Rabies

Question 1

Symptoms

Answer 1

• Starts with a bite or scratch (for humans this is always through a dog or pet)
  
  • Quite a long asymptomatic period (20-90 days or longer)
  • Once the symptoms begin, it’s too late (will die)
    Prodromal period:
  • Symptoms usually start off as a sensation around the bite, but progresses into flu-like symptoms
    Clinical presentation
  • can go 2 ways:
    ○ There’s a paralytic form in about 20% of cases
    ○ There’s a furious form in about 80% of cases (frothing of the mouth and other crazy symptoms)
  • Start getting things like fever, salivation, convulsions, hallucinations
  • A unique symptom for this disease is hydrophobia (fear of water - can’t even drink it - can’t drink a glass of water) ; not clear why this happens. Is an indication that you are losing control of your body - another is hypersexuality - unwanted advances and constant ejaculation
  • There are temporary periods of clarity, but eventually you will go into coma and death (once these symptoms start there’s 100% chance of death)

Question 2

Rabies Vaccine

Answer 2

Rabies Vaccine:

- Attenuated the virus by infecting rabbits, extracting the spinal cords of the rabbits and drying them for several days (weakens it) - dried spinal cords was crushed and used to inoculate people
- Drying caused the virus to be attenuated 
- They varied the number of days the spinal cords were dried to create a progressive series from very attenuated to less attenuated - more days the virus was dried for, the more attenuated it became
- First tested this on a 9 year old boy who had been bitten by a rabid animal (gave 13 different doses of the vaccine) and the boy survived - first person to be vaccinated
- Boy was vaccinated after he would be bitten (normally too late in other diseases) but in the case of rabies, vaccine can be given after exposure to the virus before onset of symptoms 

Newer versions:
- New versions of this vaccine are inactivated and improved
These vaccines are normally given to people that are at high risk such as vets

Question 3

Rabies: still a problem

Answer 3

• Still a lot of people dying every year (mainly in the poorer areas of the world, often rural - vaccine is expensive)
  
  • Rabies is a zoonotic disease (almost all warm blooded animals can be infected by this virus)
  • Humans get it from dogs 99% of the time
  • Reservoirs of the virus are in nature (in wild animals) so it’s almost impossible to eliminate it from the earth
  • Way to get control over rabies is to vaccinate dogs and pets and do wild-life oral baiting (wildlife get vaccinated through food - food contains vaccines)
    There’s no human-human transmission (humans are a dead end host) - humans can get rabies from animals but they can’t pass on the virus to others

Question 4

What are rabies virus

Answer 4

• Rabies virus is a type of Lyssa virus
  
  • The rabies disease that we see is not only caused by rabies virus, and some of these other Lyssaviruses can also cause rabies or rabies-like disease - Duvenhage, European Bat Lyssavirus

Question 5

Does Australia have rabies

Answer 5

• Although we don’t have rabies virus, we have our own Lyssavirus that can cause rabies-type disease
  Australian bat lyssavirus-
  
  • Zoonotic and similar to rabies viruses but it’s passed from bats
  • Have been fatalities from this in Australia
  • Treatment is same as rabies vaccine
    Prevention involves avoiding handling bats and if you get bitten by one seek medical attention immediately

Question 6

How does rabies infect host

Answer 6

• Starts with animal bite/scratch (virus is within the saliva of the animal)
  
  • It’s passed from the animal saliva into the muscle area of host where it’s thought to replicate
  • It eventually gets transmitted to the peripheral nerves and into the central nervous system
  • Virus particles travel along the neuronal axons (retrograde direction) towards cell body in vesicles
  • Vesicles transported to cell body via microtubules.
  • • At the neuronal cell body, it’s released from the vesicle and replicates
  • Newly assembled virus infects the next neuron near this neuron via synapses (trans-synaptic spread)
• Virus slowly creeps its way up the spinal cord towards the brain
  
  • This is a slow process, depending on where you’re bitten (if you’re bitten on foot, virus has a long way to travel so there’s a lot of time before onset of symptoms - allows vaccination. If you get bitten on face, it’s much closer to brain so you don’t have much time at all)
    At the brain, it causes encephalitis and spreads to other organs (this is when the typical symptoms start occurring)
  • Virus is very good at dampening immune system before it reaches brain (which is why it goes unnoticed and people are asymptomatic for so long) - eg inhibits apoptosis of neurons

Question 7

What are lyssaviruses

Answer 7

Lyssavirus:
- -ssRNA enveloped virus
- Group V of Baltimore system
- RNA viruses mutate quickly (because RNA-dependent RNA polymerase don’t have proof-reading activity like the DNA polymerases) - thus they often have small genomes, and can also adapt to situations quickly
- Since it’s negative sense, it needs to bring its RNA-dependent RNA polymerase with it in the particle itself
- Only has one piece of RNA (monopartite)
Belong to the order of mononegaviruses (contain enveloped negative sense single stranded RNA)

