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BMS2052 MICRO > WEEK 12: RABIES VIRUS > Flashcards

WEEK 12: RABIES VIRUS Flashcards

1
Q

How long do you wait until you get symptoms from first getting bitten?

A
  • 20-90 days will pass before getting symptoms
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2
Q

Does Rabies virus kill 100% of the time once the symptoms begin?

A
  • YES
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3
Q

Is there a sensation at the original bite site?

A

-YES

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4
Q

When symptoms begin, what are they similar to in general?

A
  • the flu–> flu like symptoms
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5
Q

What is the most common clinical feature of rabies occurring in 80% of cases?

A
  • The furious type
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6
Q

What clinical feature of rabies occurs in 20% of cases?

A
  • Paralytic
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7
Q

What are the 6 clinical symptoms of rabies?

A
  • Fever, moth salivates, convulsions
  • Hydrophobia–> can’t drink a cup of water
  • Hallucinations
  • Hypersexual behaviour (ejaculating once per hour)
  • Moments of clarity
  • Coma, death
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8
Q

Which clinical symptoms is UNIQUE to rabies?

A
  • Hydrophobia (can’t drink a cup of water)

- Body rejects water–> unknown why

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9
Q

Is there any record for human-human transmission?

A

-NO

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10
Q

What is the rough timeline of rabies?

A
  • Exposure–> 20-90 days
  • First symptoms –> prodrome (Early symptom) (1-2 days)
  • Clinical expression –> Acute neurological phase (1-4 days)
  • Coma –> death (1-7 days)
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11
Q

Who was the rabies vaccine developed by?

A

-Louis Pasteur and Pierre Roux in 1885

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12
Q

What was done for the first rabies virus?

A
  • Weaken a virulent rabies virus by aging and drygin spinal cords of rabies infected rabbits –> administered to 9y r old boy
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13
Q

How was the rabies vaccine improved from the first one?

A
  • It was inactivated and –> recombinant vaccines (G protein)
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14
Q

Can the rabies virus be applied post-exposure (before symptoms)?

A
  • YES

- 5 vaccination course

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15
Q

How many deaths a year roughly occur from rabies?

A
  • > 55 000 deaths
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16
Q

Which types of animals can be infected by rabies?

A
  • Zoonotic–> almost all warm blooded animals

- This includes bats, dogs, foxes, raccoons, skunks etc

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17
Q

What do 99% of rabies cases come from (which animal)?

A
  • Bats
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18
Q

How come rabies is almost impossible to eliminate?

A
  • Due to so many wildlife reservoirs
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19
Q

How can we control rabies?

A
  • Vaccination of dogs, pets

- Wild-life oral bating (herd immunity)

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20
Q

What is meant by humans being a ‘dead-end host’ for rabies?

A
  • no-human-human transmission

- they are dead, don’t pass on

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21
Q

If you were bitten by a dog, would it be better to be bitten on the face or the foot?

A
  • Better to be bitten on the foot

- Because it has more time to travel to the brain compared to already being on the face

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22
Q

Is rabies only caused by rabies virus?

A

NO

- Also caused by lyssaviruses (“greek spirit for mad rage’)

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23
Q

How many members of the lyssavirus genus is there?

A
  • About 14
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24
Q

Does Australia have rabies?

A
  • We have something very similar
  • ABLV–> Australian Bat Lyssavirus
  • 3 human cases recorded –> fatal
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25
Q

What is ABLV found in?

A
  • flying foxes and bats
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26
Q

What is the treatment for ABLV?

A
  • Same as for rabies–> rabies Ig and Rabies vaccine protects against lyssavirus
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27
Q

Is there any prevention for ABLV?

A
  • Avoid handling bats
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28
Q

What are the 6 non molecular steps of the rabies virus?

A
  1. Animal bite or scratch (virus in saliva)
  2. Infects muscle (replicates), transmitted to peripheral nerves, then CNS
  3. Virus particles transport in along neuronal axons (i.e. retrograde) in vesicles using MICROTUBULES
  4. When at neuronal cell body, released from vesicle, replicate, assemble, new virus particles, then infect the next neuron.
  5. travels up SC–> to brain–> encephalitis
  6. Spreads to other organs (e.g. salivary glands, eyes)
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29
Q

How does rabies virus travel to the CNS?

A
  • Via microtubules as part of retrograde transport
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30
Q

When rabies virus travels up the neurons, does it damage them?

A
  • NO

- Very little cytoplasmic effect

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31
Q

How does the rabies virus kill if it doesn’t ‘damage’ neurons?

