Week 3 - Lecture 1 & 2 Flashcards
What are the purposes of viral disease diagnosis?
• Surveillance of viral diseases among certain population
• Identify the causative agent of certain suspected clinical cases of viral origin
• To monitor the progress of some viral diseases
• To monitor the antigenic/genetic variations of certain virus
• To help in the design of the right vaccine against the homologous circulating strains of viruses
What is the direct approach to viral diagnosis?
Identifying the virus or viral products (such as proteins, nucleic acids) in clinical samples or after virus isolation from clinical samples.
What is the indirect approach to viral diagnosis?
Detecting an immunological response to the virus (detect antibodies)
What tests can be used to detect a virus and the virus’ effect on cells?
**Make more **
Electromicroscopy (TEM, SEM), Immunoelectronmicroscopy, Light microscopy
skipped
Make questions from this chart
I-Diagnosis of viral infections at the individual animal or individual Herd level
- Management of the animal or its prognosis is influenced by the _______
- ______ and _______ diagnosis of the causative virus can be the basis for establishing the _______ plan (?)
diagnosis, Rapid, accurate, management, Biosecurity, vaccination, antimicrobial treatment
II- Certification of freedom from specific viral life long infections or proof of vaccination (BLV, BVDV, EIAV)
-These certificates or vaccines are mandatory for animal _______, participation in certain ______, or show for ____
-Artificial ________, _____ transfer, and blood ______
-Male used for _____ collection, female used for _____ transfer, blood _____ animals should be screened for wide range of viral infections (EAV, EIAV, BLV, etc)
- _______ viruses: (RVfV, Rabies, WNV, EEEV, Hendra virus, etc)
- All these animals require _______ and _______ as well as veterinary care
traveling, exhibition, sale, insemination, embryo, transfusion, semen, embryo, donor, Zoonotic, screening, testing
III- Diagnosis of virus infection at the State, country and International level
- ____ and _____ programs for some viruses such as (MDV, EIAV, BHV-1, BVDV)
-Surveillance programs in support to _______ diseases research control activates
-Surveillance programs in support to ______ diseases research control activates
Test, removal, enzootic, exotic
IV- Prevention of new emerging and re-emerging diseases
?
If it is a respiratory virus, what types of specimens would you collect antemortem?
If it is a respiratory virus, what types of specimens would you collect postmortem?
If it is an enteric virus, what type of samples would you collect antemortem?
If it is an enteric virus, what type of samples would you collect postmortem?
If it is a genital virus, what type of samples would you collect antemortem?
If it is a genital virus, what type of samples would you collect postmortem?
If it is a nervous system virus, what type of samples would you collect postmortem?
If it is a nervous system virus, what type of samples would you collect antemortem?
If it is a systemic virus, what type of samples would you collect antemortem?
If it is a systemic virus, what type of samples would you collect postmortem?
What should be considered during sample collection?
-Proper site of collection
- Right time
-Suitable volume/quantity
What should be considered during transportation of viral samples?
-Viral transport media (VTM)
-Antibiotic/antifungal cocktail
-Sterile containers
What should be considered for sample preservation?
-Proper preservation temperature
-Avoid freezing and thawing
Light microscopy is used to/for:
1. Monitor the ____ and ______ of cell culture
2. Monitor the viral infection in ____ ______ (____)
3. Detect ____ ______ ______ (TBDL)
4. Study the _______ changes of some viral infected ____
5. Immuno-histo-chemistry: ____ virus antigen (___ ____)
growth, multiplication, cell culture, CPE, viral inclusion bodies, histopathological, tissues, Rabies, brown dots
In most other cases viruses are _______ in cell culture before they can be visualized by ____
isolated, EM
Virus is surrounded by ______ ____ (electron ____) material, including: ?
This results in electrons being ______ from regions covered with stain thus creating a _____ which outlines viral _____
electron, dense, opaque, -2% uranyl acetate, 1-3% sodium or potassium tungstate, scattered, contrast, structures
What are the advantages of EM in diagnostic virology?
