Flaviviruses - Hepatitis C Virus Flashcards
Discovery of Hepatitis C
Previously known as Non-A and Non-B
Discovered through infection of chimpanzees
Discovered through PCR and sequencing of infectious cloned RNA variant samples that led to implementation of EIA tests.
Epidemiology
Blood-borne pathogen
highest prevalence in Africa, Asia and South America
Silent epidemic - burden on the health system
Two stages of HCV infection
- Acute infection - first six months. 20% of those infected will spontaneusly clear the inection
- Chronic infection - After6 months
- 80% will progress and develop Cirrhosis and liver failure.
-5% develop hepatocellular carcinoma.
What happens to during acute infection?
Virons detectable in blod plasma in a few days.
-Peak viral loads observed 6-10 weeks post infection
Hepatocytes release alanine aminotransferase (ALT)
-Used as an indicator of extent of damage
-Normal range = 5-40 IU/L
-Elevation of 5-15 x normal range observed 4-12 weels post infection
Immune response delayed by several weeks
-Only detectable following ALT peak ( Once HCV-specific cytotoxic T cells are detectable an ALT peak is observed)
-Humoral responses may appear after 8 weeks p.i
-spontaneous clearance associated with strong CD4+ and CD8+ responses
What happens to during chronic infection?
Viral loads remain high in periphery.
ALT levels fluctuate throughout infection:
-often normal levels followed by erratic peaks.
-Indicative of progressing liver damage
-Males tend to have higher ALT levels than females.
Cellular mediated immune responses are generally ineffective:
-Humoral responses detected in 50-70% of patients.
No correlation between viral load and liver damage- reinforcing that damage is caused by active immune response.
Hepatitis C Viron Components
50 nm in diameter
Envelope E1 and E2
Core/Nuleocaspid- surrounds genome
-Enveloped virus
-Contains positive sense, single stranded RNA molecule
HCV genome - Components and function
CORE: Nucleocapsid
E1 AND E2: Envelope of virus – involved in cell entry
P7:Ion channel – needed for viral infectivity
NS2: Viral assembly and release
NS3: Helicase enzyme – unwinds RNA and DNA
NS4A: Anchors NS3 to Endoplasmic Reticulum membrane
NS4B: Membranous web essential for replication
NS5A: Genome replication
NS5B: Encodes the RNAse dependent RNA polymerase (RdRp) enzyme
Viral Life Cycle - List stages
Virus enters periphery - travels to liver
1. Viral entry into cells
2. Viral translation
3. Polypeptide processing
4. Viral Replication
5. Viral assembly and release
Viral Life Cycle - Viral Entry happens in 4 stages
- Attachment to cell (hepatocyte) surface
-Viral Envelope E2 binds to two receptors (CD81 and SRB1)
-Binding of CD81 forces viron closer to hepatocyte tight junctions
-SRB1 found only on hepatocytes and is normally used in lipoprotein uptake - Direction to tight junctions
-Purpose to separate neighbouring cells and maintain diffusion
-Two types of tight junctions present: CLDN1 and Occludin
-CLDN1 critical for viral entry
-Occludin essential for anchoring adhesion
-Exact mechanisms not known. - Uptake via endocytosis
-HCV fuses to cell forcing rearrangment of envelope (E1 and E2) proteins
-Creates fusion pore
- Endocytic vesicles formed - Clathrin-coated pits - Delivery of nucleosome
-Vesicle coat shed, delivering viral nucleocaspid (containing genetic information) into the cell cytoplasm.
Viral Life CYcle - Polypeptide Processing
Host signal peptidases cleave structural proteins:
- further processing of core forms nucelocaspid
-envelope proteins released - forms a non-covalent heterodimeric complex
P7 also released and folds into ion channel in the outer envelope
Viral Life Cycle - Viral Translation
- Inside cells ribosomes initiate translation of RNA sequences.
-Viral 5’UTR contains an IRES - positions host ribsoosme at the beginning of viral sequence (riibosome attaches there)
-Host cell then initiates protein synthesis of viral genome
-Viral polypeptide produced.
Polypeptide procesing - role of proteases
Two viral proteases: NS2/NS3 and NS3/4A
NS2/NS3 zinc metalloproteases: cleaves NS2 and NS3 proteins
NS3 is a serine protease requiring cofactor NS4A
-association prevents protease degradation
-cleaves junctions between NS3/4A, NS4A/4B, NS4B/5A and NS5A/5B
Cleavage forms the replication complex
Viral Life Cycle - Viral Replication
-Replication driven by viral NS5B RdRp enzyme
-Essential viral component
-Structural fold consisting of a palm, finger and thumb domain:
- Palm domain contains active enzymatic site
- Feeds single stranded RNA through
- Copied in 5’ to 3’ direction
Viral Life Cycle - Viral Assembly and Release - How does the Viral particle exit the cell?
Viral particle exits cell via Golgi apparatus system using secretory pathway
Viral budding: Nucelocaspid is surrounded by a phospholipid bilayer with E1 and E2 anchored into the bilayer.
Viral particles become tightly bound with VLDL during assembly.
Exact process of secretion is not known.
Viral Evasion of Immune System
HCV thought to outpace the immune response - reaches maximum titres quickly
Sequence mutations:
- HCV exsists as a quasispecies
-NS5B RdRp lacks proof reading ability during replication
-Virus able to select escape mutants - constantly evades immune system
-Hinders vaccine development (E2 HVE1 believed to mutate)