A
  • Inhibits apoptosis

- Immunosuppressive strategies (immune evasion)

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32
Q

Which two types of polymerases must host viruses provide that aren’t in host cells?

A
  • RNA polymerase –> (RNA dependent RNA polymerase)

- Reverse transcriptase (RNA dependent DNA polyemrase)

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33
Q

What type of RNA does rabies virus use?

A
  • Negative sense RNA
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34
Q

In rabies virus, because it uses negative sense RNA, what does this mean for the replication?

A
  • It must BRING and make its own RNA polymerase
  • this allows it to make the +ve sense RNA strand
  • The +ve sense RNA strand can now be used to translate the viral RdRP which can allow for replication of RNA (+ve and -ve sense produced)
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35
Q

In a normal virus such as polio that uses +ve sense RNA, what are the steps in replication?

A
  • Easier than rabies
  • Ribosome translates protein in form of RNA dependent RNA polymerase
  • This then allows for +ve RNA to be translated directly and replicated to both +ve and -ve sense
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36
Q

In rabies virus, what are the steps in the RNA replication (-ve sense RNA)?

A
  • -ve sense RNA must bring with it a viral RdRp to allow +ve sense RNA to be made
  • This then follows the same steps as polio where ribosome translates into protein and allows for replication
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37
Q

Which type of nucleic acid do Lyssaviruses use?

A
  • RNA
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38
Q

Does rabies virus have to make its own RNA polymerase?

A
  • YES
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39
Q

What are some features of the lyssaviruses genome?

A
  • Mutates quickly –> no proofreading

- Small genome

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40
Q

What strand and sense of RNA is involved in lyssaviruses?

A
  • ss -ve sense
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41
Q

How many segments does the genome of lyssaviruses have?

A
  • ONE

- Non-segmented (unlike influenza which has lots of segments)

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42
Q

Which class in the Baltimore scheme is it?

A
  • V
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43
Q

What is the rough size of the Lyssavirus genome?

A
  • 11kb
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44
Q

How many genes does the Lyssavirus genome contain?

A
  • 5

- N, P, M, G, L

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45
Q

What order is Lyssaviruses in?

A
  • Monoegavirales
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46
Q

Apart from Rabies (Rhabdoviridae), what are some other viruses that are under the monoegavirales order, and do they have similar pathways to rabies?

A
  • Ebola (Filoviridae)
  • Hendra, Measles, Mumps, Respiratory syncytial virus
  • YES they all have similar pathways; if you understan the steps of lyssaviruses then you will understand the steps of ALL Mononegaviruses
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47
Q

What shape is the rabies particle/virion?

A
  • Bullet
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48
Q

Is the rabies particle enveloped?

A
  • YES
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49
Q

What are the 5 common steps to all viral life cycles?

A
  1. Attachment
  2. Entry
  3. Synthesis
  4. Assembly
  5. Release
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50
Q

Which part of the Rabies virion is helical?

A
  • The RNP (Ribonuccelocapsid)
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51
Q

Are all 5 proteins in the Rabies particle?

A
  • YES

- N, P, M, G (on surface), L

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52
Q

What is the function of the N protein in the Rabies virion?

A
  • Binds and covers (encapsidates) RNA genome (nucleoprotein)
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53
Q

Where is the G protein present in Rabies virus?

A
  • On the surface (spikes)
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54
Q

Do viruses have their own translational machinery?

A
  • NO

- They must ALWAYS use the host cells ribosomal machinery to make proteins

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55
Q

Are proteins made by the host cell ribosomes (not the virus)?

A
  • YES
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56
Q

What is the function of the nucleoprotein (N) in Lyssaviruses?

A
  • Encapsidates the genome (nucleocapsid) –> required for transcription, replication
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57
Q

What is the function of the P protein (Phosphoprotein) in Lyssaviruses ?

A
  • Co-factor for polymerase (L), and links L to N-RNA
  • Required for tranascription and replication
  • Binds N, immune evasion
58
Q

What is the role of the Matrix protein (M) in lyssaviruses?

A
  • Required for viral assembly and budding
59
Q

What are two characteristics and the role of the G protein (Glycoprotein) in Lyssaviruses?

A
  • Outside of the particle, it is immunogenic

- Required for VIRAL ENTRY into the host cell

60
Q

What is the role of the L protein (Large protein) in Lyssaviruses?

A
  • RNA-dependent RNA polymerase (RdRp)

- Replication and transcription

61
Q

What occurs in the attachment and entry in the rabies virus?

