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Virology > Lecture 6: Diagnostics > Flashcards

Lecture 6: Diagnostics Flashcards

0
Q

viremia =

A

presence of virus in the blood.

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1
Q

what are the current techniques to diagnose a viral infection?

A 
virus isolation: gold standard
detection of viral antigens
detection of viral nucleic acids
detection of virus specific antibodies
visualization and identification of viruses by electron microscopy
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2
Q

when does the max titer of a virus often occur?

A

it will coincide with the peak fever, this may precede the onset of other clinical signs

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3
Q

virus isolation

A

slow
agent independent
cytopathic effects are indicative of the virus involved - often indicative of the virus involved
provides inexhuastible amount of virus

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4
Q

what specific cell lines are known to grow many viruses?

A

embryonated hen’s eggs

suckling mice

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5
Q

what characteristic is often times sufficient in assigning a virus to the correct family?

A

morphology

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6
Q

viral diagnosis by EM

A

useful in detection of non-cultivable viruses.
agent independent
negative staining
thick-sectioning (most cells must contain virus, expensive takes days)

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7
Q

detection of viral antigen by immunofluorescence is used on what type of samples?

A

cryostat sections

lesion smears tissue cultures

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8
Q

what is IF not compatible with?

A

formalin fixation

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9
Q

direct IF uses what?

A

FITC-labeled specific viral antiserum

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10
Q

indirect IF uses what?

A

labeled anti-species antiserum after 1st antibody

increases sensitivity but also non-specific background

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11
Q

immunohistochemical (IHC) staining: what enzyme is usually used? what color does it become once it reacts with substrate?

A

horse radish peroxidase

brown

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12
Q

what are the advantages of IHC over IF:

A

needs only a light microscope

amplifies reaction product and color; gets stronger by increasing incubation time

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13
Q

what other information can you get from IHC that you dont get from other diagnostic techniques?

A

provides evidence of antigen localization inside cells

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14
Q

“point of care”: immunochromatography

A

migration of antibody-conjugate complexes thru a filter matrix or lateral flow matrix

  • all controls are included in the membrane
  • results are seen as colored spots
  • rapid and simple assay: each test contains a positive and negative control
  • sensitivity varies
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15
Q

detection of viral nucleic acids: what types of viruses can be detected

A

detects viruses that are non-cultivable

  • allows detection of viruses that are not viable
  • detects latent infections
  • detects viruses that have been bound or complexed
  • detects virus in formalin-fixed tissues
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16
Q

detection of nucleic acids: PCR

A
  • is an in-vitro method for the enzymatic synthesis of specific DNA sequences
  • it amplifies DNA
  • primer annealing provides dsDNA for the polymerase to extend the DNA synthesis
  • primers can also degenerate to increase the test sensitivity whenever there is variability
17
Q

reverse transcription polymerase chain reaction (RT-PCR): for RNA viruses
what versions are there?

A

conventional two-tube RT-PCR

single-tube RT-PCR: all components for both reactions are contained in a single reaction tube

18
Q

PCR usually consists of 3 steps:

A
  1. denaturation of dsDNA
  2. primer annealing
  3. extension
19
Q

some PCR precautions:

A
  1. lab becomes contaminated with amplified DNA products as tubes are opened and false positives are obtained in subsequent PCRs
  2. nested PCR assays are contamination-prone
  3. real-time PCR or qPCR measures accumulation of DNA as it is amplified and has solved most of these contamination problems
20
Q

deep sequencing

A

new viruses are discovered by random nucleic acid amplification and relatively low cost sequencing.

21
Q

real-time polymerase chain reaction

A

once tubes are set, there are no longer opened - preventing cross-contamination.

  • PCR product is measured by increases in fluorescence intensity generated by reporter molecules.
  • this is more sensitive than conventional PCRs
22
Q

examples of reporter molecules

A

DNA binding dyes like SYBR GREEN I, TaqMan probes and quenchers

23
Q

advantages of PCR

A
  • useful to detect virus that wont grow in culture: papillomavirus, norovirus, astrovirus, rotavirus
  • can be used on any sample appropriate for virus isolation
  • fast
  • preferred for initial ID of dangerous viruses (rabies, hendra virus, nipah virus, influenza, ebola, etc)
24
Q

what sample defines infection status of animals?

A

serology

it decloses past infections and dtermines proper responses to vax

25
Q

convalescent serum titer must be how many times greater than paired sera?

A

four fold greater or more

26
Q

what type of viral antibodies in the acute serum may provide a presumptive diagnosis?

A

IgM !!

27
Q

Enzyme linked immunosorbent assay (ELISA): for detection of viral antibodies

A
  • rapid and cost effective
28
Q

virus neutralization is the gold standard for what

A

quantification of antibodies

29
Q

neutralizing antibodies correlates well with protective immunity from_____

A

in vivo

30
Q

what is the most widely used techniqu when neutralizing antibodies

A

constant virus-variable serum dilutions

31
Q

what does neutralizing antibody rely on?

A

binding of antibody to critical viral antigens preventing virus from attaching and infecting susceptible cells

32
Q

the neutralizing antibody titer of a serum is defined as what?

A

the reciprocal of the highest serum dilution that completly inhibits CPE or antigen production: PLAQUES or END-POINT CPE

33
Q

what are the advantages to virus neutralization

A

species independent

ideal for wildlife studies and may be validated in weeks vs MONTHS for ELISAs

34
Q

what are hemagglutinating viruses of importance

A 
orthomyoxoviridae
paramyoxviridae
coronaviridae
parvo
adenoviridae
35
Q

how do hemagglutinating viruses work?

A

they produce agglutination of RBCs by crosslinking sialic acid residues on the RBC surface

36
Q

what is hemaggluination inhibition (HI) currently used for

A

detection of antibodies against influenza, arboviruses, parainfluenza, parvo and paramyxoviruses

37
Q

how do HI antibodies work?

A

bind to virus RBC receptors and block hemagglutination

38
Q

what has been the workhouse in teh viral field for decades?

A

agar gel immunodiffusion (AGID) for detection of viral antibodies

39
Q

what are the pros to useing agar gel immunodiffusion (AGID)?

A

simple, inexpensive, doesnt require infectious antigen

40
Q

what are cons to AGID?

A

strictly qualitative, cant be autoamted, not as sensitive as ELISA

Virology (7 decks)

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