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Veterinary Virology > Diagnosis of Viral Diseases > Flashcards

Diagnosis of Viral Diseases Flashcards

1
Q

What are 5 purposes of viral disease diagnosis?

A
  1. surveillance of viral diseases among certain populations
  2. identify the causative agent of suspected clinical cases of viral origin
  3. monitor the progress of some viral diseases
  4. monitor the antigenic/genetic variations of certain viruses
  5. help in design of the right vaccine against the homologous circulating strains of viruses
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2
Q

What are the 2 approaches of viral diagnosis?

A
  1. DIRECT: identifying the virus or viral products in clinical samples or after the virus is isolated from the samples
  2. INDIRECT: detecting an immunological response to the virus and detecting antibodies (seeing the effect of the virus)
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3
Q

What are 6 common strategies to viral diagnosis? How is each done?

A
  1. detection of virus and viral effect on cells - EM (TEM, SEM), IEM, LM + histopathology
  2. isolation of virus - embryonated chicken eggs, lab animals, cell culture
  3. detection of viral antigens - IFA, ELISA, hemoagglutination test
  4. detection of viral antibodies - hemoagglutination inhibition test, ELISA, SNT
  5. virus infectivity titration - plaque assay, TCID50, LD50
  6. detection of viral genomes - PCR, qPCR
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4
Q

What antemortem and postmortem samples should be taken for respiratory, enteric, genital, nervous, and systemic/generalized manifestations?

A

RESP: AM = nasal/oropharyngeal swab, tracheal wash, whole blood/sera
PM = tissues from respiratory organs and regional lymph nodes
ENTERIC: AM = rectal swab, feces, whole blood/sera
PM = intestinal tissues
GENITAL: AM = semen, vaginal swab, whole blood/sera
PM = tissues from genital tract and testis
NERVOUS: AM = nasal swab, spinal fluid, blood
PM = brain tissue
SYS/GEN: AM = nasopharyngeal swab, urine, blood
PM = tissue samples from liver, kidney, spleen

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5
Q

What needs to be considered for sample collection, transportation, and preservation?

A

COLLECTION: proper site of collection, right time, suitable volume/quantity

TRANSPORTATION: viral transport media, antibiotic/antifungal cocktail, sterile containers

PRESERVATION: proper temperature, avoid freezing and thawing

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6
Q

In what 5 ways is light microscopy used in diagnostic virology?

A
  1. monitor the growth and multiplication of a cell culture
  2. monitor the viral infection in cell culture (CPE)
  3. detection of viral inclusion bodies (TBDL)
  4. study the histopathological changes of some viral infected tissues
  5. immunohistochemistry —> Rabies
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7
Q

How are viruses prepared for electron microscopy?

A

isolated in a cell culture and then surrounded by electron-dense (opaque) material (uranyl acetate, sodium, potassium tungstate), allowing electrons to scatted from regions covered with stain, which creates a contrast that outlines viral structures

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8
Q

What are 4 advantages and 3 disadvantages to using EM in diagnostic virology?

A
  1. can detect the virus in body secretions and excretions
  2. doesn’t require special reagents
  3. no cross reaction with other small viruses
  4. rapid
  5. less sensitive (needs high virus concentration)
  6. expensive
  7. requires expert personnel for interpretation
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9
Q

How does scanning EM and transmission EM compare in resolution, picture, specimen preparation, expense, and safety?

A
  • SEM has a lower resolution than TEM
  • SEM only shows morphology; TEM shows crystrallization, morphology, stress, etc.
  • easy to prepare specimen for SEM; TEM requires specimen to be thinned (tiring, time-consuming)
  • SEM is much cheaper than TEM
  • SEM is much safer than TEM
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10
Q

How are viruses stained to observe under EM?

A

negative scanning - briefly applying a heavy metal salt solution to a sample on a TEM grid in an attempt to surround the virus with dense material without infiltrating the virus

not as helpful for turkey coronavirus 10

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11
Q

TEM of SARS-CoV-2:

A

spike proteins = protrusions from surface of the virus that allows it to attach to host cell receptors

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12
Q

What is immuno-electron microscopy (IEM)?