A
  • Enters by receptor mediated endocytosis–> Receptor interacts with the G protein–> clathrin coated pits –> endosome forms –> travels down microtubules
62
Q

What are the three specific steps in attachment and entry of rabies virus?

A
  1. Endosome becomes acidic–> conformational change in G
  2. Viral envelope and endosomal membrane fuse
  3. Release nucleocapsid in cytoplasm
63
Q

What roughly occurs in the Synthesis step?

A
  • Nucelocapsid released into cytoplasm –> must bring with it N, P, and L for RNA synthesis
  • Transcription
  • Translation
  • Replication
64
Q

Is the -ve RNA genome to the +ve RNA antigenome done using the primary genome or seconday genome (i.e. old or new genome)?

A
  • Using the old genome (primary)
65
Q

What is the nucleocapsid and the +ve sense RNA antigenome used for?

A
  • a template to make -ve RNA
66
Q

What are the details of transcription (mRNA synthesis) in Lyssaviruses?

A
  • P cofactor for viral RNA polymerse (L)
  • P binds N-RNA and L –> links them together
  • transcriptase uses start-stop mechanism-specific signals b/w genes -> dis engages from template, then re-engages and so on
  • Generates mRNA with CAP and POLY-A tail (generated by L protein)
67
Q

Which protein generates the cap and poly A tail?

A
  • L protein
68
Q

Why is a + RNA antigenome (nucleocapsid) produced?

A
  • To act as a template so it can make more -ve RNA
69
Q

Which protein is ALWAYS trasncribed first in the Lyssaviruses?

A
  • N
70
Q

Does the P-L comlpex always re-engage with the RNA?

A
  • NO, it can fall off

- This leads to a transcription gradient forming

71
Q

In lyssaviruses, what is a transcription gradient due to?

A
  • Stop-start mechanism
  • Polymerase complex does NOT always re-engage with the genome
  • SOOO because N is always transcribed first, you are likely to have more N than L
  • More specifically–> N>P>M>G>L
72
Q

What is the order of transcription of the proteins in Lyssaviruses?

A

N, P, M, G, L

- No People Make Grapes Lie

73
Q

What is involved in translation in lyssaviruses?

A
  • Host cell ribosomes translate the N, P, M, G, L proteins

- this corresponds to the transcription gradient- so more N protein than L `

74
Q

What needs to occur for -ve sense RNA to be making more -ve sense RNA in lysaviruses?

A
  • Needs to have an antigenome (+ve RNA) to act as a template for the -ve RNA
  • the -ve RNA genome makes ANTIGENOMES (+ve RNA) to make new genomes
75
Q

What is involved in the replication step in lyssaviruses?

A
  • Genome -veRNA makes ANTIGENOMES (+veRNA) to make new genomes
  • Replication requires NEW N protein to encapsidate RNA (i.e. translation of viral proteins)
76
Q

In the translation of protein (replication) of lyssaviruses, what does the N protein function to do?

A
  • prevents skipping of the P-L complex (polymerase)
  • The N protein gets added to the new RNA to prevent skipping past proteins
  • It then encapsidates the RNA
77
Q

What would happen if a blocker was added to translation of the viral proteins?

A
  • There would be no trigger to replicating the genome (just transcription of mRNA)
  • So it would just keep on making +ve sense RNA which doesn’t allow for the virus to replciate
78
Q

Where does replication of rabies occur?

A
  • “liquid” negri bodies

- Membrane-less cytoplasmic inclusions caused by rabies infection

79
Q

What are negri bodies?

A
  • Liquid organelles
80
Q

What are negri bodies formed by?

A
  • N and P proteins
81
Q

Is it likely that most -ve sense RNA viruses use liquid organelles (negri bodies)?

A
  • YES
82
Q

Is the transcription phase of rabies virus mediated by the host or virus?

A
  • Mediated by the virus
83
Q

Is the translation phase of rabies virus mediated by host or virus?

A
  • Host
84
Q

What does no translation of protein mean for rabies virus development?

A
  • No replication
85
Q

Is the replication phase of rabies virus host or virus mediated?

A
  • Virus mediated
86
Q

Where is the rabies virus assembled in the cell?

A
  • at the plasma membrane
87
Q

What are the three main components of the rabies virus that are assembled?

A
  1. Nucleocapsid (RNA, N, P, L)
  2. M (at plasma membrane)
  3. G (glycosylated –> sugar groups added)
88
Q

What is protein M’s role in the assembly of the Rabies virus?

