Hepatitis C Virus Flashcards
Hepatitis C Virus -> General information
Family: Flaviviridae
Genera: Flavivirus, Hepacivirus (Hepatitis C Virus, HCV), Pegivirus, Pestivirus (Bovine viral diarrhea virus 1 (BVDV-1), Classical swine fever virus (CSFV))
Genome: RNA
- single stranded
- positive polarity
- size of 9.5 kb
- IRES element
- no poly-A
Gene expression: Single open reading frame -> Polyprotein
Virions: 40-60 nm in diameter, Lipid envelope
Global prevalence:
- 71 Mio. chronic cases world wide
- 399 000 death 2016
Hepatitis C Virus (HCV) -> Detection and discovery
- Formerly non A / non B hepatitis
- 1987 discovery of HCV genome in patient material (Chiron Corp., CA., USA)
- ELISA for the detection of anti-HCV antibody - Western Blot and RT-PCR detection system
-> No new infections via blood transfusions/blood products!
HCV-Infections in Western Europe
- 5 Mio chron. infected persons
- 20% of all acute hepatitis
- 70% of all chronic hepatitis cases
- 40% of all final stage cirrhosis cases
- 60% of all hepatocellular carcinomas
- 30% of all liver transplantations
- ca. 12.000 deaths each year
Risk factors for HCV-infection
60 % iv drugs
15 % sexual
10 % Transfusion (untested blood products)
5 % Else (Nosocomial, Public Health Service, Perinatal)
10 % Unknown
Sexual and perinatal communication compared to HBV far less relevant
After 20 years of sexual relationship 5 % chance of communication to the partner
Hepatitis C -> behavior
- Communication by blood, blood products, associated risk factors
- Extrahepatic manifestation (mixed cryoglobulinaemia; B-cell anomaly)
- High genomic variability of HCV
- Successive HCV infections possible
(no sufficient cross-immunity) - No vaccine
Hepatitis C: clinical features
- Incubation periode: 2-26 weeks (6-7)
- Immunity: no permanent protection (reinfection)
- Vaccine: not in sight
- Therapy: PEG-interferon + Ribavirin, FDA approved: since 2014 several directly interacting drug
Course of hepatitis C virus infection
25% apparent -> 20% Virus elimination (within 6 months)
75% inapparent -> 80% persistent infection -> ca. 60% chronic hepatitis -> (1-3 decades) -> 10 - 20% liver cirrhosis (ca 40% of LC) -> (2-10 years) -> Hepatocellular carcinoma (ca. 60% of HCC) (1-5% each year)
HCV - pathogenesis
healthy liver -> inflammation; fibrosis -> cirrhosis -> Hepatocellular cancer
Summary of HCV pathogenesis
- HCV frequently establishes persistent infections
- HCV infection causes a complex disease
- liver cell destruction caused mainly by immune response
- contribution of viral cytopathogenicity unclear
- even after successful virus elimination elevated risk for liver cancer
Problem for any study:
- Only valid animal model is chimpanzee (now also mice with human liver cells; see below)
- Consequence: Many data only from cell culture
Course of viral infections
Acute infection -> e.g. influenza virus, Rhinovirus
Chronic infection -> e.g. hepatitis C virus
Latent infection -> e.g. herpes simplex virus (HSV)
Slow infection -> e.g. HIV
Immunological reactions against viral infections
- Innate immune system
- interferone system
- macrophages
- „natural“ killer cells (NK)
-> Period between infection and activation: hours - Cellular immune response
- T-killer cells (CD8)
- T-helper cells (CD4)
-> Period between infection and activation: 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
-> Period between infection and activation: ca 1-2 weeks
HCV: Strategies of virus persistence
1. Strategy: suppression of the 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 /RNA with 5 ́triphosphate
HCV: Strategies of virus persistence
2. Strategy: very high antigenic variability
7 genotypes (8th?): - 30 -35% divergence on RNA level
- within a single genotype 20 – 25% difference (RNA)
Consequence:
- often no cross-neutralisation
- several vaccines needed
Estimated number of formed viruses/per patient and day: ca. 1011-1012 (half live of free virus 11-19 h)
Variance per genome copy: between 1 and 10 exchanges
- single treatment with Telaprevir® eliminates 99.97% of virus in patient
- still 107 viruses per patient replicating
- rebound of titer within 7 – 10 days
Preexisting resistant mutants are a 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
Antigenic variability is critical for the entire adaptive immune response
HCV: different genotypes in different countries
USA; Canada: 1a, 1b, 2a, 2b, 3a
South America: 1a, 1b, 2, 3a
Europe: 1a, 1b, 3a
Northern and Central Africa: 4
South Africa: 5a, 2, 3
Japan, Taiwan, China: 1b, 2a, 2b
Vietnam: 6, 1b, 2
Hongkong: 6a, 1b, 2a, 2b
History of Hepatitis C Virus Research
- until 1989 HAV known, HBV known but high numbers of hepatitis patients without detectable HAV or HBV infection. Terminology: nonA nonB hepatitis
- 1989: Research team of Dr. M. Houghton at Chiron Pharma cloning first piece of HCV genome as cDNA
- cloning of entire genome
- test systems for screening of blood donors (core ELISA, PCR)
But - no cell culture system for HCV isolates from patients (still today)
- no replication of viral RNA in cell culture until 1999
- 1999: Breakthrough! Small lab in Mainz develops HCV RNA replicon system
Basis for all drugs currently in clinical testing against HCV!