A

the addition of viral-specific antibodies that allow the concentration of virus particles, allowing it to be seen under EM easily

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13
Q

What is unique in the isolation of viruses from fecal samples?

A

they are not sonicated (ultrasonic vibrations)

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14
Q

What are the 3 methods of virus isolation?

A
  1. embryonated chicken egg innoculation (ECE) - yolk sac, amniotic sac, allantoic sac, CAM
  2. cell culture - primary cell culture, secondary cell culture, established cell line
  3. lab animal innoculation - IM, IV, IP, SQ, ID administration
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15
Q

How can you tell an egg has a live embryo in it?

A

put it over a light and if you can see blood vessels, the egg is live and able to be used for viral isolation

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16
Q

How are embryonated chicken eggs inoculated?

A

a needle is used to insert the virus into the chorioallantoic membrane, allantoic cavity, amniotic cavity, or the yolk sac and left to develop —> changes in the embryo based on infection are observed

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17
Q

What is the most common way to inoculate viruses in embryonated chicken eggs? What are the 4 steps of this process?

A

on the chorioallantoic membrane (CAM) —> can be difficult

  1. hole is drilled at the air space and a second one is drilled at the top
  2. suction is applied to the first hole and 0.1 mL of the sample is inoculated into the second hole
  3. incubate and look for membrane edema or focal necrosis
    4 harvest CAM
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18
Q

What are the 4 steps of harvesting inoculated embryonated chicken eggs?

A
  1. open inoculated eggs with sterile scissors
  2. remove the egg shell and membranes
  3. pour the content of the egg into a clean, sterile petri dish
  4. examine the inoculated embryos and embryonic membranes
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19
Q

What 5 pathological changes can be expected in inoculated embryonated chicken eggs?

A
  1. curling and dwarfing of embryos
  2. death of the embryo
  3. deformities
  4. hemorrhage and thinkening of chorioallantoic membranes
  5. detection of hemagglutinin in the egg fluids, as detected by the HA test
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20
Q

What are pock lesions?

A

circumscribed, white-opaque, rounded foci of necrosis on the surface of CAM of various shapes and sizes, characteristic of Poxvirus and Herpesvirus multiplication
- can be raised or depressed
- can be hemorrhagic

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21
Q

What are inclusion bodies?

A

viral components (proteins) accumulated at the site of virus replication/maturation - intranuclear or intracytoplasmic - inside of cells

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22
Q

Give 3 examples of viruses that cause the formation of intracytoplasmic inclusion bodies. 3 intranuclear? 2 both?

A

INTRACYTOPLASMIC:
1. rabies - Negri bodies
2. small pox - Gaunielr bodies
3. fowl pox - Bollinger bodies

INTRANUCLEAR:
1. adenovirus
2. togavirus
3. herpesvirus

BOTH:
1. canine distemper virus
2. measles

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23
Q

What is syncytia formation?

A

virus-induced cell fusion that facilitates the transfer of viral genomes to the neighboring cells
when the number of fusogenic proteins decorating the infected cell is sufficient to engage receptors on neighboring cells, cell–cell fusion may be triggered

24
Q

What’s the difference between primary cell cultures and established cell lines?

A

PRIMARY CELL CULTURES: cultured cells that are derived directly from tissue (often embryonic tissue)

CELL LINES: specific cell types artificially maintained in the lab for scientific purpose - a population of cultured cells or animal origin undergo a change, allowing the cells to grow indefinitely

25
Q

What is an advantage and disadvantage to primary cell cultures and established cell lines for virus isolation?

A

PRIMARY CELL CULTURE
- advantage: cells have not been modified in any way
- disadvantage: limited lifespan of the culture and potential contamination

CELL LINES
- advantage: cells grow indefinitely
- disadvantage: not all viruses replicate well in cell lines

26
Q

How are primary cell cultures prepared? How are subcultures prepared?

A
  • a piece of the tissue of interest is sectioned off
  • enzymes break down embryonic tissue into separate cells
  • those cells are put in a nutrient-rich medium
  • with time the cells grow and multiply, filling up the dish/flask and attaching to one another
  • the resulting mass of cells are broken down by more enzymes and the process is repeated
  • can only be done about 10 times
27
Q

What are 3 common cell culture media used in primary cell cultures?

