rabies and lyssavirus Flashcards
1
Q
stages of rabies
A
- Early ○ Bite ○ No symptoms (20-90 days) - Symptoms begin ○ Kills ~ 100% once symptoms begin ○ Sensation at original bite site ○ Flu0like symptoms - Clinical (furious (80%), paralytic (20%)) ○ Fever, mouth salivates, convulsions ○ Hydrophobia (unique to rabies) § Fear of water § Can't even drink water ○ Hallucinations ○ Hypersexual behaviour ○ Moments of clarity Coma, death
2
Q
Vaccine and treatment
A
- Vaccine developed by Louis Pasteur and Pierre Roux
○ Weaken a virulent rabies virus by aging and drying spinal cords of rabies-infected rabbits
○ Tested on 9 year old rabies patient- Vaccines improved (inactivated)
○ Recombinant vaccines (g protein) - At risk individuals
- Can be applied post-exposure (before symptoms)
Rabies immunoglobulin, 5 vaccine treatment
- Vaccines improved (inactivated)
3
Q
Still a problem
A
- > 55,000 deaths a year
- Under reported, under-served, poorly resourced regions, rural
- Vaccine expensive; multiple courses
- Zoonotic - almost all warm-blooded animals can be infected
- > 99% human cases come from dogs
- Almost impossible to completely eliminated due to so many wild-life reservoirs
- Control: vaccination of dogs, pets
○ Wild-life oral baiting (herd immunity)
Humans are a dead-end host (no human to human transmission)
4
Q
is rabies caused exclusively by the rabies virus
A
no, other viruses can cause rabies. e.g. Lyssavirus
5
Q
Does Australia have rabies
A
- Australian Bar Lyssavirus virus (ABLV)
- Very similar to rabies virus
- Zoonotic - flying foxes, bats
- Treatment
○ Same for rabies, rabies immunoglobulin and rabies vaccine protects against lyssavirus - Prevention
Avoid handling bats
6
Q
Rabies infection of the host
A
- Animal bite or scratch (virus in saliva)
- Infects muscle (replicates), transmitted to peripheral nerves, then central nervous system
- Virus particles transport in along neuronal axons (retrograde = towards cell body) in vesicles using microtubules
- When at neuronal cell body, released from vesicle, replicate, assemble new virus particles, then infect next neuron
- Travels up spinal cord, leading to brain, causes encephalitis
Spreads to other organs (e.g. salivary glands)
7
Q
In the neuron
A
- Virus endocytosed into the neuron from muscle
- Gets a ride on the microtubules to cell body
- Once in cell body gets released out of endosome and can start replicating
- Transports to the trans-synaptic spread
Goes to the next neuron
8
Q
rabies process efficacy
A
Slow process (depending on where bitten) - allows time to vaccinate
- If bitten on foot has to travel a lot further than if bitten on the face
Very little sign of damage to neurons (how does it kill?)
- Very stealthy - very little cytopathic effect
- Inhibits apoptosis
Immunosuppressive strategies (immune evasion)
9
Q
Lyssaviruses: the stats
A
- RNA ○ mutate quickly (no-proofreading), small genome, must make its own RNA polymerase - Single strand and negative-sense ○ Must bring RNA polymerase - One (monopartite) ○ One molecule, non segmented - Order - mononegaviruses ○ Rabies (rhabdoviridae) ○ Ebola (Filoviridae) ○ Nipah (Paramyxoviridae) Envelope - YES
10
Q
Rabies virion/ particle
A
- Enveloped
- Helical (wrapped up by M protein)
- All 5 proteins in particle (N, P, M, G, L)
- N binds and covers (encapsulates) RNA genome (nucleoprotein) forming a ribonucleoprotein
○ RNA + protein
G on surface (spikes)
11
Q
Lyssavirus Genome
A
- ~11-12 kbp long (average RNA virus long)
- 5 genes as said above
- L brings RNA-dependant RNA polymerase to make mRNA
- Then can make viral proteins (N, P, M, G, L)
mRNA made by host ribosome (host makes proteins)
12
Q
Attachment and entry
A
- The particle itself has the glycoprotein