Breakthrough: HCV replicon system
Trick:
- 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
- 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
Result: Adaptive mutations allow HCV RNA replication in cultured cells
Replicon system: Basis for all drugs currently tested against HCV!
The molecular basis for cell culture adaption of HCV
HCV WT requires low levels of PI4KA and adapts to high PI4KA expression levels in Huh7-derived hepatoma cell lines.
Model of the role of PI4KA in HCV cell-culture adaptation. Bottom left: PI4KA levels are low in vivo (normal human hepatocytes).
HCV has evolved a mechanism to activate PI4KA via NS5A (blue) and NS5B (green) to generate a PI4P-enriched microenvironment (red hexagons) essential for RNA replication.
Top right: in permissive Huh7 cells, PI4KA levels are increased. Following stimulation, massive amounts of PI4P are generated, which is harmful for most HCV WT isolates.
Bottom right: HCV adapts to increased PI4KA levels by inactivating the PI4KA-stimulating function within NS5A or NS5B via adaptive mutations.
Cell culture adaption in NS5A and NS5B
- In normal human hepatocytes HCV needs to activate the intrinsic low level of PI4Kinase III alpha (PI4KA) molecules by NS5A and NS5B; activated PI4KA recruits cholesterol and glcosphingolipids via OSBP and FAPP2, respectively. This leads to a permissive PI4 phosphate-enriched membrane microenvironment required for
RNA replication. - In hepatoma cells (Huh7) PI4KA levels are massively higher;
in this setting the activation of PI4KA by wt HCV NS5A and 5B blocks RNA replication (delicate balance!) - Cell culture adaptation in Huh7 hepatoma cells requires mutations in NS5A and NS5B to allow for high level RNA replication in the presence of high levels of PI4KA
- In hepatoma cells wt HCV (without adaptive mutations) can only replicate when PI4KA and CKIa are blocked by chemical inhibitors (these inhibitors are a functional substitute for the adaptive mutations in NS5A and 5B)
History of Hepatitis C Virus Research
1989: first HCV cDNA sequence identified from Hep nonA-nonB patient material
1999: Breakthrough! Bartenschlager Lab. in Mainz develops HCV replicon system But still no formation of infectious HCV particles in cell culture!
Observation:
- cDNA derived HCV clones with original sequence are infectious in chimp
- cDNA derived HCV clones with cell culture adaptive mutations are not!
Strong negative effect of cell culture adaptation on virion production
Similarly:
Secretion of HCV core from transfected cells is suppressed by cell culture adaptive mutations
2003
Takaji Wakita lab: First HCV replicon which replicates without cell culture adaptation
Japanese fulminant hepatitis (JFH) strain (strong RNA replicase!)
Insertion of viral structural genes into replicon allows entire replication cycle in cell culture
This strain (+ second one)! No system for standard patient virus but JFH chimeras with structural proteins of all different genotypes!