-Can detect the virus in body _______ and _______
-Do not require special _____ such as proteins standard, etc
-No ____ reaction with other ____ viruses
- _____ technique
secretions, excretions, reagents, cross, similar, Rapid
What are the disadvantages of diagnostic virology?
- ______ sensitive than other tests: requires ____ virus concentration
-The EM machine is ______
-Requires ______ _______ to do interpretation
Less, high, expensive, expert personnel
Scanning electron microscope uses a ________ resolution of tens of nm.
lower
Scanning electron microscope shows only _______ of specimens.
morphology
Scanning electron microscope is a ________ way to prepare specimens.
simple
Scanning electron microscopy is ______ and relatively _____ to use.
cheap, safe
Transmission electron microscope uses a ___________ resolution of ?
higher, 1nm or less
Transmission electron microscope shows multiple ________ of objects such as ________, _________, _____, and many more.
characteristics, crystallization, morphology, stress
When using transmission electron microscope, you must prepare your specimen by ________ your sample which is ______ and _______ consuming.
thinning, tiring, time
Transmission electron microscope is ________ and relatively _______ to human health.
expensive, harmful
What does this image show?
Transmission EM- SARS-CoV-2
Spike proteins are shown as protrusions from the surface of the virus and attach to the host cell receptors
virus is circulating and spikes embeds and attaches to receptor. Based on this –> develop vaccine, antiviral therapy, diagnosis, etc.
What does this image show?
EM
- one of the drawbacks is less viral sensitivity; need a large sample
What does this image show
Virus + Abs
IEM (Immuno-electron microscopy)
Serum specific to virus to clump viral particle together so you can better see the virus.
Ab captures virus -> clumps together
kind of modification to overcome one of the drawbacks to EM
Immuno-electron microscopy (IEM) is the _______ of viral-____ _______ will allow the ______ of virus particles that can be seen under EM easily.
addition, specific antibodies, concentration
Viral Diagnostic-Direct method-Virus isolation
1. Nasal swabs (in ________ media containing ______)
2. _____ (homogenize), ____ coat cells and _____
- Make __- ___% solution in cell culture medium – sonicate* homogenized tissues and buffy coat cells or freeze/thaw to ___ the cells (release the virus)
•_______ (3,000X g) or filter (0.2 µ)
- In most cases inoculation of sample (supernatant/ filtrate) onto cells of tissue culture – incubate at 37˚C in a CO2 incubator
* Feces – no sonication
transport, antibiotics, Tissue, buffy, feces, 10, 20, lyse, Centrifuge
**What are the methods of virus isolation?
Embryonated chicken egg innoculation (ECE)
Cell culture
Laboratory animals inoculation
What are the routes of inoculation for chicken eggs?
- Yolk sac
- Amniotic sac
- Allantoic Sac
- CAM Corio??
Each virus req. and likes certain routes compared to others.
Like/love = tropism
What are the types of cell culture used for virus isolation?
- Primary cell culture
- Secondary cell culture
- Established cell line
What are the routes of inoculation for laboratory animals?
- IM
- IV
- IP
- SC
- ID
ECE
we need to get a fertile egg.
Embryo with different tissues, live tissue, that can support viral repliction.
We need to candle egg to see if fertile or not.
If see B = live eg, C = can not support viral rep
Clean egg with 70% isopropol alcohol before puncturing
Describe the process of inoculating the Chorioallantoic Membrane (CAM) of a chicken egg.
Quite difficult, but very common
** Use older embryos (10 - 12 days)**
1. Drill hole at air space and then drill 2nd hold at top
2. Apply suction to 1st hole, then inoculate 0.1 ml of sample (to be tested) with syringe and needle in the 2nd hole.
3. Incubate & look for membrane edema or focal necrosis
4. Harvest the CAM
5. Preparation of artificial air sac
CAM is best to isolate Pox and herpesvirus
suctioning separates membranes so you can insert needle into CAM
List the methods of harvesting inoculated materials
• Open the inoculated eggs with sterile scissors
• Remove the egg shell and the egg shell membranes
• Pour the content of the egg into a clean, sterile petri dish
• Examine the inoculated embryos and the embryonic
membranes
List and describe the pathological changes of the virus on the ECE.
