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CMB1003- Microbiology and immunology > virology L12-17 > Flashcards

virology L12-17 Flashcards

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1
Q

virus structural components

A

DNA/RNA genome
surface proteins
+/- envelope
capsid protein

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2
Q

virus characteristics

A

obligate intracellular parasite

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3
Q

virion

A

purified virus particle
seen w e- microscope
simplest life-form

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4
Q

nucleopcapsied

A

genome enclosed by capsid

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5
Q

5 basic virus structural forms

A

naked icosahedral
naked helical
enveloped icosahedral
enveloped helical
complex

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6
Q

cycle of infection

A

transmission
entry
primary replication site
spread within host
shedding
REPEAT

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7
Q

3 modes of virus transmission

A

horizontal
vertical
zoonosis

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8
Q

6 steps of viral replication

A

attachment
penetration
uncoating
biosynthesis
assembly
release

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9
Q

biosynthesis requirements

A

host ribosomes, enzymes and precursors

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10
Q

2 types of internalization

A

fusion from within (only enveloped)
receptor-mediated endocytosis (both)

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11
Q

receptor-mediated endocytosis

A

fusion w endosome
conformational change in viral protein
escape through pore
fusion w vesicle

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12
Q

biosynthesis

A

replication and transcription of viral genome after penetrationa nd uncoating

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13
Q

pox virus biosynthesis exception

A

have own DNA/RNA polymerase

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14
Q

virus protein synthesis

A

genome replication
genome packaging
metabolism alteration

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15
Q

2 forms of metabolism alteration

A

non-structural (function)
structural (part of molecule)

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16
Q

viral mRNA translation Cap involvement

A

5’ terminal cap binding site w 40S ribosomal sub-unit by eukaryotic initiation factors

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17
Q

viral release mechanisms

A

lysis
budding
cell-cell spread
post-maturation step

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18
Q

bacteriophage

A

viruses that infect bacteria

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19
Q

M13 filamentous bacteriophage

A

circular ssDNA
forms own channel via budding
lytic/ lysogenic
turn bacteria virulent
phage therapy alternative
lab tool for recom DNA

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20
Q

HIV transmission

A

sexual
mechanical
vertical

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21
Q

2 HIV strains

A

HIV1> AIDS
HIV2> milder symptoms/ less infectious)

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22
Q

HIV origin

A

simmian immunodeficiency virus

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23
Q

what type of virus is HIV?

A

a retrovirus (has reverse transcriptase)

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24
Q

anti-HIV therapies

A

triple therapy (HAART)
nucleoside analogues
peptide analogues
anti-CCRS therapy

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25
Q

4 genera of coronaviruses

A

alpha (mamm>mamm)
beta (mamm>mamm)
delta (mamm>avian)
gamma (avian)

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26
Q

coronavirus chracteristics

A

single strand positive sense RNA
enveloped virion/ proteins
internal nucleocapsid

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27
Q

coronavirus key proteins

A

spike (binds to receptor)
M (virion assembly)
N (nucleoprotein binds/ protects)
E (viral release from cell)
S (immunogenic target for vaccines)

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28
Q

where does coronavirus replication/ assembly take place?

A

in the cytosol

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29
Q

coronavirus assembly location

A

golgi/ ER

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30
Q

viral attachment and entry

A

spike protein binds ACE2 on csm
TMPRSS2 cleaves spike protein
viral and cellular membrane fusion

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31
Q

SARS

A

Severe
Acute
Respiratory
Syndrome

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32
Q

SARS incubation

A

2-10 days

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33
Q

SARS symptoms

A

initial fever and cough/ shortness of breath > pneumonia

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34
Q

SARS tests

A

serological
RT-PCR
IR-thermography

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35
Q

SARS prevention

A

global cooperation
lockdowns
burnout

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36
Q

MERS

A

Middle East respiratory Syndrome

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37
Q

MERS symptoms

A

fever
cough
dyspnoea
pneumonia

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38
Q

covid-19 transmission

A

fomites
droplets
aerosols

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39
Q

covid-19 incubation

A

4-6 days

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40
Q

covid-19 common symptoms

A

runny nose
headache
sore throat

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41
Q

covid-19 diagnosis

A

mass-testing
PCR
lateral flow
nasopharyngeal swabs
RNA detection

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42
Q

lateral flow evaluation

A

quick
50% accuracy

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43
Q

covid-19 lab findings

A

lymphophenia
ARDs
^LFTs
inflam markers
coagulation abnormalities

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44
Q

covid-19 complications

A

“silent” hypoxia
ARDs preceded by pneumonia > difficult recovery and pulmonary scarring
sepsis
secondary infections

