virology L12-17 Flashcards
virus structural components
DNA/RNA genome
surface proteins
+/- envelope
capsid protein
virus characteristics
obligate intracellular parasite
virion
purified virus particle
seen w e- microscope
simplest life-form
nucleopcapsied
genome enclosed by capsid
5 basic virus structural forms
naked icosahedral
naked helical
enveloped icosahedral
enveloped helical
complex
cycle of infection
transmission
entry
primary replication site
spread within host
shedding
REPEAT
3 modes of virus transmission
horizontal
vertical
zoonosis
6 steps of viral replication
attachment
penetration
uncoating
biosynthesis
assembly
release
biosynthesis requirements
host ribosomes, enzymes and precursors
2 types of internalization
fusion from within (only enveloped)
receptor-mediated endocytosis (both)
receptor-mediated endocytosis
fusion w endosome
conformational change in viral protein
escape through pore
fusion w vesicle
biosynthesis
replication and transcription of viral genome after penetrationa nd uncoating
pox virus biosynthesis exception
have own DNA/RNA polymerase
virus protein synthesis
genome replication
genome packaging
metabolism alteration
2 forms of metabolism alteration
non-structural (function)
structural (part of molecule)
viral mRNA translation Cap involvement
5’ terminal cap binding site w 40S ribosomal sub-unit by eukaryotic initiation factors
viral release mechanisms
lysis
budding
cell-cell spread
post-maturation step
bacteriophage
viruses that infect bacteria
M13 filamentous bacteriophage
circular ssDNA
forms own channel via budding
lytic/ lysogenic
turn bacteria virulent
phage therapy alternative
lab tool for recom DNA
HIV transmission
sexual
mechanical
vertical
2 HIV strains
HIV1> AIDS
HIV2> milder symptoms/ less infectious)
HIV origin
simmian immunodeficiency virus
what type of virus is HIV?
a retrovirus (has reverse transcriptase)
anti-HIV therapies
triple therapy (HAART)
nucleoside analogues
peptide analogues
anti-CCRS therapy
4 genera of coronaviruses
alpha (mamm>mamm)
beta (mamm>mamm)
delta (mamm>avian)
gamma (avian)
coronavirus chracteristics
single strand positive sense RNA
enveloped virion/ proteins
internal nucleocapsid
coronavirus key proteins
spike (binds to receptor)
M (virion assembly)
N (nucleoprotein binds/ protects)
E (viral release from cell)
S (immunogenic target for vaccines)
where does coronavirus replication/ assembly take place?
in the cytosol
coronavirus assembly location
golgi/ ER
viral attachment and entry
spike protein binds ACE2 on csm
TMPRSS2 cleaves spike protein
viral and cellular membrane fusion
SARS
Severe
Acute
Respiratory
Syndrome
SARS incubation
2-10 days
SARS symptoms
initial fever and cough/ shortness of breath > pneumonia
SARS tests
serological
RT-PCR
IR-thermography
SARS prevention
global cooperation
lockdowns
burnout
MERS
Middle East respiratory Syndrome
MERS symptoms
fever
cough
dyspnoea
pneumonia
covid-19 transmission
fomites
droplets
aerosols
covid-19 incubation
4-6 days
covid-19 common symptoms
runny nose
headache
sore throat
covid-19 diagnosis
mass-testing
PCR
lateral flow
nasopharyngeal swabs
RNA detection
lateral flow evaluation
quick
50% accuracy
covid-19 lab findings
lymphophenia
ARDs
^LFTs
inflam markers
coagulation abnormalities
covid-19 complications
“silent” hypoxia
ARDs preceded by pneumonia > difficult recovery and pulmonary scarring
sepsis
secondary infections
Covid-19 treatment
hospitalization
remdesivir
remdesivir
viral RNA polymerase inhibitor
covid-19 threats
transmissibility
vaccine bypass
virulence
population control
virus characteristics for tools in medicine
easy to genetically manipulate/ modify
targeted delivery to specific cells
powerful gene expression
gene-carrying
can be attenuated
human attenuation
human viruses having lost properties to replicate efiiciently in human cells therefore not disease-carrying
non-human attenuation
non-human virus able to infect human cells w limited replication and some gene expression
gene expression in lab via viruses
- virus promoter cloned into DNA plasmid vector
- PCR generates cDNA from mRNA
pCDNA3.1
ori in bacteria
ori in human cells
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
transfection process
- isolate ORF of interest
- clone into expression vector
- isolate/ purify plasmid DNA
- transfect target cells and detect gene expression
why are viruses more efficient as vectors than chemical/ liposomal transfection?