Question 8

What proteins does rabies virus encode

Answer 8

Phosphoprotein, glycoprotein, nucleoprotein, RNA dependent RNA polymerase, matrix protein
- Rabies only encodes 5 proteins and these are all in the particle itself - NLPGM
- On the outside of the particle are the glycoproteins (essential for binding to cells and getting in)
- Virus has an envelope which is taken from host cell as it is leaving
- Matrix protein is required for assembly (links the glycoproteins and envelope with nucleocapsid)
The RNA genome is helical

Question 9

Rabies genome

Answer 9

• RNA genome is wrapped up by the nucleoprotein (N protein) - N protein binds to the RNA about every 9 bps and wraps it up into a helix
  
  • Size of this helix is determined by how big the RNA itself is - the longer the RNA, the longer the helix
• RNA viruses have fairly small genomes
  
  • Because it’s a negative sense virus, it needs to carry RDRP with it- RDRP is used to convert -ve RNA to +mRNA
  • Negative RNA is 3’ to 5’, whereas mRNA IS 5’ TO 3’
  • RNA carries 5 genes
  • The viral proteins can then be made from the mRNA using host machinery (ribosomes) - mRNA
    Makes N, P, M, G proteins

Question 10

Attachment and entry of rabies virus into cell

Answer 10

• Glycoprotein on virus binds to receptor on host cells, inducing endocytosis (clathrin-coated pits form) releasing particle into cell enclosed in an endosome
  Endosome hooks onto microtubules and is transported towards cell body (retrograde) - Virus takes advantage of what happens when the endosomes are travelling on microtubules along axon towards cell body (pH within endosomes becomes more and more acidic)
  
  • The acidity of the endosomes causes a conformational change in the glycoproteins (cause them to fuse into the endosomes, resulting in membrane of endosome and membrane of virus fusing together, releasing the nucleocapsid into the cytoplasm
  • The released nucleocapsid comprises the RNA genome, nucleoproteins (N), P ( phosphoprotein) and RDRP (L) - Ribonucleoprotein

Question 11

What does the nucleocapsid consist of

Answer 11

• N protein is covering whole RNA genome
  P protein and L proteins (RDRP) also seen
  Need to bring N, P, L for RNA synthesis

Question 12

Rabies replication process

Answer 12

• Once particle releases nucleocapsid into cytoplasm, firstly the genome is transcribed into viral mRNA which is translated to make viral proteins
  
  • Once we have viral proteins, replication occurs (negative RNA genome makes positive RNA antigenome which is used as a template to make more negative RNA genomes)
  • Primary replication stages ) involve using components that came in with the particle itself - template is original genome
    Secondary replication involves using proteins produced in host cell - template is newly replicated genome

Question 13

Rabies Transcription

Answer 13

• P protein is basically the cofactor for RDRP
  
  • P protein binds to RDRP (L protein) and N protein to bring them together so that the RDRP can synthesise the mRNA
  • So P, L and N form complex which bind to the start of the genome (3’ end)
  • P and L move along the RNA genome and uses a start -stop mechanism which allows it to stop at every gene and reengage with the template at the next gene produce the 5 different mRNAs for the each of the 5 viral proteins
    RDRP also mediate addition of cap and poly-A tail to mRNA

Question 14

Transcription gradient

Answer 14

• Despite there being a stop start mechanism, usually the P and L complex cannot transport all the way through the RNA genome-it falls off completely at certain points and is unable to re engage with template at next gene- has to start again from the beginning ; creates transcription gradient - this means there’s always more N protein than there’s P and so on, as the N gene is at the start of the genome (N > P > M > G > L)
  
  • This can be useful since virus needs a lot more of the N protein than the other proteins
  • There’s lowest amounts of L protein since it’s at the end, but this isn’t a problem since not a lot of RDRP is required for mRNA synthesis

Question 15

Translation

Answer 15

Translation:
- To make the proteins from the mRNA, it’s same process at what happens in our cells normally (host cell ribosomes)
Due to transcription gradient, there’s a lot more N proteins and very little L proteins

Question 16

Replication

Answer 16

• Antigenome needs to be made as a template to make more negative RNA genomes
  
  • This is still dependent on P and L complex
  • Synthesis of sufficient levels of N protein means it can start binding to the naked mRNA made by the P and L complex (starts encapsidating genome) - P and L complex no longer skips parts; goes through it all and makes entire positive sense antigenome which is used as template
  • Trigger to cause P and L complex to go from making mRNA to making the full genome is protein synthesis and presence of sufficient N protein which starts encapsidating naked mRNA
    If you have an inhibitor of translation, only mRNA would be made (no N protein made to enable replication of genome)

Question 17

Where does replication occur

Answer 17

• Replication of genome occurs in cytoplasm in bodies created by the virus called Negri bodies
  
  • Negri bodies used as a diagnostic marker for infection
  • Negri bodies are in the cytoplasm but don’t have membrane around them- have been identified to be liquid bodies - virus these liquid organelles for replication
    These bodies are formed through expression of N and P proteins

Question 18

How to block Rabies virus replication?