Two major breakthroughs
- HCV replicon system
- JFH infectious virus
HCV entry: receptor-mediated endocytosis
Receptors:
CD81
Low density lipoprotein (LDL) receptor Scavenger (SR-BI) receptor
Claudin1 (CLDN1)
Occludin (OCDN)
HCV replicative cycle
Entry: receptor mediated endocytosis
Protein translation, processing, RNA replication (in cytoplasm/ at ER membrane)
Membrane topology of HCV proteins
- All proteins are directly or indirectly linked to the membrane
- Membrane topology is critical for RNA replication and processing by cellular ER localized proteases
- Viral proteins are located not only to the membraneus web / ER, but also at mitochondrial membranes (important for the manipulation of host functions and pathogenesis; virion formation?)
NS5A: Membrane incorporation by amphipathic helices -> only into the outer sheet of the bilayers
Model: Oligomers of viral membrane proteins induce membrane bending
Upon release of viral genomic RNA into the cytoplasm of the infected cell, the viral genome is translated into a poly protein that carries the structural and non-structural proteins. The viral non-structural protein NS4B induces the formation of membrane alterations, which serve as a scaffold for the assembly of the viral replication complex (RC). The RC consists of viral non-structural proteins, viral RNA and host cell factors. Within the induced vesicles, viral RNA is amplified via a negative-strand RNA intermediate.
HCV-induced membrane remodeling
Expression of NS3-5A is sufficient to induce formation of Membrane web at the ER
Location of viral RNA replication
Virion morphogenesis:
Infectious virions shown very low density in preparative density gradients!
Why?
Association with apoB apo- lipoprotein B (apoB) and triglyceride rich lipoproteins (TRL)
lipo-viro-particles (LVP)
Architecture of membrane rearrangements induced 16 h after HCV infection
- Membranes are derived from the ER, like in the case of Dengue virus
- However, not by invagination; vesicles are protrusions of the ER membrane (in Dengue infected cells vesicles are invaginations not protrusions of ER)
- More similarity between HCV, Coronaviruses, Nidoviruses and Picornaviruses
- Main components of the membraneous web are single and double membrane vesicles (DMV)
- DMVs are the predominant form
- The vesicles are frequently connected to the ER membrane via a neck-like structure
Model of the predicted role of the Core-coated lipid droplet in the production of infectious HCV
The association of the core-coated lipid droplet with NPC-and RC-rich ER may enhance the interaction of HCV and VLDL.
VLDL is generated frequently and enhanced in the microenvironment where lipid droplets associate with the ER.
This increased concentration may increase the frequency of the association of HCV and VLDL. HCV/VLDL is released as an infectious particle with a low density, whereas HCV particles that are not associated with VLDL are secreted into the culture medium as noninfectious, dense particles.
However, this model does not discriminate the possibility that noninfectious virus particle also associates with or is integrated with some lipoprotein like structure.
HCV: Virion morphogenesis at ER/Lipid droplets interface
- Lipid droplets are loaded with viral and cellular proteins
- Viral proteins at LD membrane mediate interaction between LDs and ER membrane
- Unloading of viral proteins from LDs to generate virions
„Replication organelles“
- membrane spherulae/memb. web
- sites of RNA synthesis
- transfer of genomic RNA to core
Loading of virion with ApoE
- „Lipo-viro-particle“
- Budding into ER, secretory pathway
HCV exploits liver specific miRNA122
- 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
Liver-specific expression of miRNA122 may contribute to the tissue tropism of HCV
Silencing of miR-122 as an anti-HCV therapy?
HCV therapy with antisense miR-122 could have two-fold effect:
1. Sequestration of miRNA122 would reduce HCV RNA abundance
2. Would affect lipid metabolism in the liver by reducing host steatosis
LNA technology: short modified RNA molecule
-> ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2’ oxygen and 4’ carbon
Benefits of the LNA technology:
- Increases the thermal stability of duplexes
- Resistant to exo- and endonucleases resulting in high stability in vivo and in vitroapplications
Therapeutic silencing of MicroRNA-122 in Primates with Chronic Hepatitis C Virus Infection
- treatment of chronically infected chimpanzees with a locked nucleic
acid (LNA)–modified oligonucleotide (SPC3649) complementary to miR-122
Findings:
* leads to long-lasting suppression of HCV viremia, with no evidence of viral resistance or side effects in the treated animals.
