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Lectures for biomedical students. Hepatitis Virus, Hemorrhagic fever viruses, Arbovirus 肝炎病毒、出血热病毒和虫媒病毒. Dr. Jing Qian Dept. of Medical Microbiology and Parasitology Medical school, Zhejiang university Email: jingqian@zju.edu.cn 2014.5.29. Hepatitis & Hepatitis Virus. 肝炎和肝炎病毒. - PowerPoint PPT Presentation
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Dr. Jing Qian
Dept. of Medical Microbiology and Parasitology
Medical school, Zhejiang university
Email: jingqian@zju.edu.cn
2014.5.29
Lectures for biomedical students
Hepatitis Virus, Hemorrhagic fever viruses,
Arbovirus
肝炎病毒、出血热病毒和虫媒病毒
肝炎和肝炎病毒
Hepatitis & Hepatitis Virus
• Hepatitis- Inflammation of the liver, destruction of hepatocytes
• Acute Infection-Icteric ( 黄疸的 ) phase
• Chronic Infection-May progress to:Hepatic fibrosis, cirrhosis, liver failureIncreased risk of hepatocellular carcinoma
Definitions and Introduction
Icteric phase/Jaundice
* Skin, sclera ( 巩膜 ), underneath tongue
AA“ Infectious”
“ Serum”
Viral hepatitis
Entericallytransmitted
ParenterallytransmittedF, G, TTV
? other
EE
NANBNANB
BB DD CC
Viral Hepatitis - Historical Perspectives
• Focus on the viruses
– Viral structure, biology– Transmission and epidemiology
– Pathogenesis and immunity– Clinical findings
– Laboratory diagnosis– Treatment and Prevention
• HAV• HBV• HCV• HDV• HEV
Hepatitis A Virus (HAV)
• “Epidemic jaundice”• “Infectious hepatitis” (1912)• Hepatitis A
Biological properties• Picornavirus classified as Enterovirus 72 in 1980s, later classified
Heparnavirus
• 27 nm naked (non-enveloped) icosahedral capsid
• Extremely stable capsid
mature particle
Immature 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: 非编码区IRES: 内部核糖体进入位点
3B-VPg/primer protein病毒基因组连接蛋白
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
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
• Extremely stable capsid
• 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
• 1 serotype and 7 genotypes
1 个血清型 主要抗原决定簇 VP1
7 个基因型( VP1 )
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
• Extremely stable capsid
• 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
• 1 serotype and 7 genotypes
• Animal model and cell culture
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
• Extremely stable capsid
• 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
• 1 serotype and 7 genotypes
• Animal model and cell culture
Fecal-Oral spread
contaminated water or food ( shellfish, green onions)
Risk factors:
poor sanitation and hygiene, overcrowding, daycare
Transmission of HAV
Epidemiology
Enters bloodstream through gastrointestinal epithelium
Replicates in hepatocytes and Kupffer cells
Released by exocytosis, not cell lysis in cultured cells
It is likely that attack by cytotoxic T cells causes the demage to the hepatocytes
Goes into bile, intestine, excreted in feces
Shedding of virus for 10 days prior to any symptoms
PathogenesisNot fully understood
Immunity
无论显性感染或隐性感染 均能产生抗 -HAV 的 IgM 和 IgG 抗体
抗 -HAV 的 IgM 在急性期和恢复早期出现 阳性可作为甲肝的确诊依据
抗 -HAV 的 IgG 在恢复后期出现 有保护作用,维持终身
May be mild to asymptomatic in children
Abrupt onset of disease in adults
“Self-limited”-controlled by immune system
Low overall mortality from fulminant ( 暴发性的 ) hepaptitis
Higher risk with simultaneous liver disease such as cirrhosis due to alcohol or chronic Hepatitis B or C
Clinical findings
Acute Hepatitis A Infection
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
Diagnosis of HAV
No antiviral therapy is available
Treatment and Prevention of HAV
Passive immunizationPolyclonal anti-HAV antibodies that persist for 6 monthsOnly effective for 2 weeks prior to exposureExpensive, painful, IM injection site reactions
Active immunizationa killed HAV vaccine
a live attenuated HAV vaccine
Sanitation
Avoidance of questionable food and water in endemic regions
Hepatitis B virus (HBV)
• Baruch Blumberg, 1963: ‘Australian antigen - Au‘.• 1968: Au was a viral antigen = HBsAg (surface antigen)HAA• Dane, 1970: Discovered 42nm 'Dane particles‘ HBcAg (core antigen). • 1973: HBeAg discovered (endogenous antigen = a truncated