Answer 18

Block translation. No translation means no replication as translation of N gene to produce N protein is what triggers replication

Question 19

Assembly

Answer 19

Assembly:
- Occurs at plasma membrane
- 3 main components that are assembled together
a. Nucleocapsid
- Contains the RNA genome that’s been replicated from antigenome, along with N, P and L proteins
b. Glycoproteins
-Is on outside of particle - is trafficked through the ER and the Golgi where is glycosylated.
c. M protein
- In plasma membrane
- Brings everything together and enables assembly process
- Selectively binds to complete negative sense RNA genomes and facilitates putting nucleocapsid in the particle and glycoproteins on the outside of particle
In budding process, section of the host cell membrane is incorporated into the particle (envelope)

Question 20

Innate immune defense against viruses

Answer 20

• PAMP such as DS RNA recognised by PRR of the innate immune system such as Rig-1 (Rig-like helicases) triggers signal cascade that leads to production of cytokines like interferon
  
  • Interferon is released and can signal to the same cell (autocrine manner) or other cells (paracrine manner) to turn on antiviral defenses - such as switching on genes to make antiviral proteins
    Viruses need to be able to counter this to be effective

Question 21

How was interferon discovered

Answer 21

• Took influenza virus and infected chicken cells plated on media - Resulted in replication and production of more virus
  
  • They took inactivated virus and put that into the cells, no replication occurs so no virus is made;
  • When they took the media in which these infected cells were on and put it on top of some fresh cells and then infected these cells with the active virus, a lot less virus was produced
  • Suggested there was something in the media from inactivated virus that was interfering with virus
    Something was secreted out into the media by cells which was interfering with viral replication

Question 22

Type 1 interferon

Answer 22

Involved in anti viral responses - two types: alpha and beta
- Production of interferon alpha/beta is very rapid (is made within a couple hours after infection)
- Interferon binds to interferon receptors on cells, causing a signal cascade that leads to gene expression (it switches on interferon stimulated genes - ISGs)
Mechanisms of most ISGs known

Question 23

IFN INDUCTION AND SIGNALLING

Answer 23

IFN induction:
- Virus releases its genome into cell, which is recognized by the PRR RIG-I
- RIG-I is activated and changes conformation after recognition, causing signal transduction cascade which leads to activation of protein IRF3 (interferon regulatory factor 3)
- IRF3 gets phosphorylated and forms a dimer
- Dimer can be transported into nucleus and bind to promoter of type I IFN gene
- IFN mRNA is made and is translated into IFN protein which is secreted out of the cell
IFN Signaling:
- IFN binds to IFNa receptors which phosphorylates the receptor
- STAT proteins can bind to phosphorylated IFN receptor causing the STAT proteins themselves to be phosphorylated
- STAT1 and STAT2 proteins then form a dimer which is transported into nucleus
STAT dimers bind to the promoters of hundreds- thousands of genes and switch on a whole lot of mRNAs, making lots of different proteins (many of them antiviral)

Question 24

How viruses combat IFN

Answer 24

How viruses counter this:
3 mechanisms:
1. Inhibition of host gene expression (so IFN can’t be made by host cells - shuts off IFN signalling)
2. Sequester/mask the PAMP (virus can hide from PRR and avoid activating it, thus the IFN response isn’t triggered)
3. Sequestration/modification of signaling components (can interfere with different parts of pathway and prevent phosphorylation at some parts)

IFN antagonists (which block interferon function) are viral proteins that usually have some other role but are also involved in interfering with interferon response

Question 25

Interferon antagonist in Rabies virus?

