Dr. Jing Qian Dept. of Medical Microbiology and Parasitology Medical school, Zhejiang university

Dr. Jing Qian

Dept. of Medical Microbiology and Parasitology

Medical school, Zhejiang university

Email: [email protected]

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

mailto:[email protected]



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


Heparnavirus


• 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


Heparnavirus


• 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


Heparnavirus





• 1 serotype and 7 genotypes

1 个血清型主要抗原决定簇 VP1

7 个基因型（ VP1 ）


Heparnavirus






• Animal model and cell culture

Biological properties

• Picornavirus classified as Enterovirus 72 in 1980s, later classified Heparnavirus






• 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


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


• S region: capsid protein

– S gene: HBsAg

– preS1 gene: preS1Ag


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


• C region

– C gene: HBcAg

– preC gene + C gene : HBeAg


• P region: DNA polymerase

(RDDP,DDDP, RNase H)(RDDP,DDDP, RNase H)


•X region: HBxAg

0.8 kb mRNA

trans-activation factor


• 4 open reading frames

• S region: capsid protein

– S gene: HBsAg



• 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


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).



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


Mature particle

Immature particle

Single Strand (+) RNA

7.2-7.6 kb


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

Documents

Dr. Jing Qian Dept. of Medical Microbiology and Parasitology Medical school, Zhejiang university