○ Binds to the receptor of the host cell- Induces endocytosis
- Clathrin-coated pits form allowing virus into cell but enclosed in endosome
- Endosome hooks onto microtubules
- Transported to cell body
○ As the endosomes head towards the cell body the inside of the virus becomes more acidic
○ Inducing a conformational change
○ The glycoprotein fuses in, brings the membrane from the virus and the membrane from the virus together so they can merge
○ Then release out the nucleocapsid
Contains L protein, M protein and P protein
13
Q
Synthesis
A
- Nucleocapsid is a ribonucleoprotein complex
○ RNA strand + N, P and L proteins- Nucleocapsid released into cytoplasm
- Transcription - make viral mRNA
- Translation - make viral proteins
- Replication - -ve RNA genome -> +ve RNA antigenome
Used as a template to make more -ve RNA genome
14
Q
Transcription
A
- We need the RNA encapsidated by N protein
- We need the P protein to act as the intermediate
○ Binding to L protein and N protein
○ Brings them together
○ So polymerase can start - The P&L complex starts at 3’ end
- Transcribing across using a stop start mechanism
○ There’s a signal sequence in the RNA telling the complex to disengage and re-engage and make next gene - Sometimes the PL complex falls off as it moves across the genome
○ Therefore we will always have more of the first proteins (N>P>M>G>L)
Called the transcriptional gradient - as sometimes the complex fails to re-engage
- We need the P protein to act as the intermediate
15
Q
Translation
A
- Uses host ribosomes
Has transcriptional gradient of course
16
Q
Replication
A
- We need to make +RNA antigenome to be a template to make more -RNA genomes
- Replication requires new N protein to encapsidate RNA
○ If no viral translation = no trigger to replicating the genome (just transcription of mRNA) - Replication occurs in “liquid” Negri bodies
○ Membrane-less cytoplasmic inclusion caused by rabies virus
○ Rabies diagnostic marker
○ Site of replication
○ Recently shown to be liquid organelles
○ Formed by N and P
Likely most -ve sense RNA viruses use liquid organelles
- Replication requires new N protein to encapsidate RNA
17
Q
host vs virus mediations
A
Transcription - virus mediated
Translation - host mediated
Replication - virus mediated
No translation = no replication
18
Q
Assembly and release
A
- Assembly at plasma membrane
- 3 main components assmebled:
○ Nucleocapsid (RNA, N, P, L)
○ M (at plasma membrane)
○ G (glycosylated - sugar groups added) - M mediates assembly, ‘selects’ nucleocapsids (complete, not antigenomes)
- Triggers budding from host cell
Gain membrane envelope from host cell as buds from cell
- 3 main components assmebled:
19
Q
summary of lyssavirus infection cycle
A
1.G binds cellular receptor and endocytosed into endosomes.
- Low pH of endosome causes conformational change in G, triggering fusion with
endosomal membrane, releases of viral RNP into cytoplasm (nucleocapsid, N, P, L). - Transcription occurs (requires viral proteins), making mRNA
• Transcription stop-start gradient causes different levels of viral mRNA. - mRNA translated by ribosomes
- G protein translated into ER/secretory pathway
- Replication begins when enough N is made, N coats new viral RNA (make
antigenomes) - Replication occurs in Negri Bodies in the cytoplasm.
8. Antigenomes make new viral genomes. New genomes used for: - 8. Transcription - 6. Make more antigenomes - 12. Packaging into new particles.
Viral genomes are packaged at the plasma membrane.
11 & 12. M associated with membrane, packages genomes into
particles.
- G passed through secretory pathway, glycosylated (9
and 10) at the plasma membrane packaged on outside of
particle, and particles released from cell by budding from
membrane (gain envelope).
