Clinical virology Flashcards
Types of infection
i) acute infection
ii) latent infection
iii) progressive infection
iv) chronic infection
Where is the next emerging virus going to come from?
animals
What is the one health concept?
Look into animal health and see how it interacts with human health
How can animals spread zoonotic virus to ppl?
i) direct contact
ii) vector (mosquito)
iii) indirect contact
iv) food
How to detect the level of desired DNA formed?
use probes
—> higher cycle threshold value is equivalent to less DNA —> less virus
What does high Ct value infer?
less nucleic acid —> fewer viruses
Molecular testing used for viral infection
q-PCR of RT-PCR (For RNA viruses)
Advantage of using PCR
i) able to test multiple viruses at once (multiplex)
ii) little hands on time
iii) gd turnover time
iv) available in many labs
v) lots of samples can be done at once
What does turnover time mean?
time needed to get the results
Drawbacks of using PCR for viral infection
i) costly
ii) cannot test for immunity (only test the virus)
iii) highly dependent on specimen quality
iv) need to go to reference centre for confirmation
Example of probes used in detect of DNA in PCR
- non specific (Sybr Green)
- specific ones (Taqman)
Steps to design PCR for viral infection
i) design primers to target specific region of virus
- target one or more viruses?
ii) include primers and probes (control)
iii)determine the sensitivity and specificity levels
iv) check with clinical samples
Why primers and probes are needed in PCR?
as a control to make sure the sample is not degraded
Why we need to check for specificity of PCR test designed?
make sure it specifically tst for this virus
—> not detecting other closely related virus
What can u do if u cannot get the samples u want for PCR?
test to see if other methods of getting samples can also be used for testing
Why is important to know viral genome?
used in diff. categories
- develop molecular diagnostics
- template for vaccine
- help with epidemiology studies
- understand viral evolution and transmission
Two types of whole genome sequencing
i) metagenomics
ii) amplicon based
Factors to consider when using sequencing
i) coverage (how much of the genome should be sequenced)
ii) depth (the level of detail in sequencing)
What happens if you prioritize depth for genome sequencing?
discover rare variants….
Major steps in whole genome sequencing
i) library prep (prepare DNA samples needed + tags to identify them)
ii) sample amplification
iii) get sequence through identifying signals
iv) bioinformatic analysis (see if it belongs to any known family)
How can bioinformatic analysis help in whole genome sequencing?
- characterize the virus
- reduce epidemiologic uncertainty
metagenomic vs amplicon based sequencing
i) amplicon one order the genome in order after sequencing
ii) metagenomic just sequences the whole thing without order
Why metagenomic sequencing is preferred over amplicon sometimes?
i) no info known to the virus
—> cannot do amplicon based sequencing
Prep before doing whole genome sequencing
i) generate cDNA or genomic DNA into smaller parts
ii) include barcodes (tags for multiplexing)
What are barcodes in wgs?
unique DNA tags to certain virus
how is barcodes used in whole genome sequencing?
tag DNA which allows sequencing of multiple types of DNA in same cycle
How does whole genome sequencing determines sequence?
i) records fluorophore signal at each position
ii) use this to identify and order the bases
—> each base have different color
How is WGS used in Covid?
i) identify different strains
ii) determine if patients are infected by same or different strains of Covid
What is serological testing?
detect specific antibody against infecting organism
What can serological testing used to identify viral infection?
see if it’s acute, recurrent or past infection depending on their ability to bind
advantage of serological testing
i) cheap and easy
ii) can go through lots of samples at once
drawbacks of serlogical testing
also indicate previous exposure, not just active infection
which antibody indicates acute infection
IgM
Which antibody indicates resolving infection
igG
Confounding factors for serology testing
i) antibody cross-reactivity
ii) co-infection
iii) immunosuppression
iv) pregnancy
What makes it hard to diagnose active infection through serological testing?
i) need to be able to differentiate between IgM and IgG
ii) depends on time where samples are collected
Types of antibody detection assays
i) direct assay
ii) indirect assay
iii) capture assay
In which virus can serology be used in clinical situaitons?
EBV, West nile virus, HIV, HBV, Hepatitis a, Hepatits E
Example of direct assay for antibody
CMV immunohistochemistry
–> only primary antibody bind to target and causes rxn
Example of capture assay
HIV p24 detection assay
–> have capture antibody + primary antibody bind to antiibody
Example of indirect assay
Anti-VZV IgG ELISA
–> use 2nd antibody for rxn that bind to 1st antibody
When does antibody appear in HIV infection?
peak during asymptomatic infection
–> start appearing around 3 weeks after infection
How does serological testing work for EBV?
testing different antibodies against different antigens
–> differentiate primary from past infections
In EBV, what shows that it is past infection?
positive for EBNA1 IgG
In EBV, what shows that it is primary infection?
negative in EBNA1 IgG
+ in VCA IgG and IgM
In EBV, what shows that it is seronegative?
