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immunobiotechnology > Lecture 10 > Flashcards

Lecture 10 Flashcards

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1
Q

What was the mortality rate of SARS 2003?

A

9.6%

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2
Q

What was the unique symptom of SARS 2003?

A

Temporary loss of smell (anosmia) in early phase without nasal obstruction.

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3
Q

What are the structural proteins on Coronavirus?

A

Spike protein
Membrane protein
Envelope protein
Nucleocapsid protein

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4
Q

How many open reading frames does TMV have?

A

4

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5
Q

For the protein produced by ORF1 of TMV, what are the 2 domains?

A

Methyltransferase and helicase (replicase)

replicase: an enzyme which catalyses the synthesis of a complementary RNA molecule using an RNA template

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6
Q

ORF1 + ORF2 of TMV = which protein?

A

Methyltransferase , helicase and
RNA-dependent RNA polymerase

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7
Q

What is ORF3 of TMV for?

A

Movement protein: to facilitate the virus to move through the thick cell wall of the plant cells

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8
Q

What is ORF4 of TMV for?

A

capsid protein

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9
Q

Which ORF of TMV are subgenomic RNAs?

A

ORF 3 and ORF 4

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10
Q

What is the genetic material of coronavirus

A

+ssRNA (+gRNA)

size of 30,000 nts

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11
Q

What are the three classes of proteins that are produced by coronvirus genome?

A

16 nonstructural proteins
4 structural proteins ( S,E,M,N)
6 accessory proteins

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12
Q

What is an open reading frame?

A

ORF is open-reading frame, meaning that the DNA (or RNA) sequence appears to code for a protein – of reasonably length before a stop-codon

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13
Q

What are the ORF of Cov-2?

A

ORF1a and ORF1b

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14
Q

Describe the genome replication of SARS-CoV-2

A

More ss(+) RNA (or +gRNA) were
generated from the –gRNA.

In turn, subgenomic ss(-) RNA (or –sgRNA) were generated by loop forming.
+sgRNA were transcribed.

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15
Q

What proteins are produced from +sgRNA of CoV-2?

A

structural and accessory proteins

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16
Q

Describe how coronavirus replication takes place step by step.

A
  1. S protein bind to ACE2 receptor, TMPRSS2 cleave S protein –> mediate membrane fusion and release of genome into cell
  2. translation of +gRNA to express nsp1-16 in cytoplasm
  3. genome replication in DMV by continuous replication of +gRNA by -gRNA through RTC
  4. generation of -sgRNA and +sgRNA by discontinuous transcription RTC
  5. expression of structural proteins and accessory proteins from +sgRNA
  6. Virion assembly and release
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17
Q

Where does replication of CoV-2 genome take place?

A

In double membrane vesicle (DMV)

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18
Q

Name one non-structural protein of SARS CoV-2 that is important for its genome replication

A

RNA dependent RNA polymerae

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19
Q

What is spike protein for?

A

Spike protein (trimer) for binding to receptor ACE2 (angiotensin converting enzyme 2) and facilitates membrane fusion after cleavage by TMPRSS2 (transmembrane serine protease 2).

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20
Q

What is envelope protein for?

A

Envelope protein (pentamer), an ion channel to induce membrane curvature for viral assembly.

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21
Q

What is membrane protein for?

A

inhibits type-1 interferon (anti-viral cytokine) production.

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22
Q

What is nucleocapsid protein for?

A

Nucleocapsid protein that stabilize the ss RNA genome.

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23
Q

What are the major targets of Covid-19 and why?

A

alveolar (lung) epithelial (surface) cells because of high levels of the recptor ACE2 and TMPRSS2

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24
Q

What is the full form of ACE2 and TMPRSS2?