version of HBcAg). • 1983: members of Hepadnaviridae
Biological properties
Electron microscopy of hepatitis B virus-positive serum reveals 3 morphologically distinct forms of particles
spherical particle
(HBsAg)
Dane particle
(complete virion )
tubular particle
(capsid) (capsid) detergentdetergent HBeAgHBeAg
(also found in the soluble forms in virus-positive sera)
Dane particle-structure
Dane particle-antigen
HBsAg •surface (coat) protein •“ a ” antigenic determinant : 124-147aa •4 phenotypes adw, adr, ayw, ayr
HBcAg •inner core protein •a single serotype
HBeAg •secreted protein•function unknown
5’- TTCACCTCTGC DR 1: direct repeat (+)DR 2: direct repeat (-)
DNA POLYMERASE
LEADING SEQUENCE
cohesive terminus
粘性末端
Genetic structure
dsDNA(-) 3200 nt(+) 50-90% of (-)
Genetic structure(L-)
• 4 open reading frames
S, C, P, X regionsS, C, P, X regions
P region overlap with S C PP region overlap with S C P
Genetic structure(L-)
• S region: capsid protein
– S gene: HBsAg
– preS1 gene: preS1Ag
– preS2 gene: preS2Ag
SP1 promoter , 2.4kb mRNA , large protein/ 大蛋白 SP2 promoter , 2.1kb mRNA , middle protein/ 中蛋白 & main protein 主蛋白
HBV envelope proteins :
− 主蛋白: HBsAg , S gene , 226aa
− 中蛋白: HBsAg+PreS2Ag , S+PreS2 gene , 281aa
− 大蛋白: HBsAg+PreS1Ag+PreS2Ag , S+PreS1 gene+PreS2 gene,400aa
Genetic structure(L-)
• C region
– C gene: HBcAg
– preC gene + C gene : HBeAg
Genetic structure(L-)
• P region: DNA polymerase
(RDDP,DDDP, RNase H)(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
• X region: HBxAg
(trans-activation factor)
(RDDP,DDDP, RNase H)(RDDP,DDDP, RNase H)
1. absorption, uncoating Pre S1, Pre S22. L-DNA → dsDNA DDDP3. 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→HBxAg5. packaging of pre-genome and inner caspid
& mRNA(pre-genome) →DNA(-)6. DNA(-) → DNA(+) RNase H7. virion packaging and release budding/exocytosis
replication
Genotype
Variation >=8% nt seq A-H Genotype vs. serotype
A adw B adw C adr, adw, ayr D ayw
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 PreCgene )
e supression(1762/1764 ntmuationin 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 °C 10min, pH 2.4 6h
Sensitive to detergent
Pathogenesis
Transmission Routes
Concentration of Hepatitis B Virus
in Various Body Fluids
High ModerateLow/Not
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
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 HBV Infection
Balance between virus clearance and liver injury
• Virions released by exocytosis, not cytolytic
• Ab-mediated immune responses – Type II hypersensitivity– Type III hypersensitivity
• Cell-mediated immune responses– Type IV hypersensitivity
Immunopathogenesis
– Immature responses– mild symptoms, chronic infection (90%)
HBV infection in infants and young children
HBV mutation and disease
HBV pre-C mutation and chronic hepatitis
HBV C mutation and fulminant 暴发性的 hepatitis
HBV mutation and drug resistance
Immune responses
CTL mediated anti-virus immune responses vs. liver injury
Antigen/Antibody Responses
HBsAg & anti-HBs
•HBsAg
–major sign of HBV infection
–acute infection
–chronic infection or carrier
–Hepatocellular cancer patient
•anti-HBs
–neutralization antibody
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
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
大三阳 小三阳
Prevention and Treatment
Treatment• Interferon - for HBeAg +vs carriers with chronic active hepatitis. Response rate is
30 to 40%.
– alpha-interferon 2b (original)
– alpha-interferon 2a (newer, claims to be more efficacious and efficient)
• Lamivudine - 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 – less likely to develop resistance than Lamivudine and may be used to treat Lamivudine resistance HBV. However more expensive and toxic
• Entecavir – most powerful antiviral known, similar to Adefovir
• Successful response to treatment will result in the disappearance of HBsAg, HBV-DNA, 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.
If you have never had hepatitis B,you can get 3 shots . . .