A
  • Mediates the assembly –> “selects” nucleocapsids (complete, not antigenome)
  • Triggers BUDDING from the host cell
  • Gain membrane envelope from host cell as buds from cell
89
Q

Which one of the proteins acts as qa cofactor?

A
  • P protein
90
Q

What is the 6th step in the virus life cycle?

A
  • Immune Evasion
91
Q

What is the role of the proteins in the Lssa viruses in terms of the immune evasion?

A
  • Counteract the immune response
92
Q

What are examples of immunity that is continuous?

A
  • Anatomical and chemical barriers

- e.g. Mucous, Saliva, Stomach Acid, Skin

93
Q

What are examples of immunity that is immediate (present) and what is this known as ?

A
  • Autophagy (recycling of cellular components), Apoptosis, RNAi
  • Known as intrinsic immunity
94
Q

What are examples of induced immunity and what is this known as?

A
  • NK cells
  • Complement cells
  • Cytokines
  • Innate immunity
95
Q

What is an example of tailored immunity and what is this known as?

A
  • B and T cells

- Acquired immunity

96
Q

What are cytokines?

A
  • Small proteins secreted by cells, involved in signalling/communicating with other cells. e.g. interferon
97
Q

Why are the cytokines called interferons termed that way?

A
  • Because there was an experiment done where influenza virus was inactivated (showed no virus) and when the media (with no virus) was transferred to viral cells, there was reduced production of the virus
  • Therefore the protein ‘interfered’ with the infection hence interferon
98
Q

What are the three main types of interferon and what do they each consist of?

A
  • Type I –> IFN alpha and IFN beta –> direct response to infection broad cellular expression and receptors
  • Type II (IFNgamma) –> Immune cells
  • Type III (IFN lambda)
99
Q

Is the production of IFNalpha/beta rapid?

A
  • YES

- Within hours of infefction and declines by 10 hours

100
Q

What does IFN binding to IFN receptors lead to?

A
  • The synthesis of >1000 cell proteins –> called IFN stimulated genes (ISGs)
101
Q

Are the mechanisms of ISG well known?

A
  • NO
102
Q

What is the rough pathway for induction of IFN type I normally?

A
  • RIG-1 (PRR) recognises either the dsRNA or lack of cap and becomes active
  • Signal transducton occurs (includes phosphorylation of IRF3 –> Interferon regulatory factor 3 and dimerisation)
  • This then enters the nucleus, binds to type I IFN genes
  • This allows IFN mRNA to be transcribed
  • Protein is produced and exits cell
103
Q

What is the rough pathway for IFN signalling in a cell normally?

A
  • IFN binds to IFNAR (Interferon type I receptor)
  • This causes a heterodimer of STAT1 and STAT2 to form
  • These enter the nucleus to allow for ISGs to be produced (Interferon stimulated genes)
  • This then allows for antiviral proteins to be produced
104
Q

What are 3 broad strategies for allowing viruses to inhibit the IFN production and action (IFN antagonists) ?

A
  1. Generals inhibition of host gene expression
  2. Sequestration/masking of PAMPs
  3. Sequestration/modification of signalling components
105
Q

Are IFN antagonists often multifactorial viral proteins?

A
  • YES
106
Q

What does sequestration/masking of PAMPs involve?

A
  • Blocking recognition of virus pamp
107
Q

What is sequestration/modification of signalling components involved in for IFN induction and signalling respectively?

A
  • Blocking PRR activation and blocking of STAT activation
108
Q

What does the general inhibition of host gene expression involve in IFN induction and IFN signalling respectively?

A
  • IFN expression and ISGs
109
Q

How does the rabies P protein block IFN induction?

A
  • By inhibiting phosphorylayion of IRF3 –> so IRF3 can NOT be activated
  • BUT mechanism not clear
110
Q

How can the P protein block IFN signalling?

A
  • Binds PHOSPHORYLATED STAT1/2–> this prevents nuclear import
  • Blocks activation of ISGs likely using SEVERAL ways
111
Q

What do ISGs stand for?

A
  • Interferon stimulated genes
112
Q

Which protein in the Rabies virus acts as the IFN antagonist?

A
  • P protein
113
Q

What is the structure of the P protein?

A
  • Has section for binding L protein
  • Has a NES sequence (for nuclear export)
  • Has C terminal domain –> contains NLS ( nuclear localisation sequence) and section for binding N protein as well as P-STAT proteins
114
Q

In the P protein, what is the C terminal domain known as and what does this mean?

A
  • The Globular region

- Binds lots of host proteins

115
Q

What are the specific functions of the P protein in terms of acting as IFN antagonist?