Answer 25

P protein

Question 26

P protein mode of action

Answer 26

Bind to phosphorylated STAT 1 and STAT2 dimer and prevents it from getting into nucleus, and prevents phosphorylation of the interferon regulatory factor 3

Question 27

P protein

Answer 27

Has 297 amino acids
Has a C terminal globular domain which it uses to bind to Interferon regulatory factor 3 + phosphorylated STAT 1 and Stat 2 proteins
Has a nuclear import sequence and nuclear export sequence - so it can be trafficked between nucleus and cytoplasm

Question 28

How does P protein inhibit STAT proteins

Answer 28

• P protein can also use its nuclear localisation sequence to get into nucleus and bind to STAT dimers within nucleus and traffic them back out again into the cytoplasm through their nuclear export sequence
  P protein only binds to phosphorylated STAT, if P protein binds to STAT the whole time P protein can’t perform its role in viral replication so only when STAT is activated does P-protein to come into action

Question 29

Ribosomal leaky scanning

Answer 29

• Rabies only has 5 proteins, so these proteins have to be highly multifunctional - it has to get the best out of its small genome
  Ribosomal Leaky Scanning:
  
  • Normally, ribosomes bind to cap of mRNA and travel along until they see a start codon AUG, from where they begin translation
  • In ribosomal leaky scanning, the nucleotides surrounding the start codon (Kozak sequences) determine whether the ribosome will start translating at that particular start codon or at a different one next to it
    Ribosomes skip over weak Kozak sequences and start translation at strong Kozak sequences to make proteins

Question 30

P protein isoforms

Answer 30

There are 4 start codons in the P protein
Ribosomal leaky scanning means that the ribosome can start translation at the first codon to produce entire P protein, can start translation at second codon to produce truncated version of P protein, can start translation at third codon to produce truncated version of P protein, or can start translation at 4th codon to produce truncated version of P protein.

Altogether, this results in 5 different versions of the P protein, P1 full length is cytoplasmic whereas P3 is in the nucleolus

Question 31

P3 protein function

Answer 31

• P3 protein can traffic into nucleus and bind to STAT dimer
  It bundles microtubules and holds the STAT proteins in them (sequesters the STAT proteins to prevent them from getting into nucleus and activating the genes)

Question 32

How important is P protein inhibition of STATS

Answer 32

P protein’s ability to inhibit STAT is crucial to rabies pathogenesis
Took pathogenic rabies genome and passaged it through chicken cells
The virus started mutating over time, giving us an attenuated version of the rabies virus
The attenuated virus is unable to kill the mice (the pathogenic rabies virus can kill the mice)
If they took the P-protein from the pathogenic virus and added it to the attenuated rabies virus, then the attenuated virus strain became pathogenic and could kill the mice

Pathogenic rabies can replicate inside cells and give rise to pathogenic rabies
Attenuated rabies virus is unable to replicate inside cells, no virus is obtained, however it is able to replicate inside cells with no interferon - thus attenuated rabies is unable to counteract interferon
Attenuated rabies with Pathogenic P protein is able to replicate inside cells and give rise to pathogenic rabies strains- thus P protein can counteract interferon

Question 33

The many tricks of RNA viruses

Answer 33

• RNA viruses have a small genome by necessity because their RNA polymerase doesn’t have proofreading activity and therefore makes errors
  
  • They can utilize tricks to increase what their small genome can do such as
    
    • Ribosomal leaky scanning (making different truncated versions of same protein)
  • Cutting up one protein into smaller bits
  • Proteins can perform multiple functions
• Ribosome frameshifting

Question 34

How to manipulate rabies virus

Answer 34

• For RNA viruses we use reverse genetics (we have an RNA virus genome and we make a DNA version of it)
  
  • Insert DNA copy of RNA genome into DNA plasmid
  • Can easily mutate things when they’re on plasmid
  • Easy to grow +ssRNA (doesn’t need anything else with it, just needs genome)
  • Since rabies is a -ssRNA virus and requires RDRP to come with it, it’s not so easy
  • Take -ssRNA genome and put it into DNA plasmid (if we just insert this plasmid into cell, the RNA will be made but nothing else happens because it’s -ssRNA and isn’t recognised by ribosomes)
  • Based on knowledge or rabies virus we know it needs RDRP (L), nucleoprotein (N) and P protein ; this results in production of viral mRNA and rest of the steps occur
  • So instead of just the genome plasmid being put into cell, N, P and L plasmids are also added in separately or together
    Can now produce the virus

Question 35

How to use Rabies Virus for good

Answer 35

• Neurotracer: can map whole neural network using green fluorescent protein attached into virus (doesn’t kill the neurons due to manipulation)
  
  • Vector for vaccines: people have taken out G gene from rabies and inserted other immunogenic proteins from other viruses - virus is attenuated so it is able to replicate but not cause disease
  • Pass BBB: usually hard to create drugs that can pass BBB and target brain, but rabies can pass BBB due to G protein on surface (specifically a 28 amino acid peptide of G) ; when this peptide is fused onto other things, you can get them through BBB- this has been used to create drugs that can treat encephalitis caused by West Nile Virus
    Alzheimer’s: bit of a stretch, but since neural inflammation is responsible for this condition, we might be able to use P protein to learn how it shuts down this pathway as neural inflammation is dampened during rabies infection (do cells treated with P protein prevent neural inflammation)