20
Q
cytokine affect
A
○ Autocrine = affects same cell
Paracrine = affects different cells
21
Q
Type 1 interferon
A
- IFNa and IFN b - direct response to infection broad cellular expression and responses
- Production of IFNa/b is rapid: within hours of infection, declines by 10h
- IFN binding to IFN receptors leads to synthesis of >1000 cell proteins (IFN stimulated genes = ISGs)
Many antiviral
22
Q
IFN induction
A
- Virus releases its content into the cell
- The PRR, RID-I is inactive
- It picks up a signal
- Changes conformation -> signal transduction
- Phosphorylates IRF3 leading into the nucleus
- Binds to promotor of type 1 interferon genes
IFN mRNA is made and secreted out of cell
23
Q
IFN signalling
A
- IFN binds to cell receptors
- Causes phosphorylation, that allow stat proteins to bind and form dimer
- Trafficked into nucleus
ISGs switches on many mRNA to make antiviral proteins
24
Q
Viruses encode IFN antagonists
A
- 3 broad strategies
○ General inhibition of host gene expression
○ Sequestration/masking of PAMPS
○ Sequestration/modification of signalling components- Often multiple strategies
IFN antagonists often multifunctional viral proteins
- Often multiple strategies
25
Q
Rabies antagonises the IFN response
A
- At least one protein must act as IFN antagonist
○ P protein
§ Stops phosphorylation of IRF3 (inhibits RIG-I signal transduction)
§ Inhibits the STAT proteins from getting into the nucleus
□ Binds to STAT1/2, preventing nuclear import
Blocks activation of ISGs
26
Q
Rabies P protein
A
- N-terminus binds to L protein
- C-terminal domain (CTD) binds to N protein
- CTD binds phosphorylated STAT1/2
- Traffics between nucleus and cytoplasm
○ NLS = nuclear localisation sequence (import into nucleus)
○ NES = nuclear export sequence (export out of nucleus)
Can use NLS protein to get into the nucleus and bind to STAT1/2 bringing it out of nucleus
27
Q
Ribosomal leaky scanning
A
- mRNA with cap and tail
- Ribosome binds to cap goes along until start codon, translates until tail
- Kozak sequence = nucleotides around the start codon determine likelihood the ribosome initiates translation at that start codon
○ Strong Kozak = will start at the AUG
○ Weak Kozak = can skip AUG - Sometimes the weak AUG signal will allow the ribosome to keep going until a strong Kozak is seen
○ This will make the sequence shorter
○ This can make 5 different truncated (shorter) variations of the P proteins with different functions
E.g. If the L protein is at the start, but the sequence starts later due to weak Kozak the part that binds to L protein wont be made, thus will serve a different function like IFN interference
28
Q
P-protein inhibition of STAT is critical to pathogenesis
A
Nuclear P3 binds STATs in nucleus, prevents binding DNA
Uses microtubules to sequester STAT
- When p protein is activated in attenuated rabies, pathogenesis occurs
- With pathogenic rabies the virus was present
- With attenuated rabies no virus was present
○ If in cells with no IFN the virus was present
§ If IFN was added on top of these cells no virus was present
With attenuated rabies + pathogenic P protein the virus was present
29
Q
RNA have to use clever tricks to increase coding capacity - due to small size
A
- Each protein has multiple job
○ Ribosomal leaky scanning
○ 1 RNA -> polyprotein, cleaved (cleaving 2 proteins and combining to make a new protein)
○ Multifunctional proteins
○ Ribosome frameshifting
○ Suppression of termination
Proteins are regulated (trafficking signals, conformational change, etc.)
30
Q
How to manipulate rabies virus
A
- Use reverse genetics => DNA copy of RNA virus genome
○ Insert DNA copy of RNA into DNA plasmid
§ Easy to manipulate - mutate, insert gene, delete etc.- -ve RNA isn’t easy
○ We can take virus genome from rabies virus plasmid and put it into a cell
○ -ve RNA wont do anything, we need polymerase
○ We need N, L, P proteins to make viral mRNA
We put N plasmid, L plasmid and P plasmid with rabies plasmid into cell
- -ve RNA isn’t easy
31
Q
Manipulating rabies genome
A
- Mutant genome
- Delete gene
○ If deleted G gene, it can’t infect new cell
○ If we put it on outside, it can infect new cell but can’t create more G
Insert gene
- Delete gene
32
Q
Using rabies for good
A
- Neurotracer
○ Map neural network (trans-synaptic)
○ Label virus
○ Replicates, therefore labels each neuron similarly
○ Low cytopathic effects- Vector for vaccines
○ Use highly attenuated rabies virus
○ Engineered to express immunogenic proteins of other viruses
○ Highly immunogenic - Pass blood-brain barrier
○ Rabies passes BB by binding receptor on nerves (G protein)
§ Found 28-resifue peptide of G when fused to cargo, delivers to brain
○ Treat west Nile virus - Curing Alzheimer’s?
○ Neuroinflammation major factor for disease
○ Rabies P is good at impairing immune/inflammatory response
Can we use P to learn how to target these pathways to tackle these disorders
- Vector for vaccines
33
Q
All mononegaviruses have similar genome organisation
A
- They all have similar proteins in the same sequence that function the same
If you understand one you can understand them all