* transcriptome and histological analyses of liver biopsies demonstrated derepression of target mRNAs with miR-122 seed sites, down-regulation of interferon-regulated genes, and improvement of HCV-induced liver pathology.
Reduced concentrations of cholesterol and low-density lipoprotein in the high dosis group
But: viral RNA concentrations increased again in serum and liver after the therapy
The prolonged virological response to SPC3649 treatment without HCV rebound holds promise of a new antiviral therapy with a high barrier to resistance.
miR-122 is an attractive target for antiviral HCV therapy!!
Factors that determine the host cell tropism of HCV
Approach: introduce genes for known host factors into non-permissive cell line 293 T
- receptor
- micro RNA
- lipid metabolism
Therapy against hepatitis C -> Importance of HCV genotype on the success of IFN-alpha therapy (in %)
IFN alpha (genotype 1 -> 6-11 / genotype 2/3 -> 28-37)
IFN alpha + Ribavirin (genotype 1 -> 28-31 / genotype 2/3 -> 64-69)
peg IFN alpha (genotype 1 -> 14-28 / genotype 2/3 -> 47-56)
peg IFN alpha + Ribavirin (genotype 1 -> 40 (65% by addition of Telaprevir - but side effects, 4 x more dropouts during therapy) / genotype 2/3 -> 80)
-> Still a big problem until 2014
-> Medical costs ca 15 000 € per patient (without Telaprevir!); 400.000 pat in Germany! Costs including protease inhibitors about 60 000 €
Therapy against HCV
- 60% of the patients with HCV genotype 1 can not be cured by the pegINF and Ribavirin therapy
Reasons:
- Genotype of virus is critical; to date no association to specific virus genes; to date all cloned virus isolates can be inhibited with IFN in vitro
- Genetics of the host: Different response in Caucasians and others Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance
upstream region of IL28B gene = IFN-l3 = IFN class 3 molecule
Now in the clinic:
- Inhibitors of viral serine protease NS3/4A (first two approved)
- Inhibitors of RNA Pol (NS5B)
- NS5A antagonist (very potent; mechanism unclear)
Daclatasvir plus Sofosbuvir for Previously Treated or Untreated chronic HCV Infection
98% of 126 previously untreated patients
Advocates Protest the Cost of a Hepatitis C Cure -> Sofosbuvir
- works better than anything on the market
- each pill (one a day!) costs 1000$
- cures roughly 90% of genotype 1, 2 and 4 infections in 12 weeks with relatively minor side effects, when given with Ribavirin and for genotypes 1 and 4, interferon injections
- drug’s performance led Gilead in January 2012 to pay a staggering 11.2$ billion to purchase the small company that first made it
- Combination therapy since 2015 -> Harvoni
Small animal for HCV
-> Pathogenesis of hepatitis C virus infection in Tupaia Belangeri
-> Northern tree shrew (Nördliches Spitzhörnchen)
- can be infected
- mild hepatitis
- intermittent viremia
- develop chronic infection
Towards a small animal model for hepatitis C
Yet not existing!
But recent progress:
- all receptor proteins identified which are required for viral entry
- still low RNA replication efficiency in mouse cells
Small animal model for HCV -> Human liver-uPA-SCID mouse
- uPa+/+-SCID mice, overexpress urokinase-type plasminogen activator (uPA) genenunder control of mouse albumin enhancer/promotor (homozygous!)
- death of mouse liver cells allows repopulation by donor cells
- genetic background: Swiss athymic (nu/nu) Alb-uPA mice -> allograft accepted!
- transplanted early after birth with primary human hepatocytes: injection of human hepatocytes into mouse spleen, cells migrate via splenic vein and portal vein into the liver
- human hepatocytes integrate in the parenchyma
- progressively repopulate diseased mouse liver
- maintain normal metabolic functions
Successful use in antiviral drug testing!
Completion of the entire hepatitis C virus life cycle in genetically humanized mice
- transgenic mice stably expressing human CD81and OCLN allow virus entry into mouse hepatocytes
- but innate and adaptive immune responses restrict HCV infection in vivo Solution:
- use of partially immunodeficient mice (deficient for STAT1, IRF1 and IRF7)
Problem:
What can we learn from HCV replication in severely immunocompromised mice?
