Flaviviridae Flashcards
Name the genera and characteristics of Flaviviridae
Genera:
Flavivirus
Hepacivirus (Hepatitis C Virus, HCV)
Pestivirus
Characteristics:
(+)strand RNA virus
Lipid envelope
Single ORF - polyprotein
Why is it hard for the immune system to recognise HCV?
High genomic variability
What are the stages of HCV pathogenesis?
- inflammation/fibrosis of the liver
- cirrhosis
- hepatocellular cancer
Damage is mostly done by immune system.
What are the immunological reactions against virus infections?
- Innate immune system
-interferon system
-macrophages
-NKC
~hours - Cellular immune response
-T-Killer cells (CD8)
-T-helper cells (CD4)
~days - Antibodies
- neutralizing Abs are important for the protection against reinfection - main determinant for success in vaccination
- does not allways protect against infection with identical pathogens
e.g. influenza virus, hepatitis C virus, HI virus
~1-2 weeks
What are HCV‘s strategies of virus persistence?
- suppression of innate immune system
- NS3 needs NS4A as cofactor for full protease activity; is essential for viral replication
- NS3/4A complex cleaves TRIF:
- no signaling through TLR 3 (dsRNA)
- no signaling through TLR 4 (viral glycolipids)
- NS3/4A complex cleaves Cardif (= MAVS; VISA; IPS-1)
- no signaling through RIG-I or Mda-5
- no recognition of cytoplasmatic dsRNA /14 RNA with 5 ́triphosphate - very high antigenic variability
- 6 genotypes: 30 - 35%divergence on RNA level and within the same genotype 20-25% difference
- often o cross-neutralisation
- several vaccine needed
-pre existing resistant mutants are major problem:
- footrace: Antigenic main species is attacked via clonal propagation of B- and T- cells with matching antigen receptors
- minor representatives of quasispecies are not eliminated and thus spread
What was the breakthrough of the HCV replicon system? And how was it achieved?
Allows HCV RNA replication in cell culture through adaptive mutation
Workflow:
- reduction of genome size to minimal set of genes required for RNA replication - integration of a selectable marker (drug resistance gene)
- propagation transfected hepatoma cells (Huh7) under selection pressure:
cells without HCV replication die;
only cells with replicating HCV RNA express resistance gene and survive - surviving cells form colonies (Fig. B)
- replicating HCV RNA can be isolated, sequenced and compared to input RNA
- cell culture adaptive mutations have to be cloned back into original replicon cDNA - RNA replicons dervied thereof can replicate their RNA in Huh7 cells
Which receptors are part of the receptor-mediated endocytosis of HCV?
CD81
Low density lipoprotein (LDL) receptor Scavenger (SR-BI) receptor
Claudin1 (CLDN1)
Occludin (OCDN)
Why could miRNA-122 silencing be a therapeutic strategy for HCV?
- miRNA122 is preferentially expressed in the liver
- miRNA122 regulates fatty acid and cholesterol synthesis - central to liver function
- HCV RNA replication depends on cellular miRNA-122
- Direct interaction of miR-122 with two target sites in the 5‘UTR of the HCV genome (third interaction site in 3`UTR)
- Binding of miR-122 to the 5‘UTR increases the stability and translation of HCV RNA
What are the therapeutic targets for antiviral therapy’s in HCV?
- Entry inhibitors
•neutralising/blocking Abs
•SB-B1 inhibitors
•carbohydrate binding proteins
-Anti-miRNA122 LNA
-virus-assembly inhibitors
•p7 and NS2
- polymerase inhibitors
- protease inhibitors
How does BVDV achieve life long persistence in cows?
There are two biotypes of BVDV: noncp and cpBVBD
The noncpBVBD is transferred to the embryo of cows where it acquires immunetolerance due to the fetus recognising it as „own“ antibody.
What are the 4 strategies of BVDV to achieve virus persistence?
- intrauterine infection → no adaptive immune response due to acquired immunetoleranze in fetus
- suppression of the innate immune system (Npro and Erns block INF induction and innate immunity
- suppression of apoptosis/innate immunity: stringent regulation of NS3 release and therefore RNA replication due to limiting cellular protease cofactor jiv (only NS2-3 autoproteolysis with bound jiv) →leads to the non cytopathic nature of BVDV
4.High antigenic variability of the viral surface antigens
Reinfection of pregnant cows despite of previous BVDV infections
What is needed for the development of a BVDV vaccine?
- Recombinant virus with ncp biotype
- Virus with deletion of Npro and inactivating mutation in RNase of Erns
- no blocking of IFN induction / innate immunity - is not able to establish persistence in the fetus
- should include 2 variants with the envelope protein E2 of BVDV subtypes 1 and 2 (covering a wide range of the antigenic variability)
- safe live vaccine
Still missing: DIVA
marker for the discrimination between infected and vaccinated animals
What it the reason for the reappearance of cpBVDV in cows infected with noncpBVDV?
RNA-recombination:
Identification and characterization of fragments of cellular mRNAs inserted in the viral RNA genomes!
Those insertions encode often substrates for cellular proteases
Impact of the identified genome variations
additional cleavages in the polyprotein, deregulation of replication cytopathogenicity, letal diseases in the PI animal
Effect of ubi-insertion:
- deregulated, complete NS2-3 cleavage
- upregulated RNA replication
- viral cytopathogenicity and disease
What are the two mechanisms of RNA-recombination of BVDV
- template switching: during viral RNA replication switching to a cellular mRNA →hybrid RNA of Viral and cellular RNA
- breakage and ligation: replication independent
How come flaviviruses are transferred to humans as the normal cycle is between ticks/mosquitos and birds?
The decrease of rain Forrest leads to loss of habitat of mosquitos and birds. Mosquitos migrate to cities where they infect humans. Viruses adapt to humans