A
  • Binds N and L protein for transcription and replication
  • CTD binds MANY host proteins (unressolved functions)
  • CTD binds PHOSPHORYLATED STAT1/2
  • It traffics b/w nucleus and cytoplasm via NLS and NES
116
Q

When P protein is trying to inhibit IFN signalling, does it bind when STATS are phosphorylated or not phosphorylated?

A
  • When the STATs are PHOSPHORYLATED (active)
117
Q

How can the P protein allow for the virus transcription and replication?

A
  • P binds to STAT1/2 and prevents nuclear import

- P traffics INTO THE nucleus to bring the STATs that snuck in back out

118
Q

What does the Kozac sequence do?

A
  • Nucelotides around the start codon determines the likelihood of the ribosome initiating translation
  • Ribosomal leaky scanning
119
Q

If there is a STRONG kozac sequence where will the roibosome start translation?

A
  • At AUG

- e.g. gccaccAUGgcg

120
Q

If there is a WEAK kozac sequence, where will the ribosome start translation?

A
  • Can SKIP AUG

- tttttAUGcta

121
Q

Does the P protein have many isoforms?

A
  • YES
122
Q

Are some of the P protein isoforms truncated?

A
  • YES
123
Q

What does the P protein use to make alternative versions of itself?

A
  • Kozack sequences
124
Q

What are 4 possible ways that the P protein and inparticular the P3 isoform (in 3 and 4) inhibits IFN signalling?

A
  1. P binds and prevents nuclear import
  2. P traffics into nucleus and brings STATs that have snuck in back out
  3. Nuclear P3 binds STATs in the nucleus, prevents binding DNA
  4. P3 binds STATs and arrests them on Microtubules (MTs)
125
Q

What is CRITICAL to the pathogenesis of Rabies virus?

A
  • The P protein inhibition of STATs
126
Q

What is currently under investigation in terms of pathogenesis of rabies and what implications can this have?

A
  • Where specifically does STAT bind?

- Possible vaccine strategy

127
Q

Which protein si the primary IFN antagonist?

A

P protein

128
Q

In what ways is P-protein IFN antagonism multipronged?

A
  • Impairs IFN induction (mechanisms uncle–> inhibits phosphorlyation of IRF3
  • Impairs IFN signalling –> binds phospohrylated STAT proteins , many ways to prevent the transcription og ISGs
  • P-STAT interaction is associated with pathogenesis
129
Q

In what ways do RNA viruses use clever tricks to increase their coding capacity even though they have such small genomes?

A
  • Robosomal leaky scanning
  • 1 RNA –> polyprotein (cleaved)
  • Multifunctional proteins
  • RIbosomal frameshifting
  • Suppression of termination
  • Proteins regulated (trafficking signals, conformational, PTM,etc)
130
Q

How do we manipulate the rabies virus?

A
  • using REVERSE GENETICS–> DNA copy of the RNA virus genome
  • Insert a DNA copy of the RNA genome into DNA plasmid (easy to manipulate, insert gene, delete etc)
131
Q

What is a limitation in manipulating rabies virus?

A
  • Mutating it
132
Q

What do you need to make the rabies virus?

A
  • Rabies genome
  • N, P, L plasmid –> these are REPLICASE PROTEINS
  • SO must transfect all 4 different plasmids
133
Q

When making a modified rabies virus, can anything happen without the replicase?

A
  • NO

- Kind of like jump starting a car–> “jumpstart replication with replicase proteins”

134
Q

What are the replicase proteins known as?

A
  • N,P,L
135
Q

What are 4 potentially good things that the rabies virus can be used for?

A
  • Neurotracer–> Mapping neural networks
  • Pass blood-brain barrier (BBB)—> treating West Nile virus
  • Vector for vaccines –> highly immunogenic
  • Curing Alzheimer’s –> P exceptional at imparing the inflammatory response, can we use P to learn how to target these pathways to tackle these disorders??
136
Q

Is rabies one of the most lethal viral diseases?

A
  • YES
137
Q

Do all mononegaviruses such as Rabies, Ebola, Marburg, Hendra, Mumps, borna disease all have similar genome organisation?

A
  • YES
138
Q

What does the N mRNA code for?

A
  • The nucleocapsid
139
Q

What does the P mRNA code for?

A
  • Polymerase co factor, immune evasion
140
Q

What does the M mRNA code for?

A
  • Matrix
141
Q

What does the G mRNA code for?

A
  • The Glycoproteins
142
Q

What does the L mRNA code for?

A
  • The Polymerase

BMS2052 MICRO (10 decks)

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