1-Curling: and dwarfing of embryos: IBV
2-Death: of the embryo: some viruses induce the death of the embryo such as NDV
3-Deformities: dwarfing, and hemorrhage of the embryo such as IBV
4-Hemorrhage: and thickening of the Chorioallantoic membranes as in case of Pox and Herpesviruses
5-Detection of hemagglutinin: in the egg fluids as in the case of NDV and AIV, which can be detected by the HA test.
Pock lesions are ________, _________ foci of _____ on the surface of _____. Varies in shape and size.
circumscribed, rounded, necrosis, CAM
The term pock is designated to the _____, _____, ______ ____ resulting from virus multiplication of the ____ as in the case of ____ and ______.
These ______ foci vary in ____ (large, medium, large), _____ (circular, oval, irregular). It may with _____ or ______ centers and may be ____ in some
cases.
white, opaque, necrotic, foci, CAM, Pox, Herpesviruses, necrotic, size, shape, raised, depressed, hemorrhagic
Pock lesions
Inclusion bodies are viral components (most likely _______) accumulated at the site of virus _________. They produced due to some replication/maturation process of viruses inside the infected cells.
proteins, multiplication
The position of the inclusion bodes depends on the ____ of viral ______ either in the _____ or in the _______.
site, assembly, nucleus, cytoplasm
List some examples of viruses with Intracytoplasmic IBs
• Rabies: Negri bodies
• Small pox- Gaunielr bodies
• Fowl pox Bollinger bodies
List some examples of viruses with Intracytoplasmic and intranuclear IBs
• Canine distemper virus
• Measles
List some examples of viruses with intranuclear IBs
• Adenovirus
• Togavirus
• Herpesvirus
Intracytoplasmic IB
Intracytoplasmic IB
Intracytoplasmic IB
Rabies: Negri bodies
Intracytoplasmic and Intranuclear IB
Intranuclear IB
Describe synctium formation.
Virus infects cell —> fusion of two cells —> multinucleated giant cell —> synctium formation
This is a way for the virus to spread to adjacent cells
Primary cell culture is made up of cultured cells that are derived ______ from _____ (often ______ ______).
directly, tissue, embryonic tissue
What are the advantages of primary cell culture?
Advantages: cells have not been “modified” in any way
What are the disadvantages of primary cell culture?
limited lifespan of the culture potential contamination problems
Established cell lines are made up of ______ cell types ______ maintained in the _____ (ie, in vitro) for scientific purposes.
A population of _______ cells, of _____ origin,
undergone a change allowing the cells to grow ______.
specific, artificially, laboratory, cultured, animal, indefinitely
What are the advantages of an established cell line?
• Advantages cells grow indefinitely
What are the disadvantages of an established cell line?
• Disadvantage not all viruses replicate well in
cell lines
Several ways in order to make individual cells:
Manual with scissors or by enzymes (trypsin, etc) dissolve the intracellular junctions. Put cells in incubator + anti bacterial + antifungal cocktail + 37 —> make sheet.
Subculture = do this in case of primary cell culture up to 10x. In case of tumor cell we can do it indefinitely.
List some examples of common cell culture media
Describe preparing primary kidney cell culture
Cut tissue with scalpel
Cut into small pieces
Make cell juice
Count number of cells
Calculate number of cells in each flask
Incubate in CO2 incubator
Look under microscope to make sheet
What are the causes of CPE?
1-Effect of virus on the cell membrane during _____ or ______.
2-Effect of the virus on the host cells ____, _____ or ______
3-Transformation of ____ cells by some viral _____.
4-Accumulation of the ____ proteins and the new _____ viruses.
attachment, release, DNA, RNA, proteins, host, genomes, viral, progeny
What are some examples of CPE in cell culture?
1-Cell lysis: FMDV
2-Cell rounding: IBDV on Vero Cell line
3-Giant cells : Muti-nucleated cells: Syncytial formation: NDV,
Ex:Avian Reovirus
4-Transformation of cells: MDV, Avian Leucosis
5-Haemadsorption: Influenza, PI-3
A = area of necrosis
True or False: Not all viruses cause CPE.
