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Virology > general viro plenary > Flashcards

general viro plenary Flashcards

1
Q

virus as obligatory cell parasites mean:

A

they can not survive or multiply in the environment. They need living cells to do that.

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2
Q

Need of specified laboratories in case of/It is also obligatory in case of:

A
  • Regulations or rules, notifiable diseases e.g: FMD, ASF, BSE
  • Suspected zoonosis e.g: rabies
  • Eradication programs, or in order to control the eradication and declare the free status of the herd (Bovine Lucosis Virus, Infectious Bovine Rhinotrachetitis virus etc.)
  • Certifications: free status, SPF herd, transport, competitions
  • Clinical signs, pathological findings are not sufficient to establish the diagnosis
  • Herd diagnosis: endemic viruses present in the herd
  • In the need of official certifications of free status, specified pathogen free (SPF) herd: before exhibitions/animal shows/competitions, transport, export, travel.
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3
Q

Methods of laboratory diagnosis:

A
  • Direct virus demonstration

- Indirect processes (virus serology)

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4
Q

Direct virus demonstration consists of?

A
  • Whole virus (isolation)

* Its components: proteins, Nucleic acids

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5
Q

Indirect processes (virus serology) consists of?

A

• Antibody detection from the infected animals

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6
Q

Aim of the examination (i.e diagnosis) determines:

A
  • The type of sample
  • Its amount
  • Shipping
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7
Q

Aspects of the sample collection and transport

A
  1. Sample type an timing of sampling
  2. Unambiguous mark
  3. Letter of basic information = accompanying letter
  4. Period and circumstances of the samples shipping to a diagnostic institute
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8
Q

Necessary onformation for documentation for packaging:

A
  • Location and contact information
  • Case information
  • Epidermiological information
  • Submitted samples
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9
Q

Sampling for virological investigation

- Direct method:

A

Direct method (in early phase): Virus shedding is the highest in this phase!

  • Corpse, organ, tissue (cadaver)
  • Secretion: ventricle (thorax), organ (udder)
  • Blood anticoagulant treated (leucocyte separation)
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10
Q

Sampling for virological investigation

- Indirect method:

A

Indirect method (generally in late phase)

  • Blood coagulated, serum
  • Milk, liquor
  • Ventricle and organ secretion
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11
Q

Why do you use samples for serological detection of viral pathogens?

A

In order to be able to see an increase in antibody titres, whereas an increase of min 4 x is considered significant: sera pair investigation

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12
Q

Types of Laboratory investigations:

A
  • Direct virus demonstration

- Indirect virus demonstration

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13
Q

Direct virus demonstration:

A

The whole virus or its components (proteins, nucleic acids) can be investigated by propagation of viruses in cell culture (virus isolation) or more specific tests such as:

  • Antigen test (ELISA, haemagglutination, peroxidase or immne-fluorescence (IF))
  • Protein detection (Western blot)
  • Nucleic acid detection (i.e Nucleic acid hybridization, PCR)
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14
Q

Indirect virus demonstration:

A

Serological methods (ELISA, virus neutralization test, indirect IF, haemagglutnination inhibition (HAI)) by which the virus-induced antibodies from the blood or body fluids of the infected animals can be detected.

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15
Q

Virus isolation:

- period of the diagnosis?

A

2-3 weeks

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16
Q

Virus isolation

- Prerequisite of in vitro propagation?

A

infective virion –> early phase (acute stadium) –> virus shedding

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17
Q

Virus isolation:

Sample may be taken from?

A
  • Ante-morten: animal alive (buffy coat, body fluids, faeces)
  • Post-mortem: animal dead (organ samples)
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18
Q

what type of sample preparation of virus isolation is used in Direct virus demonstration?

A

Sample preparation in PBS (Ab-Am supplemented) 1:10 dilution

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19
Q

The method of sample preparation in PBS:

A

(Ab-Am supplemented) 1:10 dilution
- Organ pieces: cutting and homogenization in potter/seramic mortar
- Swabs: rinsing for 1-2 hours
1st centrifugation: cell debris quartz sand (1000 x g, 10min)
2nd centrifugation: purification (300 x g, 10min) or filtration

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20
Q

How is Buffy coat (WBCs) separated from non-coagulated blood?