Is it possible to select in those mice for HCV variants capable of replication in normal mice?
Bovine viral diarrhea virus (BVDV)
- Polyprotein
- plus-strand RNA genome
- Model for lifelong virus persistence without detectable adaptive immune response: Infection with noncp BVDV (day 30-100 of gestation) -> Persistent infection with noncp BVDV (immunotolerance!) -> Mutation (1-2 years) -> Isolation of noncp and cp BVDV (virus pair) -> Mucosal Disease (100% lethal)
Affects 1-2% of the cattle population world-wide!
BVDV: A model for virus persistence
-> 1. Strategy of intrauterine infection
- Infection of a cow with the virus (ncp) between day 30 and 100 of pregnancy leads to an infection of the fetus, so it‘s immune system (while maturing, thymic education) will recognize the virus as „self-antigen“
this means that for the life span of the animal no adaptive immune response develops against the persisting virus
Acquired pathogen specific immunotolerance
BVDV: A model for virus persistence
-> 2. Strategy of suppression of the innate immune system
Npro: N-terminal protease
- cleaves itself in an autocatalytic way and so separates itself from the polyprotein - binds to interferon-regulatory factor 3 (IRF3)
- leads to degradation of IRF3 in the proteasome
- consequence: No interferon synthesis in BVDV inf. cells
Erns: Envelope protein ribonuclease secreted
- essential envelope protein of the virion
- is secreted by infected cells (detectable in the blood plasm of p.I. animals) - has RNase-activity (T2 type; prefered cleavage downstream of U)
- binds dsRNA and blocks IFN induction via dsRNA in the supernatant of cell cultures; natural substrate unknown!
BVDV: A model for virus persistence
-> 3. Strategy for suppression of apoptosis / the innate immune system
Essential for persistent infection:
- non cytopathogenic biotype of the virus (no apoptosis!)
- RNA replication / protein translation on a low level
-> Stringent regulation of NS3 release required
Mechanism of regulation of the NS3 release / efficiency of replication:
- viral autoprotease in NS2 needs a cellular cofactor for its activity
- this cofactor is a chaperon (Jiv = J domain protein interacting with viral protein; also termed dnaJ-C14) and is required in a 1:1 ratio
- cofactor becomes depleted during infection by binding to NS2; afterwards hardly any NS2-3 cleavage/replication; slow reproduction of cofactor
Uncleaved NS2-3: No part of replicase
Free NS3: Essential part of replicase
NS2 is an autoprotease: Catalyzes NS2-3 cleavage into NS2 and NS3
NS3 amount regulates efficiency of replication
Temporal regulation of NS2-3 cleavage correlates with decrease of viral RNA synthesis
RNA synthesis is limited by the amount of free NS3
Longer NS2-3 cleavage period as consequence of elevated cofactor level
MDBKTetOnJiv cells:
- allow regulated overexpression of Jiv by addition of inductor (tetracyclin or analogs)
-> Consumption of cellular cofactor pool leads to inhibition of NS2-3 cleavage
Temporal regulation of pestiviral NS2-3 cleavage
Findings:
- NS2 autoprotease requires cellular co-factor (Jiv/DNAJC14) to become active
- Limiting co-factor amounts in the host cell lead to temporal downregulation
of NS2-3 cleavage
early phase:
NS2+Jiv -> NS3 -> efficient RNA-replication
late phase (cellular Jiv pool depleted: NS2+NS3 -> low level RNA replication / vision morphogenesis
Different functions for uncleaved NS2-3 and cleavage poducts (free NS3):
- Free NS3 is required for replicase assembly
- Uncleaved NS2-3 required for virion assembly and production of infectious pestiviral progeny
-> Remarkable difference between BVDV and HCV with respect to NS2-3 requirement for virion morphogenesis
NS2-3-independent vision morphogenesis of pestiviruses requires only two mutations: NS2/E440V and NS3/V132A
NS2/E440V and NS3/V132A are essential and sufficient for NS2-3-independent virion morphogenesis
Conclusions
Mutations NS2/E440V and NS3/V132A allow viral particle assembly in the absence of uncleaved NS2-3
> gain of function mutations in virion morphogenesis for NS3/4A
Mechanistic basis for the gain of function in virion assembly?