True.
IC = intracerebral
IN = intranasal
There are different routes for innucleation of lab animal.
Virus induces some pathological changes in innucleated animal —> mass on neck due to accidental trauma or virus producing lesion
Virus can also cause paresis
List and describe the factors that affect hemagglutination test principles.
1-Temperature: Each virus require certain temp for Hemeagglutenin (clumping together)
-NDV: 37 ºC
-Enterovirus: 4 ºC
2- PH
-NDV: PH (7.3)
-Enterovirus PH(5.8)
3-Salt concentration: NaCl: important for binding of
virus & RBCs
4-Presence of non-specific inhibitors in sera
RBC and virus clump together —> lattice formation
HA is not a serological test - serologic test has serology; it is an HA test
Serial dilution
Fixed amount in each
Start with 100% of virus, second tube 50, 25, 12.5, 6.25, and so on.
titer = 8
Same as Hemeagglutination but on cell culture.
Infected call; HA projections on cell. If you add ?, causes clumping or hemeagglutination.
In the AGIDT or AGIP test, both soluble ______ and _____ interact in an aqueous solution to form a _____ (insoluble visible precipitate)
antigens, antibodies, lattice
Gold standard in detecting equine infectious anemia virus
Coggins test
Make rosette = puncturing = one central well
Antigen is in the center well and the serum samples in peripheral wells
Incubate overnight
AG and AB interact. When specific to one mother, there is a line of precipitation
Different possibilities for this test
Picture on right = spur formation
ID these figures
In the process of radial immunodiffusion, _____ solution diffuses into ____ in which a specific _____ has been incorporated, and a ____ of _____ will form around the antigen well. The ____ of this ___ is ______ to the amount of antigen in the well.
antigen, agar, antiserum, ring, precipitate, area, ring, proportional
What is the principle of the complement fixation test (CFT)?
Principles: Complement that fixed by Ag/Ab reaction
thus, lysis of RBCs will not occur.
In the absence of a specific Ag/Ab reaction or absence of
either Ag or Ab, the complement is free and lyses the
RBCs
What are the principles of the fluorescent antibody techniques (FAT)?
Direct FAT =
- viral infection in tissue
We need to know if this tissue is infected with virus or not
Antigen inside tissue + fluorescent AB specifics to this antigen
Take reaction under fluorescent microscope
Indirect FAT =
- sometimes you take antibodies from swine and inject it into rabbit
AB can act as antigen if infected into other species
Serum sample from pigs inject into rabbbit. Rabbit will detect it as antigen even though it is antibody. Anti=species gl;o Bulin. Use anti species globulin against any pathogen of swine origin.
AB from a ny species —> put in another host —> immune system of second host deals with AB and antigen -> makes AB. Withdraw aB and use It as anti species globulin
Swab sample —> buffer —> well
HA inhibition = Ab specific to virus + Ab + RBC. RBCs settle down. based on presence of serum. Serum inhibits viral agglutination
HA = only virus and RBCs. if virus specific to RBC = clumping
Describe qualitative ELISA
Positive or negative results
Describe quantitative ELISA
The optical density (OD) of the fluorescent dye is
directly proportional to the concentration in the Ag or
Ab in the tested samples
Each enzyme has a substrate. Enzyme + substrate —> hydrolysis —> color produced
Intensity of color is directly proportional to the antigen concentration.
Use ELISA ready to measure optical density.
Turns intensity of color into number
Seroconversion: development of detectable ______ ___ to microorganisms in the blood ____ as a result of _____ or ______
specific, Abs, serum, infection, immunization
With seroconversion, collection of __ ____ samples (____ and ______) with __ weeks apart
•If the convalescent sample is greater than the acute sample with t least 4 folds: _____ _____.
2 serum, Acute, convalescent, 4, Recent infection
What are the principles of polymerase chain reaction?
Principles: A reaction that uses the enzyme DNA polymerase to
catalyze the formation of more DNA strands from
an original one by the execution of repeated cycles of DNA
synthesis
What are the main steps of PCR amplification?