A

ACE2: angiotensin converting enzyme 2

TMPRSS2: transmembrane serine protease

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25
What do damaged cells do at the first instance of Covid-19 infection?
trigger local and systemic cytokine synthesis and release --> cytokine storm Chemoattraction of cells of the immune system including neutrophils, B and T cells, and NK cells.
26
What are the downstream damages of Covid-19 infection when leukocytes are recruited to site of infection?
widespread tissue damage, thrombus formation, multiple organ failure (due to impaired lung function to deliver oxygen), and death
27
What are the 'severity' types of Covid-19?
Asymptomic Mild (fever, sore throat, fry cough) Moderate (persistent fever and cough) Severe (pneumonia, low O2 in blood) Critical (organ failure) Death
28
How are Sars-CoV-2 infected cells killed by cytotoxic T cells?
through the release of perforin and granzymes
29
What antibody can neutralize the virus by blocking its binding to the receptor fusion?
IgA
30
How else can phagocytosis of virus occur?
Activation of lectin pathway or the alternative pathway, leading to C3b and iC3b deposition --> virus phagocytised through the C3 receptors --> MAC assembled
31
What structural protein of coronavirus contributes majorly to B cell response? Why?
S protein because of no of amino acids of M and E exposed on surface is small note that M and E also contribute to B cell response, just that not 'majorly'
32
What proteins on covid-19 are targets of our immune response?
S,M,E ----> B cell -----> T cell N, nsp, acc -------> T cell
33
How does omicron variant differ?
more infectious, less virulent And alternate cell entry
34
What is the mechanism through which omicron variant mediate its entry into a cell and how does it differ from the original strain?
Omicron need only ACE2, making cells without TMPRSS2 available of infection. The variant is encapsulated in a bubble called an endosome, drifts into cells and then breaks out
35
What cells in our body does omicron variant infect?
cells in the airway above the lungs Other variants: infect cells in the lungs
36
State the reasons as to why the omicron variant is more infectious
infects the upper airway where binds to ACE2 receptor tightly--> virions are air borne --> longer range of infectivity Other strains are release from coughing and are carried by sputum ---> heavier and shorter range ---> less infectious than omicron
37
Why is the omicron variant less virulent when compared to other strains?
Omicron: cytokine storm leads to sore throat, lung function not imparied Other variants: infect epithelial cells of the lung ---> cytokine storm --> inflammation of lung tissue and impairment of O2 uptake --> widespread tissue damage, multiple organ failure or death
38
What are the 3 steps of PCR?
1. Denaturation (94C) 2. Annealing 3. Extension (72C)
39
What molecules involved with annealing and extention step of PCR?
Annealing: Primers forward primer: 5'-->3' reverse primer: 3' --> 5' Extention: DNA polymerase and 4 dexoynucleotides
40
What happens to sample after running PCR?
run on agarose gel and the expected size would confirm the presence of DNA in original sample
41
Where are the DNA polymerases for PCR from?
Taq polymerase from archaebacteria because stable at the denaturing temperature of 94 ̊C, and active at the elevated temperature of 72 ̊C
42
How does qPCR differ from PCR?
qPCR has a probe Probe: reporter + quencher Quencher absorbs the the photon emitted by the reporter when THEY ARE IN THE SAME MOLECULE
43
What is the purpose of quencher on the probe of qPCR?
Quencher absorbs the the photon emitted by the reporter when THEY ARE IN THE SAME MOLECULE
44
What happens in real time PCR?
-probe binds to denatured DNA -probe degraded as the Taq polymerase displaces it from the DNA template -reporter and quencher not in close proximity anymore -fluorescent emission of probe not quenched -fluorescence measured --> amount of reporter proportional to amount of template
45
Describe the mechanism of rapid antigen test
1. analyte move through capillary action 2. comes into contact with SARS-CoV-2-specific antigen antibody (with gold) 3. antigen-antibody complex migrates across nitrocellulose membrane 4. complex comes into proximity with 2nd SARS-CoV-2 antigen antibody (diff epitope) ---> test line 5. diffusion of excess complex + unbound antibody and bound to first antibody specific antibody --> formation of antibody-antibody complex (ctrl line)
46
Describe antibody testing
1. viral proteins with histidine tag 2. histidine have high affinity for metals ions (nickel) on nickel coated nitrocellulose membrane 3. serum added 4. secondary antibody with detectable reagent added 5. + result --> person has been infected or vaccinated