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45
Q

Covid-19 treatment

A

hospitalization
remdesivir

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46
Q

remdesivir

A

viral RNA polymerase inhibitor

47
Q

covid-19 threats

A

transmissibility
vaccine bypass
virulence
population control

48
Q

virus characteristics for tools in medicine

A

easy to genetically manipulate/ modify
targeted delivery to specific cells
powerful gene expression
gene-carrying
can be attenuated

49
Q

human attenuation

A

human viruses having lost properties to replicate efiiciently in human cells therefore not disease-carrying

50
Q

non-human attenuation

A

non-human virus able to infect human cells w limited replication and some gene expression

51
Q

gene expression in lab via viruses

A
  1. virus promoter cloned into DNA plasmid vector
  2. PCR generates cDNA from mRNA
52
Q

pCDNA3.1
ori in bacteria
ori in human cells

A

has pCMV promoter and BGH polyadenylation signal
ampicillin and pUC ori allow replication and selection in bacteria
neomycin and SV40 ori allows replication and selection in human cells

53
Q

transfection process

A
  1. isolate ORF of interest
  2. clone into expression vector
  3. isolate/ purify plasmid DNA
  4. transfect target cells and detect gene expression
54
Q

why are viruses more efficient as vectors than chemical/ liposomal transfection?

A

virus-own entry mechanism
affect more complex cell lines

55
Q

advantages of adenoviral vectors

A

small genome
easy to manipulate/ purify
infects many cell types
^ gene expression

56
Q

disadvantages of adenoviral vectors

A

immunogenic
limited to <8kb ORF’s
transient expression

57
Q

adenoviral gene vectors

A

mostly missing E1 gene region for replication
mostly missing all viral proteins
helper coat proteins required

58
Q

adenoviral gene vector functions

A

can amplify virus vectors
^ yields
hit nearly all cells
modify promoter for cell-type specific gene expression

59
Q

lentiviral vectors

A

commonly HIV1 only LTRs (promoters and polyA signals) packaged, no surface protein, other retroviruses used
virus supplied in trans
no gp160 attachment protein

60
Q

lentiviral vector advantages

A

small genome
^level of gene expression
many cell types
expressed in non-dividing cells
stable gene-expression over time
integrating into genome of target cell

61
Q

lentiviral vector disadvantages

A

<10kb limit
danger if inserted near to proto-oncogene

62
Q

types of vaccine

A

live
killed whole virus
subunit
gene delivery

63
Q

types of live vaccine

A

attenuated
heterologous

64
Q

attenuated live vaccine process

A
  1. isolated and cultured virus on host
  2. incubation on cells from other host
  3. spontaneous growth and mutation on alternate host
  4. won’t grow on original host
65
Q

subunit vaccine

A

components purified from whole virus
recombinant proteins

66
Q

advantages of attenuated vaccine

A

mimic wild-type
strong humoral and cellular response
induces innate immunity
fewer doses required for complete immunity

67
Q

disadvantages of attenuated vaccine

A

reversion can occur to wild-type
not viable for immunosuppressed
careful handling/ storage
virus can’t be cultured

68
Q

virotherapy

A

replacement of defective gene function by introducing ‘normal’ gene

69
Q

types of virotherapy

A

ex vivo (cells removed and reintroduced to body)
in vivo (replacement gene introduced directly into body via gene vector)

70
Q

CAR-T gene therapy

A

T cell collection from chimeric T cell receptors
lentiviral genetic modification to target cancer cells in transfection
adoptive transfer
patient monitoring > disease response/ CAR-T cell persistence

71
Q

oncolytic virotherapy

A

manipulation of lytic virus to destroy Cancer cells (not non-cancerous cells)
viral tropism change
causes inflammation, lysis and more tumour cell death exponentially

72
Q

oncolytic

A

destroying tumour cells

73
Q

oncogenic

A

promoting tumour cell development

74
Q

oncolytic virotherapy barriers

A

vector neutralisation overcome
enriching T-cell response
^spread
^tumour uptake

75
Q

oncolytic virotherapy advantages

A

combination with other cancer treatments
safe
overcomes resistance
dual mode of action

76
Q

oncolytic virotherapy disadvantages

A

resistant cancer cells
antiviral immune response
limited replication and spreading