NS2/E440V:
NS2 protease domain; cytoplasmatic; no structural information
NS3/V132A:
NS3/4A protease domain; cytoplasmatic;
novel structural information gained
Amino acid 132 of NS3 is located at the NS3/NS4A kink region interaction surface
- NS4A central domain intercalates stably into ß-barrel structure of NS3 protease domain
- The C-terminal NS4A domain interacts with NS3 surface including NS3 amino acid 132 that is critical for the gain of function phenotype
CSFV: M132
BVDV: V132
Do mutations of NS3 V132 or NS4A L45 / Y47 modulate the NS3/4A surface interaction?
The gain of function mutation in NS3 as well as mutations NS4A/L45/Y47 modulate the NS3/4A surface interaction which is critical for RNA replication
Single mutations: NS3/V132A, NS4A/L45A or 4A/Y47A allow for RNA replication in a BVDV- replicon
The NS4A-double mutation NS4A/L45-Y47-AA impairs RNA replication
Conclusion
1. A slight reduction in NS3/4A surface interaction is tolerated, a severe reduction interferes with RNA replication
2. Mutations that modestly weaken the NS3 surface interaction lead to a gain of function in virion morphogenesis
The NS3/4A surface interaction is critical for RNA replication
Conclusion
The surface interaction between NS3 and the C-terminal part of NS4A is essential for RNA replicase assembly
Weakening of this interaction results in a gain of function in virion morphogenesis/ the use of the protein complex in an alternative pathway
Functional switch:
Alternative coformations in the NS3/4A complex result in alternative functions in the viral replication cycle
Regulation of RNA replication and virion morphogenesis
Pestiviruses use an autoprotease which is regulated by the abundance of a cellular cofactor for temporal modulation of RNA replication efficacy (critical for persistence!)
Gain of function mutant:
Modulation of the NS3/4A surface interaction determines whether the complex is active in RNA replication or virion morphogenesis
-> Viruses can use regulated proteolysis as well as alternative conformations of protein complexes to enlarge their functional repertoir
BVDV: A model for virus persistence
-> Summary
- Strategy of intrauterine infection
Acquired pathogen specific immunotolerance / no adaptive immune response - Strategy of suppression of the innate immune system
Npro Erns
Block of IFN induction / innate immunity - Strategy of suppression of apoptosis/ the innate immune system
-> Stringent regulation of NS3 release/ RNA replication due to limiting cellular protease cofactor
Requirement for ncp biotype and absence of IFN response - High antigenic variability of the viral surface antigens
Reinfection of pregnant cows despite of previous BVDV infections
BVDV: A model for virus persistence
-> Vaccine development
- 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)
MD and BVDV biotypes
“Mucosal disease”
- detection of virus-pair
- noncp and cp Biotyp
- NS3- hallmark of cp biotyp
- deregulated RNA replication -> death of animal
BVDV a model for virus evolution?
RNA recombination in pestiviruses
-> Characterization of recombinant cp genomes
A Northern Blot analysis
B RT-PCR
C Nucleic acid sequencing
Identification of cellular mRNA sequences in viral RNA genomes!
RNA-recombination in pestiviruses
-> Analysis of cp pestiviruses
Identification and characterization of fragments of cellular
mRNAs inserted in the viral RNA genomes!
Those insertions encode often substrates for cellular proteases, like
- ubiquitin
- NEDD8
- GATE-16
–> common feature: 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
RNA-recombination – cytopathogenicity - mucosal disease
Ubiquitin-insertion serves as processing signal for cellular ubiquitin-specific proteases
Further cellular insertions with analogous function:
- Ubiquitin-like proteins (e.g. SUMO)
- Proteins with ubiquitin-like fold
NS2-3: no replicase component
NS3: essential replicase component
Effect of ubi-insertion:
- deregulated, complete NS2-3 cleavage
- upregulated RNA replication
- viral cytopathogenicity and disease
Mechanism of RNA Recombination
-> Models
- “template switching”: during viral RNA-replication
- “breakage and ligation”: independent of viral replication!
noncp BVDV replication and dissemination over years
generation of variants = molecular lottery
- In the course of persistence cp variants may be generated by RNA recombination
- When RNA recombination hits jackpot: cpVirus is generated and kills host - stops spread of virus!
- cpBVDV: No evolution! Dead-end street!