47
In antibody testing, what are the viral proteins tagged with?
histidine because histidine has high affinity for metal ions
48
What are the four types of Covid-19 vaccines?
1. RNA vaccines 2. Viral vector vaccines 3. Inactivated vaccines 4. Recombinant proteins vaccines
49
In RNA vaccines, how are the RNAs/nucleic acids delivered?
-packaged into liposome -package fuses with host cell for expression (secreted or membrane)
50
What are subunit vaccines?
Proteins (peptides) are expressed as a fusion protein with a self- assembling core
51
What is a popular vector for viral vector vaccine?
Adenovirus --> broad tissue tropism, --> expresses PAMP (pathogen-associated molecular patterns) through the innate immune system, to trigger the production of cytokines for the maturation of antigen-presenting cells
52
Are viral vectors for viral vector vaccines able to undergo replication?
No, the virus is engineered to be replication defective
53
What major discovery leads to the development of effective mRNA vaccines?
Uridine to pseudouridine --> significant reduction on the inflammatory response induced by the RNA
54
How does an RNA vaccine work?
1. virus genetic code turn into vaccine and injected 2. vaccine enter cell and produce spike protein 3. immune system activated --> antibodies and T cells 4. If infected later, antibodies and T cells triggered to fight the virus
55
What happens to the cells that express the products of the mRNA after the injection of mRNA vaccine?
They become the target of the immune system and are killed
56
What are the common side effects of mRNA vaccine?
1.development of abnormal blood clotting – thrombocytopenia (low platelet counts) 2.Myocarditis (inflammation of the heart muscle) 3.pericarditis (inflammation of lining around the heart 4.palpitation and chest pain
57
What protein does the mRNA in mRNA vaccine normally express?
S protein
58
What is assessed during during the pre-clinical or clinical trials?
- pharmacokinetics - therapeutic - efficacy and side effects
59
What 2 experiment groups in clinical trials
Vaccinated group and control group
60
What are the pros of nucleic acid vaccines?
- inexpensive - can cope with mutation --> modify sequence easily
61
What are the cons of nucleic acid vaccines?
- RNA fragile --> require low temp storage - short shelf life -transportation problem -only focus on epitopes of spike protein --> less epitopes for our immune system to work against -novel approach
62
What are the pros of inactivated vaccines?
- protocol establish for other vaccines - epitopes come from M,E and N proteins in addition to S protein - less stringent storage conditions --> 2-8C a few years -administrations can be carried out in smaller clinics
63
What are the cons of inactivated vaccines?
- need to grow viruses, purification, inactivation, re purification --> require high biosafety facilities --> time consuming and costly
64
What is the reporting system for reporting adverse effects of vaccines called?
Vaccine Adverse Event Reporting System (VAERS)
65
State some adverse effects caused by inflammation of major nerves
- Anosmia - Tinnitus - Deafness - Migraine headache - Nausea - inflammation of heart muscle -heart attack - thrombosis (blockage of blood vessel developed locally)
66
What are bivalent vaccines?
mRNA would have a combinatorial sequences based on the original sequence and those of a few common variants at the time
67
RNA viruses are prone to mutations. true or false
True due to limited proof reading
68
What mutation in the genome of RNA viruses may not be tolerated?
mutations in proteins involved in viral genome replication
69
State some reasons as to why people may not be vaccinated
1. Political stance 2. religious reasons 3. perceived allergy to vaccines 4. conflicting advice from experts 5. Value freedom and choice over public health
70
Can the nucleic acids (RNA) in the vaccines integrate into the genome of your cells and turn them cancerous?
Only DNA can integrate into our genome *RNA needs the enzyme Reverse Transcriptase to make DNA copies. The SARS-CoV-2 (or any coronavirus) does not have the enzyme.
71
Does SARS-CoV-2 have reverse transcriptase?
No
72
Can the viral vector vaccines integrate into the genome of your cells and turn them cancerous?
They have DNA in the vaccines Still chances are low
73
What viruses can integrate their genome into our own?
HIV, RNA virus that has its own reverse transcriptase and has dsDNA intermediate in its replication cycle also HBV (a DNA virus)

immunobiotechnology (8 decks)

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