virus-own entry mechanism
affect more complex cell lines
advantages of adenoviral vectors
small genome
easy to manipulate/ purify
infects many cell types
^ gene expression
disadvantages of adenoviral vectors
immunogenic
limited to <8kb ORF’s
transient expression
adenoviral gene vectors
mostly missing E1 gene region for replication
mostly missing all viral proteins
helper coat proteins required
adenoviral gene vector functions
can amplify virus vectors
^ yields
hit nearly all cells
modify promoter for cell-type specific gene expression
lentiviral vectors
commonly HIV1 only LTRs (promoters and polyA signals) packaged, no surface protein, other retroviruses used
virus supplied in trans
no gp160 attachment protein
lentiviral vector advantages
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
lentiviral vector disadvantages
<10kb limit
danger if inserted near to proto-oncogene
types of vaccine
live
killed whole virus
subunit
gene delivery
types of live vaccine
attenuated
heterologous
attenuated live vaccine process
- isolated and cultured virus on host
- incubation on cells from other host
- spontaneous growth and mutation on alternate host
- won’t grow on original host
subunit vaccine
components purified from whole virus
recombinant proteins
advantages of attenuated vaccine
mimic wild-type
strong humoral and cellular response
induces innate immunity
fewer doses required for complete immunity
disadvantages of attenuated vaccine
reversion can occur to wild-type
not viable for immunosuppressed
careful handling/ storage
virus can’t be cultured
virotherapy
replacement of defective gene function by introducing ‘normal’ gene
types of virotherapy
ex vivo (cells removed and reintroduced to body)
in vivo (replacement gene introduced directly into body via gene vector)
CAR-T gene therapy
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
oncolytic virotherapy
manipulation of lytic virus to destroy Cancer cells (not non-cancerous cells)
viral tropism change
causes inflammation, lysis and more tumour cell death exponentially
oncolytic
destroying tumour cells
oncogenic
promoting tumour cell development
oncolytic virotherapy barriers
vector neutralisation overcome
enriching T-cell response
^spread
^tumour uptake
oncolytic virotherapy advantages
combination with other cancer treatments
safe
overcomes resistance
dual mode of action
oncolytic virotherapy disadvantages
resistant cancer cells
antiviral immune response
limited replication and spreading
phage therapy
use of bacteriophage to kill bacteria
phage therapy advantages
specific to bacteria
can evolve to adapt resistance
self-amplifying until all bacteria dead
phage therapy disadvantages
individualised
cocktail of phage
theoretical potential for Ab resistance
g - lysis can release endotoxins (sepsis)
lysins/ endolysins
enzymes produced by some bacteriophages to cleave host cell wall in final stage of lytic cycle
> purified is more potent than antibiotics
> negligible resistance
uses of phage therapy
FDA approved phage therapy for P.aeruginosa in CF patients
trials for burn infections
biofilm treatments
2 methods of detection/ counting viruses in the lab
cell-based assays
protein-based assays
cell-based assays
plaque assay
tissue culture infectious dose assay (TCID50)
protein-based assays
haemagglutination assay
electron microscopy
immunofluorescence
enzyme-linked immunosorbent assay (ELISA)
functions of detection/ quantification of viable virus particles in lab
diagnosis
prognosis
research
virus growth in laboratory
grow cells in tissue culture
infect cells with virus
incubate infected cells
harvest cells
quantify virus yield
how to observe for effects of viral infection
cytopathic effect (CPE)
multiplicity of infection (moi)
number of infectious viral particles used to infect 1 cell
poisson distribution
chance of any 1 cell being infected by 0 or more viral particles
assumes normal distribution
moi importance
infection synchronization
different viral characteristics
stock viability testing
infection cellular response observation
plaque assay process
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
PFU
Plaque forming unit
plaque
clearing of the cell monolayer
plaque assay method to calculate viable virus concentration
use dilution factor/ amount of diluted virus stock added/ no. plaques produced
plaque assay limitations
virus may cause visible c.p.e
time required can be significant
sterility must be maintained
focus forming assay
plaque assay variation
no agar overlay
24h can fix and immunostain w flourescence antibody
ffu
focus forming unit
end-point dilution assay
simple
sequential dilution of virus stock in micro-titre plate (96 well)
multiple replicants of each dilution
count under microscope
TCID50
tissue culture infectious dose
virus concentration that kills 50% of cells in a culture system
infectious titre
endpoint dilution assay
TCID50 method
96 well plate format
serial dilution across plate
leave to infect and kill cells
score wells as infected +/-
TCID50 infection rate
1/2 cells per unit
TCID50 functions
drug development
allows non-plaquing virus quantification
TCID50 limitations
time-consuming
medium needs to be changed regularly
prone to drying out
sterility (incubation length)
electron microscopy types
scanning
transmission
electron microscopy
best for virus particle identification physically
compare to pfu
measure of particle to infectivity ratio
haemagglutination
measurement of both viable and non-viable virus particles
generic effect
uses viral ability to link RBC’s
relative quantification
how to quantify viral particles absolutely with haemagglutination?
compare results to a standard virus suspension containing known number of virus particles/ ml
immunofluorescence
stain virus antigens on cell surface/ in sections of virus-infected host cells
immunofluorescence process
add virus
wash cells to remove unabsorbed antibody
add FITC conjugated anti-rabbit IgG antibody
wash again
examine w UV microscope
FITC
fluorescein isothiocyanate
ELISA
enzyme linked immuno-sorbent assay
types of ELISA
direct (primary antibody conjugate)
indirect (secondary antibody conjugate)
sandwich (capture antibody)
ELISA pros
quicker than traditional titrations
in clinic (HIV )
can be related to viral titre w standards
ELISA pros
quicker than traditional titrations
in clinic (HIV )
can be related to viral titre w standards