Different viruses modify different intracellular membranes
Often ER-membranes:
- Poliovirus
- HCV
- Coronaviridae:
-> Mouse hepatitis virus
-> SARS virus
- Arterivirus
-> Equine arteritis virus
- Tobacco mosaic virus
ER/Golgi/Intermediate compartment:
- Kunjin virus (Flavivirus)
Lysosoms:
- Rubella virus
- Alphavirus: Semliki forest virus
Mitochondria:
- Flock house virus
First identified human virus: Yellow fever virus (flavivirus)
Wide spread in tropical regions
e.g. 1853 epidemic in New Orleans (28% mortality)
Observation: No patient to patient transmission!
Mosquito as the vector
In 2013 still 45.000 persons died from YF!
Genome Organisation of the genus flavivirus
Unique for the Flaviviridae family:
- 5’ cap
- uncleaved NS5 with capping activity (methyltransferase in NS5)
NS1:
-> essential for RNA replication
-> secreted from infected cells
-> induces leakiness of blood vessels (virus can move from blood into tissue)
Flavivirus Life-Cycle
1 Virus infection
2 Fusion and virus disassembly
3 Polyprotein translation, transit to ER and processing
4 Transport through the Golgi
5 Virus maturation (fusion to the plasma membrane and ejection from the cell)
Dengue virus
The most important, by mosquitos transmitted, viral infection for humans (CDC 2005)
4 serotypes!
- > 2.5 billion live in areas with risk of infection
- 100 million cases / year
- 500.000 cases /year DHF and DSS
- 25.000 fatal cases / year
Dengue virus - Transmission
1: Virus transmission via salvia
2: Local reproduction, transport into local lymph nodes
3: Infection of leukocytes a.o. lymphatic tissue
4: Viremia
5: Virus uptake
6: Virus replication in the gut
7: Infection of salivary glands, replication and transmission
Possible consequences of dengue infection
- Symptomless
- Dengue Fever (DF) (low- to high-grade fever)
- Dengue hemorrhagic fever (DHF)
- Dengue shock syndrome (DSS)
-> DHF and DSS are restricted to secondary infection with heterologous serotype
Enhanced Infection of Liver Sinusoidal Endothelial Cells in a Mouse Model of Antibody-Induced Severe Dengue Disease:
Virus uptake/infection mediated via interaction of non-neutralizing antibodies (Fc-part) and cellular FC-receptors
Lethal Antibody Enhancement of Dengue Disease in Mice is Prevented by Fc Modification
Genetically modified anti-DENV antibody that fails to bind the Fcγ receptor has prophylactic and therapeutic efficacy against lethal DENV challenge in vivo.
Dengue hemorrhagic fever - pathogenesis
- Mostly high viremia
– high T-cell activation
– high cytokine level (TNF-α) - viral virulence factors (DENV2)?
- dependent on HLA-haplotype
- antibody-mediated increase of infection efficiency (antibody dependend enhancement - ADE)
Dengue hemorrhagic fever - pathogenesis
-> Antibodies
Mechanism:
- Boost infection
- Immune-complexes
- Cross-reactivity with cells of endothelium
Postulated effects:
- Higher viral load
- Complement activation
- Bleedings, endothelial dysfunctions
Dengue hemorrhagic fever - pathogenesis
-> T-cells
Mechanism:
- proinflammatory cytokines
- Lysis of bystander cells
Postulated effects:
- Increased vascular permeability
- Liver damages
Antibody dependent enhancement of infection (ADE)
- Virus uptake/infection mediated via interaction of non-neutralizing antibodies (FC-part) and cellular FC-receptors
- Viremia and spread throughout body by infected phagocytes
- Vasoactive mediators (e.g. TNF-alpha) -> Severe course of disease: Dengue hemorrhagic fever, Dengue shock syndrome
Dengue virus - immune response to infection
- Ab against E
– inhibit binding of viruses to target cells, neutralizing function
– binding to non-neutralizing epitopes leads to ADE - NS1 is expressed on infected cells and is secreted from infected cells; decoy for immune system?
- T-cells/macrophages produce increased amounts of INF-γ, TNF-α and other chemokines
Non-neutralizing antibodies causing ADE and DSS/DHF may arise by primary infection or may be transmitted from mother to child!