– in EBNA1 IgG and VCA IgG
What does dual assay for HIV test for?
test for HIV IgG or IgM
plus HIV p24 antigen
Antibody progression for hAV
first IgM ard 5 weeks
then IgG keeps increasing
Advantage of using electron microscopy
i) don’t require viral specific reagents
ii) detectable virus = high viral load (Causative agent)
iii) virus directly detected from sample
iv) get insight into viral attachment
Process of electron microscopy
i) centrifuge samples to remove debri and bacteria
ii) place sample on grid/ undergo ultracentrifugation
–> negative staining with 2% uranyl acetate
What is virus stained with in EM?
2% uranyl acetate
Function of negative stain in EM?
view small details like spikes, envelope
morphology of varicella?
rectangular with shape, some coated with mucus
morphology of varioila
brick like shape, elemental bodies like vaccinia
Why do we use EM?
i) know morphology of virus –> similar to families
ii) viral attachment
iii) localization of virus (by using antibodies)
Four types of cell culture
i) primary cell culture
ii) semicontinuous cells
iii) continuous cell lines
iv) hematopoietic ceells
Features of semicontinuous cells
limited no. of passages
–> senescence
Feature of continuous cell lines
immortalized cell lines
—> unlimited passages
virus that use primary cell culture
HSV
virus that use semicontinuous cell culture
RSV
virus that use continuous cell culture
human coronavirus NL63
virus that use hematopoietic cells
HIV
why is cell culture important for understanding the virus?
i) test for vaccines, antivirals….
ii) characterize viral pathogenesis
iii) detemine which samples are infectious and how long is patient infectious
iv) link genotypic changes to phenotypic ones
v) help with cleaning and decontamination procedure
First type of cell lines to go for isolating virus
immortalized cell lines
What affects the results on cell culture
i)viral tropism
ii) cell lines used (some might not have cytopathic effect)
iii) viral entry, receptor expression level
iv) host response
signs of cytopathic effect in viral infection
inclusion bodies (mass of virions), multinucleated cells, syncytia
how to monitor cytopathic effect in infected cells
with light microscope
how does rapid cell culture work?
i) put specimen with cell monolayer in shell vial
ii) centrifuge to enhance infection
iii) immune antibodies are present to detect virus presence
How much does centrifugation in rapid cell culture shorten the time for positive culture
weeks to 24-72 hrs
Technique sometimes used with rapid cell culture
Direct immunofluorescent antibody staining (DFA)
How does direct immunofluorescent antibody sensing work?
use virus specific antibody with fluorchrome
–> see under fluorescent microscope
–> able to detect virus even if CPE not present
What is TCID50?
50% of wells show CPE
–> 50% tissue culture infectious dose
How to perform TCID50?
i) dilute samples with different dilutions
ii) add them to confluent cells
iii) visualize with dye or immunohistochemical methods for CPE
What is TCID50 used for?
quantify the amount of virus present in sample
How does plaque assay work
i) make 10fold dilutions
ii) add dilution to cell monolayer
iii) remove dilutions and overlay with agarose + media
–> each plaque = 1 virus
What is multiplicity of infection?
ratio of infectious virions to cells present
–> higher –> more cells infected
Pros of using TCID50
i) easier to test more samples
ii) don’t need agarose overlay
iii) can use more automated methods to evaluate cytopathic effect
Pros of using plaque assay
i) size of plaque an indicate virulence (larger –> more virulent)
ii) gold standard for isolating a purified virus
Con of using TCID50
i) cannot identify more virulent clones
ii) cannot purify a single virus
Con of using plaque assay
i) hard to use in high containment labs when agarose overlay is needed
ii) more labor intensive (counting plaques)
iii) not able to test many samples at a time
Method to measure viral growth
1 step growth curve
What happens if use different cell lines to measure viral growth
growth curve looks different
–> cells express different receptor levels or other host factors
How does 1-step growth curve work?
i) add a certain virus conc into cells
ii) collect cells and supernatant at mutliple time pts
iii) evaluate viral titers with TCID50 or plaque assay
iv) see when viral replication reaches plateau
What cell lines are used as the first to test with new virus
Vero cells
–> lack IFN response
How does plaque reduction neutralization test work?
i) dilute antibodies and add to virus
ii) add mixture to cell layer and overlay with agarose _ media
iii) see which dilution has 50% less plaques than antibody free control
what is plaque reduction neutralization test used for?
test the effectiveness of antibodies or antivirals against the virus