Main steps in PCR amplification:
1- Denaturing of ds- DNA template
2- Annealing of the primer
3- Extension of ds-DNA molecules
How do you visualize PCR products?
Gel electrophoresis
DNA gel
Sequencing
What are the advantages of PCR?
- Highly sensitive can detect one copy of the viral genome per sample
- Easy to set up
- Fast time (close to 2 hrs)
What are the disadvantages of PCR?
- Prone to contamination
- Requires high skilled personnel to conduct the experiment
- -Requires optimization of all parameteres in some cases
(annealing temp, primer design, etc) - -Difficulty in the interoperations os some positive results especially the latent infection with some viruses
Virus quantification: counting the _____ of viruses in a specific _____ to determine the virus ________.
number, volume, concentration
An example of a Physical Quantitative assay is?
Counting of viral particles by EM
II- Biological assays (TBS)
- ____ assay
- _____ assay
- _____50
III- Quantitative Assays :
-_______ _____ Reaction (PCR)
-_____ and ___-PCR
HA, Plaque, TCID, Polymerase Chain, qPCR, qRT
Real-Time PCR a specialized technique that allows a PCR reaction to be visualized “___ ____ ____” as the reaction progresses
• As we will see, Real-Time PCR allows us to measure _____ amounts of DNA sequences in a sample!
in real time, minute
produces a graph, not gel, earlier in the graph = greater the concentration?
What is real time PCR used for?
- Diagnosis/Detection
- Quantification
- Analysis
How does real time PCR help you diagnose/detect a viral infection?
Use specific primers and probes to amplify desired viral gene in your samples.
To diagnose certain viral infection based on some conserved viral genes amplification using specific
probes and primers.
How does real time PCR help you quantify your sample?
To measure the concentration of the target
virus/gene in a given sample based on the concentration/load of the virus/gene in the tested
samples
How does real time PCR help you analyze your sample?
To analysis the variants by studying the
melting curve of or comparing the temperature with the known sequences in the databases
Plaque assay:
Measures viral _____, specifically the ______ of the virus
______ dilution –> Pink plate = Cell culture in well + infected them with ___
Restrict ______
_ of virus before end of replication cycle by adding ___. Each virus replicating inside the cell does not have access to leave and go into another cell. Each dot represents population of ___ virus replicating in cell culture. From here, calculate the _____ of viruses.
particle, infectiousness
Serial, virus, movement, agar, one, number
In a virus neutralization assay/serum neutrailization assay, the presence of ______ against a ____ virus can be detected by the ______’ ability to prevent ______ effects (CPE)* of the reference virus in cell cultures
Most informative _______ assay about “Protective _______”
Important tool used to characterize “ ________ ”
antibodies, known, antibodies, cytopathic, serological, Antibodies, serotypes
This assay detects antibody that is capable of inhibiting virus replication (or in other words, antibody that can neutralize virus infection). Virus neutralization is a specialized type of immunoassay because it does not detect all antigen–antibody reactions. It only detects antibody that can block virus replication.
List and describe the steps of a Viral Neutralization Assay
- Serially dilute serum 1/2 1/4 1/8 1/16……….1/512
- Add equal amount of virus (100 plaque forming units) to each tube-incubate 1 hr. - infect cultured cells- incubate at 37 ̊C for plaque formation.
Reciprocal of the highest dilution that can prevent ___% _____
formation is the serum ____.
50, plaque, titer
Acute vs. Convalescent serum
_____- fold rise in neutralization titer of ______ serum relative to acute serum indicates ______ (virus ______)
Four, convalescent, seroconversion, infection
What does tissue culture of infectious doses (TCID-50) measure?
• Measure the CPE other than the lysis
• Concentration of a virus is taken to produce CPE in 5p% of the inoculated cell culture vessels
Embryonic infectious dose 50/cell culture
infect cell culture dilution with virus
Count CPE: +=presence of CPE, - =no presence of CPE
Do 50% in order to have a standard/fixed unit.
A = area of necrosis