A
  1. Dulbecco’s modified eagle medium (DMEM)
  2. Roswell Park Memorial Institute-1640 (RPMI)
  3. Ham’s F12 nutrient mixture (F12)
28
Q

What is a cytopathic effect (CPE)? What are 4 causes?

A

structural changes in host cells that are caused by viral invasion, causing lysis or cell death

  1. virus affects the cell membrane during attachment or release
  2. virus affects host cell’s DNA, RNA, or proteins
  3. transformation of host cells by some viral genomes
  4. accumulation of viral proteins and new progeny viruses
29
Q

What are 5 examples of the cytopathic effect (CPE) in cell cultures? Do all viruses cause CPE?

A
  1. cell lysis
  2. cell rounding
  3. giant cell formation (multi-nucleated cells, syncytial formation)
  4. transformation of cells
  5. hemabsorption

NO

30
Q

CPE:

A
31
Q

What is the hemagglutination test?

A

applies the process of hemagglutination, in which sialic acid receptors on the surface of red blood cells (RBCs) bind to the hemagglutinin glycoprotein of the virus and create a network, or lattice structure, of interconnected RBCs and virus particles

positive result = clumping of RBCs, not a small pellet in the bottom of the tube

32
Q

This is a hemagglutination assay. Which dilutions show a positive result? Negative? What is the titer?

A

POSITIVE: 1-3
NEGATIVE: 4-7
CONTROL: 8, no virus

titer is the last positive result = 8

33
Q

Are hemagglutination assays considered a serology test? Why?

A

NO - there is no serum involved and no antibodies present

34
Q

What 4 factors affect hemagglutination test results?

A
  1. temperature
  2. pH
  3. salt concentration (NaCl is important for binding of virus and RBCs
  4. presence of non-specific inhibitors in sera
35
Q

What is the hemadsorption test (HAd)?

A

adherence of red blood cells to the surface of cells infected by cells in a culture - typically infected cells will exhibit hemagglutinin on their cell membrane and normal ones will not

36
Q

What is the agar gel immunodiffusion test? What is the precipitation line represent?

A

wells are punctured in agar gel and an antigen is placed in the middle well, surrounded by 3 wells with serum containing antibodies and 3 wells with negative sera

antibodies and antigens being attracted to one another

37
Q

What are the possible results in agar gel immunodiffusion test? What is a common test using this type of identification?

A

line of identity = positive, spur formation
line of partial identitiy = antigenic relationship
line of non-identity = negative

Coggins test: gel diffusion test used to detect antibodys for the equine infectious anemia virus in horse sera

38
Q

What is radial immunodiffusion?

A

antigen solution diffuses into agar in which a specific antiserum has been incorporated and a ring of precipitate will form around the antigen well - the area of the ring is proportional to the amount of antigen in the well

39
Q

What is the complement fixation test (CFT)?

A

in the absence of a specific Ag/Ab reaction (presence of virus), the complement is free and will lyse RBCs in a sample

positive = no RBC lysis
negative = RBC lysis

40
Q

What is the difference between direct fluorescent antibody test vs the indirect fluorescent antibody test?

A

DIRECT: a primary antibody tagged with a fluorescent ion will bind to a specific antigen and fluoresce

INDIRECT: a secondary antibody tagged with a fluorescent ion will bind to a specific antibody that has already bound its antigen target and fluoresce

41
Q

How does immunochromatography work?

A
  • a sample containing antigen (saliva) is applied to a sample pad
  • antigens bind to antibodies on the conjugate pad
  • antigen-antibody complexes are immobilized further down by antibodies in the antigen capture pad, showing up as a colored line
  • unbound/excess reagents pass through

(COVID rapid antigen test)

42
Q

What is the hemagglutination-inhibition test?

A

serum containing antibodies, the virus, and RBCs are placed in a tube and serially diluted - the antibody should bind to the virus and keep hemagglutination from happening, resulting in RBCs settling to the bottom of the tube and forming a pellet

43
Q

What is ELISA? What are the 2 types?

