Hepatitis C Virus 1

Question 1

HCV estimated number of carriers?

What diseases does it cause?

Answer 1

123million-170 million carriers of virus

85% of infected individuals don’t clear virus and develop chronic liver disease
—Fibrosis,cirrhosis and hepatocellular carcinoma

HCV infection now main reason for liver transplant surgery

No vaccine- but effective new combination therapy
— direct acting antiviral

Question 2

HCV pathogenesis

Answer 2

Acute hepatitis leads to chronic liver disease <85% of cases

Vigorous immune response fails to clear virus( only clears 15% of cases)
May contribute to liver damage

HCV infections is major risk factor for developing chronic liver disease
–strong association between HCV and cirrhosis/hepatocellular carcinoma maybe 20-30 years later

Jaundice symptoms visible

ALT levels increasing liver damage

Question 3

Epidemiology of HCV

Answer 3

Common route of transmission is blood transfusion

• since 1991 all blood donations are screened
• intravenous drug abuse is associated to spread of this disease

Sexual and vertical transmission is uncommon but does happen
-5% of infants born to HCV-positive mothers are infected

Nucleotide sequence highly variable

• isolates grouped into 7 genotyoes
• types 1-3 most common in Western Europe

Question 4

HCV in Egypt

Answer 4

Very high incidence - approximately 15%

Mainly genotype 4 -> 90% of all infection in Egypt- but rare elsewhere

Due to mass treatment campaigns against Schistosomiasis 1960s
-Antimony potassium tartrate (emetic tartar)
-Poor sterilisation regimes
-reuse of glass syringes 504 patients only 20-30 syringes used
Syringes washed through and boiled for 1-2 minutes before filled with drugs and put back in in tray for use(quote from hospital worker)

Question 5

History of discovery of HCV

Answer 5

1970s evidence for NANBH non-A, non-B hepatitis causing chronic hepatitis

Early 80s-passage of NANBH agent in chimpanzees

Shown to be filterable and inactivated by chloroform-small envelope virus?
Fragment of genome clones
Complete genome cloned and sequenced

Question 6

Steps of HCV genome

Cloning

Answer 6

High titre human serum ultracentrifuged to concentrate virus
|
Nucleic acid extracted and reverse transcribed
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DNA fragments cloned into bacteriophage ygt11
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Expressed in e.coli
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Immunoscreened with NANBH patient serum
|
Clone identified not recognised by control sera

Question 7

HCV genome and polyprotein

Answer 7

Member of the flaviviridae- linear, single stranded +RNA, 9.5kb

Encodes single polyprotein
-encodes for 10 proteins

Genome is flanked by UTRs –> complex shaped RNA

Question 8

Non primate hepacivirus NPHV

Answer 8

Originally isolated from respiratory samples of dogs by random high-throughout sequencing

Most closely related to HCV- diverged 500-1000 years ago

Subsequently identified in 30% of horses
Infectious clones now available
- caused milf hepatitis in one horse

Hepacivirus also in mice,bats,monkeys and cattle

Question 9

Polyprotein processing in HCV

Answer 9

Signal peptides mediates cellular cleavage

NS2/3 cleavage is automatic

NS3 cleaves additional non-structural proteins

All targets for antiviral therapy

Structural proteins core-e1-e2-p7 mediated by cellular protease

Non structural proteins ns2/ns3
NS3 cleaves ns4a ns4b ns5a ns5b

Question 10

HCV replication

Answer 10

Binds
Enters by receptor mediated endocytosis

Membrane fusion and uncials

Translation and polyprotein processing

Virus budding into intracellular vesicles

Transports to cell membrane and fuses for release

(RNA replication is negative strand turned to positive strand then replicated from that template

Question 11

Molecular clones of HCV used to study HCV. What is that l?

Answer 11

A full cDNA copy of HCV was made using reverse tramscription

T7 promoter was upstream the ORF to induce RNA transcription in vitro- generated the full HCV genome

After transferring into human cell line transcription would occur(but no replication)

They tried it out on chimps after modifying the genome
-increased viral titre–>infectious system that generates replicated viruses

Question 12

Use of HCV molecular clone

Answer 12

Allowed studies on individual viral components to proceed with confidence.

Demonstrated that all identified enzymatic activities are required for viral replication

UTR are critical for replication

However- this RNA derived clones didn’t work in culture but it worked in chimp livers
—limited use since cell cultures are the ideal examination sample

Question 13

Subgenomic HCV replicon

Answer 13

All structural proteins are removed to generate subgenomic replicon

Structural genes replaced with selective marker neomycin phosphotransferase

The non structural proteins were expressed under a second ribosomal entry site MCV

Cells can be examined under confocal microscopy–>show replication in cell cultures

Question 14

What does HCV subgenomic replicon tell us

Answer 14

5 non structural proteins at essential for replication and sufficient for HCV RNA replication

NS3-5 form a multiprotein replication complex

Replicates RNA containing UTRs

RNA replication occurs in cytoplasmic membrane bound compartment

Question 15

Problems with HCV replicon

Answer 15

Only work in one cell type-huh7

Replicon with full length genomes don’t replicate

The virus adapts to the cell by mutation- produces more efficient replicons

Only allle the study of part of the viral life-cycle–> can’t isolate the virus though. You can see translation and poly protein processing and RNA replication

Selection in cell population-loads of variables come into play

Question 16

Use of replicons in drug development?

Answer 16

An upgrade was generated that was neomycin out for luciferase
-you can measure replication more efficiently-can see the difference in replication

Identify compounds that block replicon replication to identify direct acting antivirals (DAA)

Go to hep 3 document and see replicons structures