
Typical Serologic Course
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Progression to Chronic Hepatitis B Virus Infection
Typical Serologic Course
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Interpretation of Serologic Markers of HBV
infection
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Prevention and Treatment
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– α-interferon 2b (original)
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, HBV- DNA, and seroconversion to HBeAg. •
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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 healthcare 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
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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.
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for Hepatitis B if the mother has Hepatitis B 1 - 2 months old Birth +
Baby Shots for Hepatitis B if the mother has Hepatitis B Birth + 1 - 2 months old Hepatitis B Vaccine Hepatitis B Vaccine H-BIG 6 months old Hepatitis B Vaccine
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Hepatitis D virus (HDV)
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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
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Gene structure Hepatitis delta agent. Three RNA forms. Adapted from Wagner and Hewitt.: Basic Virology. Blackwell Publishing
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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
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Pathogenesis of HDV • Viral replication causes hepatocyte cell death
• Additive to HBV-induced host inflammatory response • Antibodies to HDVAg not likely to be protective
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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
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Diagnosis • Detection of HDV antigen or antibody • Clinical setting
– Acute fulminant disease – Chronic co-infection
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Hepatitis C virus (HCV)
•“Non-A Non-B Hepatitis” •Identified in 1989 by molecular methods
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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”
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Prevention • No vaccine • Prevention of HBV
• Prevention of further exposure risks in HBV chronic infection
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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.
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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).
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Biological Properties Highly varied genome of HCV
Quasispecies& strains Quasispecies
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• Parenteral – Injected drug use
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%
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Epidemiology
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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
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Vast majority are asymptomatic
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 •
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Diagnosis • Acute infection • Chronic 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
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Behavioral interventions to reduce risk
Prevention No vaccine available No immune globulin Behavioral interventions to reduce risk Treatment: Recombinant IFN-α alone or with ribavirin •
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Viral Hepatitis - Overview Type of Hepatitis
feces B blood/ C blood/ D blood/ E feces Source of virus blood-derived blood-derived blood-derived body fluids body fluids body fluids Route of transmission fecal-oral percutaneous percutaneous percutaneous fecal-oral permucosal permucosal permucosal Chronic no yes yes yes no infection Prevention pre/post- exposure pre/post- exposure blood donor screening; pre/post- exposure ensure safe drinking immunization immunization risk behavior immunization; water modification risk behavior modification 经由皮肤的 >>粘膜的
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