A

enzyme-linked immunosorbent assay

  1. qualitative ELISA - positive or negative result
  2. quantitative ELISA - optical density (OD) of the fluorescent dye is directly proportional to the concentration of Ag or Ab in the tested sample
44
Q

What are the 5 steps of the direct ELISA sandwich test?

A
  1. polystryene plate is coated with primary capture antibody specific to a viral antigen
  2. specimen (virus) binds to the antibody
  3. primary enzyme-labeled antibody binds to viral specimen
  4. enzyme substrate is added and converted into a detectable form
  5. color change is induced for a positive reaction
45
Q

Positive ELISA:

A
46
Q

What is seroconversion? How is it measured and how are results interpreted?

A

development of detectable specific antibodies to microorganisms in the blood serum as a result of infection or immunization

  • collection of 2 serum samples 4 weeks apart: acute, then convalescent
  • if the convalescent sample is has a higher antibody titer than the acute sample of at least 4-fold, it indicates a recent infection
47
Q

What is polymerase chain reaction (PCR)? What are the main 3 steps?

A

reaction that uses DNA polymerase to catalyze the formation of more DNA strands from an original one by the execution of repeated cycles of DNA synthesis

  1. denaturing of dsDNA template
  2. annealing of the primer
  3. extension of dsDNA molecules
48
Q

How are PCR products visualized?

A
  • new amplified DNA samples are isolated and put into a gel electrophoresis apparatus with a dye
  • once this has run, dyed bands will run down the gel according to their size and can be seen with UV light
  • bands can be sequenced and confirmed
49
Q

What are 3 advantages to using PCR? 4 disadvantages?

A
  1. highly sensitive and can detect one copy f the viral genal per sample
  2. easy to set up
  3. fast
  4. prone to contamination
  5. requires skilled personnel
  6. requires optimization of all parameters (annealing temperature, primer design)
  7. difficulty in the interpretations of some positive results, especially latent infection
50
Q

What is virus quantification? What are 3 ways this is done?

A

counting the umber of viruses in a specific volume to determine the virus concentration

  1. PHYSICAL ASSAYS: counting viral particles by EM
  2. BIOLOGICAL ASSAYS: HA assays, plaque assay, TCID50
  3. QUANTITATIVE ASSAYS: PCR, qPCR, qRT-PCR
51
Q

What is real time PCR (rtPCR)? What does it allow the measurement of?

A

specialized PCR technique, where a fluorescent tag on the TaqMan probe begins to fluoresce when it is displaced from the DNA molecule by DNA polymerase that is in the middle of replicating

measurement of minute DNA sequences without the need of gel electrophoresis —> fluorescence can be observed immediately

52
Q

What are the main 3 clinical uses of rtPCR?

A
  1. diagnosis/detection - diagnose certain viral infection based on some conserved viral genes amplified using probes and primers
  2. quantification - 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
  3. analysis - analyze variants by studying the melting curve and comparing the temperature with the known sequences in the databases
53
Q

What is a plaque assay?

A

quantitative method of measuring viruses by quantifying the plaques formed in cell culture upon infection with serial dilutions of a virus specimen

54
Q

What do viral neutralization tests measure? Why is it so important?

A

the presence of antibodies against a known virus that is detected by the antibody’s ability to prevent cytopathic effects of the reference virus in cell cultures

most important serological assay about protective antibodies and used to characterize serotypes of the viruses
(NOT RAPID)

55
Q

How are virus neutralization assays set up in 5 steps?

A
  1. serially dilute serum
  2. add equal amounts of virus to each tube
  3. incubate for an hour
  4. infect cultured cells and incubate for plaque formation
  5. reciprocal of the highest dilution that can prevent 50% plaque formation is the serum titer

(four-fold rise in neutralization titer of convalescent serum relative to acute serum indicated seroconversion)

56
Q

What do tissue culture infectious dose-50 (TCID50) measure?

A

measure all CPE other than lysis (cells remain alive) to calculate the concentration of a virus needed to produce such effects in 50% of the inoculated cell culture vessels

Veterinary Virology (25 decks)

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