. . . and get long lasting protection.
321
Hepatitis B can be prevented!
6 months old
Hepatitis BVaccine
Baby Shots for Hepatitis B
if the mother has Hepatitis B
1 - 2 months old
Hepatitis BVaccine
+
Birth
H-BIGHepatitis B
Vaccine
Hepatitis C virus (HCV)
•“Non-A Non-B Hepatitis”•Identified in 1989 by molecular methods
Biological Properties
• Related to flaviviruses 黄病毒 and pestiirvuses 瘟病毒
• 40-60 nm particle, spherical• an enveloped virion• Genome: (+)ss RNA• Six genotypes, regional prevalence• Great heterogeneity, many “quasispecies 准
种”
Biological Propertiesicosahedral, positive strand RNA viruses
gain an envelope from host cell virus particle is about 30 to 60nm across
Biological Propertiesgenome of 9,600 bases codes for ten proteins
viral RNA does not have a 5’ cap or 3’ poly A tract translation of the viral RNA is mediated by the internal ribosome entry site (IRES)
Biological PropertiesHCV 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 PropertiesHighly varied genome of HCV
Biological PropertiesHighly 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 PropertiesHighly varied genome of HCV
Biological PropertiesHighly varied genome of HCV
Quasispecies& strains
Quasispecies 准种:同一感染者体内同时存在同一基因亚型的不同变异株
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 nonexistent 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-αalone or with
ribavirin
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– Rare in the West
Epidemiology
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
Prevention
• No vaccine• Prevention of HBV• Prevention of further exposure risks in HBV
chronic infection
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
Hepatitis E virus (HEV)
Used to be called “Enteric” or “Epidemic” or “Water-borne”
Non-A Non B Hepatitis
Identified in India in 1955
Non-enveloped virus
Calcivirus
Biological properties
Mature particle
Immature particle
Single Strand (+) RNA
7.2-7.6 kb
Biological properties
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 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
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
Prevention
Viral Hepatitis -
OverviewViral Hepatitis -
Overview
AA BB CC DD EESource ofvirus
feces blood/blood-derived
body fluids
blood/blood-derived
body fluids
blood/blood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention pre/post-exposure
immunization
pre/post-exposure
immunization
blood donorscreening;risk behaviormodification
pre/post-exposureimmunization;
risk behaviormodification
ensure safedrinkingwater
Type of HepatitisType of Hepatitis
出血热和出血热病毒
Hemorrhagic fever viruses&
viral hemorrhagic fever
出血热病毒 · 汉坦病毒 HANTAVIRUS
布尼亚病毒科
单负链 RNA 、有包膜
在我国引起流行性出血热( EHF)
自然宿主 啮齿类动物 黑线姬鼠、田鼠、家鼠
唾液、尿、粪便污染
呼吸道、消化道、接触传播
流行季节 10 ~ 12 月
虫媒病毒 Arbovirus(arthropod-borne-viruses)
节肢动物为媒介 transmitted by means of an infected, blood-sucking, arthropod vector (arthropod borne = arboviruses)
单正链 RNA ,有包膜 , 20 面体对称
我国主要流行流行性乙型脑炎病毒、登革病毒、森林脑炎病毒
有较广的宿主范围,能在脊椎动物及非脊椎动物体内繁殖,节肢动物
可长期储存和传播病毒
致病具有明显的季节性和地方性,主要引起发热、脑炎、出血热等
流行性乙型脑炎病毒( epidemic type B encephalitis virus )
一、生物学性状黄病毒属,单正链 RNA 、有包膜、二十面立体对称
二、所致疾病引起流行性乙型脑炎 动物蚊人
储存宿主 幼猪 传播媒介 三带喙库蚊
传播途径 蚊虫叮咬 流行季节 6 ~ 7 月(南方)
8 ~ 9 月(北方)
病毒 蚊子叮咬 毛细血管内皮细胞内增殖 入血 ( 第一次病毒血症)流感样 3 ~ 7 天 血液播散 ( 第二次病毒血症)痊愈 全身症状
血 - 脑屏障痊愈 CNS 症状
恢复、死亡、后遗症
三、致病过程
四、抗原性 稳定,只有一个血清型
五、免疫性 依赖体液和细胞免疫
免疫力稳定持久,隐性感染也可获免疫力
六、预防 灭蚊防蚊 ---- 关键
人群免疫 ---- 乙脑死疫苗
幼猪免疫 ---- 乙脑死疫苗
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