77
Q

phage therapy

A

use of bacteriophage to kill bacteria

78
Q

phage therapy advantages

A

specific to bacteria
can evolve to adapt resistance
self-amplifying until all bacteria dead

79
Q

phage therapy disadvantages

A

individualised
cocktail of phage
theoretical potential for Ab resistance
g - lysis can release endotoxins (sepsis)

80
Q

lysins/ endolysins

A

enzymes produced by some bacteriophages to cleave host cell wall in final stage of lytic cycle
> purified is more potent than antibiotics
> negligible resistance

81
Q

uses of phage therapy

A

FDA approved phage therapy for P.aeruginosa in CF patients
trials for burn infections
biofilm treatments

82
Q

2 methods of detection/ counting viruses in the lab

A

cell-based assays
protein-based assays

83
Q

cell-based assays

A

plaque assay
tissue culture infectious dose assay (TCID50)

84
Q

protein-based assays

A

haemagglutination assay
electron microscopy
immunofluorescence
enzyme-linked immunosorbent assay (ELISA)

85
Q

functions of detection/ quantification of viable virus particles in lab

A

diagnosis
prognosis
research

86
Q

virus growth in laboratory

A

grow cells in tissue culture
infect cells with virus
incubate infected cells
harvest cells
quantify virus yield

87
Q

how to observe for effects of viral infection

A

cytopathic effect (CPE)

88
Q

multiplicity of infection (moi)

A

number of infectious viral particles used to infect 1 cell

89
Q

poisson distribution

A

chance of any 1 cell being infected by 0 or more viral particles
assumes normal distribution

90
Q

moi importance

A

infection synchronization
different viral characteristics
stock viability testing
infection cellular response observation

91
Q

plaque assay process

A

perform serial dilution
add to cells in appropriate volume
absorb
remove inoculum
add fresh media w agar
overlay w medium
wait
fix and stain
count number of PFU

92
Q

PFU

A

Plaque forming unit

93
Q

plaque

A

clearing of the cell monolayer

94
Q

plaque assay method to calculate viable virus concentration

A

use dilution factor/ amount of diluted virus stock added/ no. plaques produced

95
Q

plaque assay limitations

A

virus may cause visible c.p.e
time required can be significant
sterility must be maintained

96
Q

focus forming assay

A

plaque assay variation
no agar overlay
24h can fix and immunostain w flourescence antibody

97
Q

ffu

A

focus forming unit

98
Q

end-point dilution assay

A

simple
sequential dilution of virus stock in micro-titre plate (96 well)
multiple replicants of each dilution
count under microscope

99
Q

TCID50

A

tissue culture infectious dose
virus concentration that kills 50% of cells in a culture system
infectious titre
endpoint dilution assay

100
Q

TCID50 method

A

96 well plate format
serial dilution across plate
leave to infect and kill cells
score wells as infected +/-

101
Q

TCID50 infection rate

A

1/2 cells per unit

102
Q

TCID50 functions

A

drug development
allows non-plaquing virus quantification

103
Q

TCID50 limitations

A

time-consuming
medium needs to be changed regularly
prone to drying out
sterility (incubation length)

104
Q

electron microscopy types

A

scanning
transmission

105
Q

electron microscopy

A

best for virus particle identification physically
compare to pfu
measure of particle to infectivity ratio

106
Q

haemagglutination

A

measurement of both viable and non-viable virus particles
generic effect
uses viral ability to link RBC’s
relative quantification

107
Q

how to quantify viral particles absolutely with haemagglutination?

A

compare results to a standard virus suspension containing known number of virus particles/ ml

108
Q

immunofluorescence

A

stain virus antigens on cell surface/ in sections of virus-infected host cells

109
Q

immunofluorescence process

A

add virus
wash cells to remove unabsorbed antibody
add FITC conjugated anti-rabbit IgG antibody
wash again
examine w UV microscope

110
Q

FITC

A

fluorescein isothiocyanate

111
Q

ELISA

A

enzyme linked immuno-sorbent assay

112
Q

types of ELISA

A

direct (primary antibody conjugate)
indirect (secondary antibody conjugate)
sandwich (capture antibody)

113
Q

ELISA pros

A

quicker than traditional titrations
in clinic (HIV )
can be related to viral titre w standards

114
Q

ELISA pros

A

quicker than traditional titrations
in clinic (HIV )
can be related to viral titre w standards

CMB1003- Microbiology and immunology (3 decks)

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