Comparison of DF and DHF/DSS
Dengue Fever (DF)
- Mortality Rate =0
- First reported epidemics: 1770 Egypt
- Annual Reported Cases: 10 Millions
Dengue Homorrhagic Fever (DHF) or Dengue Shock Syndrome (DSS)
- Mortality Rate: 10-50%
- First Reported Epidemics: 1953 Burma
- Annual Reported Cases: 10-100 Thousands
Dengue hemorrhagic fever in Brazil
10 April 2008 - As of 28 March, 2008, the Brazilian health authorities have reported a
national total of 120 570 cases of dengue including 647 dengue hemorrhagic fever (DHF) 114 cases, with 48 deaths.
Cryo-EM structure of an antibody that neutralizes dengue virus type 2 by locking E protein dimers
DENV serotype 2 (DENV2)–specific human monoclonal antibody (HMAb) 2D22 is therapeutic in a mouse model of antibody- enhanced severe dengue disease. HMAb 2D22 binds across viral envelope (E) proteins in the dimeric structure, which probably blocks the E protein reorganization required for virus fusion. HMAb 2D22 “locks” two-thirds of or all dimers on the virus surface, depending on the strain, but neutralizes these DENV2 strains with equal potency.
Important:
The recombinant antibody used is unable to bind to Fc receptors due to two mutations in the heavy chain: no ADE induction!
Structure-Guided Design of an Anti-dengue Antibody Directed to a Non-immunodominant Epitope
A structure-based approach allows for the development of a monoclonal antibody that targets a non-immunodominant epitope to effectively neutralize all four serotypes of the dengue virus.
This antibody treats several symptoms of severe infection in animal models and may provide strategies for treatment in humans.
Ab513 Neutralizes DENV Despite
Fc Receptor-Mediated Phagocytosis!
Zika virus
Flavivirus (genus) isolated for the first time in 1947 in the Zika forest in Uganda.
- causes usually only mild symptoms, but leads to microcephaly when fetuses are infected and in rare cases to Guillain-Barré
syndrome in adults
Vector: Aedes species mosquito (see Dengue and Chikungunya viruses)
2013-2014: Epidemic outbreak in French Polynesia From 2015: South/central America
October 2019: First cases in South of France
West Nile Virus Outbreak
- 1,500 fatalities
- Europe, peak in WNV circulation in 2018, in total 1,503 cases
- WNV is now endemic in Italy and Greece
- First cases in Germany
Vaccine candidates in clinical phase II
Flaviviral RNA Replication
- RNA genome with 5 ́cap but without polyA at 3 ́end
- communication between genome ends via base pairing
-> cyclisation - random coiled RNA
- discrimination between 5’ and 3’ end by apparent volumes
Cyclisation sequences
SL: stem loop
cHP: capsid region hairpin
UAR: upstream of AUG region
CS: cyclisation sequence
RNA replication of Dengue virus
- no initiation at 3 ́UTR fragment!
- annealing of RNS genome ends allows RdRp to initiate RNA synthesis at both 3 ́ends (two products)
- replication initiation in trans: binding of RdRp to 5 ́UTR 130 allows initiation at 3 ́end
- Deletion of polyU stretch upstream of 5 ́UAR eliminates initiation at 3 ́end of 3 ́genome end
- spacer required for flexibility
RNA replication of Dengue Virus (in 4 steps)
- Cyclisation of the genome via base pairing
- Binding of RdRp to 5’ end
- Transfer of polymerase from 5’ to 3’ end
- Start of RNA synthesis at 3’ end
Flaviviral RNA replication
- RNA genome without polyA at 3’ end
- communication between ends via base pairing
-> cyclisation
Compare:
- genome segments of influenza virus (panhandle structure, cork screw)
- poliovirus: protein bridge for communication between genome ends; (most likely also the case for other positive RNA viruses)
Conclusion:
- Communication between genome ends is a common theme
- viruses evolved different solutions
Infecting the mosquitos with a bacterium to stop disease transmission produces “staggering” reduction in cases
The trial in Yogyakarta released Wolbachia-infected mosquitoes into randomly designated portions of the metropolis. Rates of dengue in these places were 77% lower, over several years, compared with areas that did not receive the mosquitoes.