A

by haemolytic resistance or buoyant density

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21
Q

Production of primary monolayer cell culture:

- Organ of origin

A
  • Rich in epithelial cells (- virus multiplication)
  • Actively dividing cells – from young animal (a few days old)
  • Kidney, testicles, thymus, embryo, etc.
  • Aseptic removal, processing within a few hours
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22
Q

Production of primary monolayer cell culture:

- Processing in aseptic circumstances

A
  • Removal of outer membranes, connective tissue
  • The tissue is cut into small pieces
  • Separation of the cells by digestion with trypsin – EDTA (versen) solution
  • Cell-containing suspension is repeatedly removed and replaced with trypsin solution
  • Blocking the effect of trypsin in ice bed (0C)
  • Sedimentation of the cells by centrifugation, removal of trypsin
  • Suspension of cells in culturing medium
  • Cell couting (in a Bürker chamber)
  • The cell suspension is transferred into a sterile culturing flask (Plate, Roux-flask, Petri-dish, etc)
  • Incubation (usually at 37 *C, 5% CO2)
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23
Q

What is used for Suspension of cells in culturing medium which gives Optimal environment?
(Production of primary monolayer cell culture)

A

MEM: Minimal Essential Medium.

  • Isotonic, isoionic, isosmotic (salts, buffer systems)
  • Nutritive (amino acids, carbohydrates)
  • Antibiotics, antimyotics, indicator
  • Foetal (neonatal) calf serum (FCS)
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24
Q

What is true for Foetal (neonatal) calf serum (FCS)?

A

 Protein source + mediators for cellular division
 Taken from colostrum-free calves
 Growth medium: 5-10%
 Maintenance medium: 2% FCS

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25
Q

What happens during incubation?

Production of primary monolayer cell culture

A

 cells settle down, attach and divide
 within 3-5 days they cover the bottom of the flask
 contact inhibition: when the cytoplasmic membranes meet, the cells stop their division
 a primary monolayer culture is developed

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26
Q

When do we use monolayers?

A

–> inoculation: virus is added into the maintenance fluid

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27
Q

Maintenance of cell cultures:

monolayers

A

• Aging of the cells – degeneration within 7-14 days
• Cell division – young cells are formed
–> Subculturing (passage)
• Removal of the medium
• Removal of the cells from the wall of the flask (trypsin digestion)
• Dilution (2-3x), putting into fresh culturing flask
–> secondary culture
• Fresh, homogenous, increased amount
• Further 2-3x passages are possible
• The cell content changes in subsequent passages (fibroblast increase), hence sensitivity might decrease

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28
Q

What is cellular cloning?

A

Cultivation of a single cell, production of permanent cell line
• Dilution of the cell suspension – 1cell/well
• Propagation, selection of quickly dividing clones

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29
Q

Types of cellular cloning:

A
  • Diploidic (i.e: PK-15, MDBK, RK-13)

* Aneuploidic – tumor cells (ie. HeLa, BHK-21, Vero)

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30
Q

Advantages of Cellular cloning:

A
  • Genetically homogenous, standard
  • Unlimited number of passages
  • Long term storage (-80C, liquid nitrogen)
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31
Q

Disadvantages of cellular cloning:

A
  • Sensitivity for virus infections varies (- presence of cellular receptors)
  • Contamination may occur (virus, mycoplasma, leptospira)
  • Presence of active oncogene
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32
Q

Adsorption is used to?

A

avoid CPE caused by toxins

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33
Q

Suspension is used for?

A

viruses which need dividing cells

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34
Q

What is Co-cultivation?

A

isolation of cell-associated and latent viruses

- Simultaneous processing and mixing of virus-infected and healthy cells

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35
Q

Suspension cultures:

A

continuous stirring, no adhesion: vaccine production

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36
Q

Microcarrier cultures:

A
  • Cells adhere on the surface of microcarrier beads (Cytodex), continuous stirring
  • Aim: increased surface (vaccine production)
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37
Q

Shell vial assay:

A 
Herpesviruses (CMV, HSV, VZV)
Adeno-, entero-, flavi-, orthomyxo-, paramyxoviruses
- Inoculation
- Centrifugation (700 x g, 45min) 
- Incubation 16h
- Mab staining
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38
Q

What is important characteristics for the egg during inoculation?

A
  • Embryonated
  • SPF (specific pathogen free)
  • White shelled – easier transillumination
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39
Q

Characteristics for the yolk sac?

A
  • Picorna- (i.e avian encephalomyelitis virus) reo-adenoviruses
  • Rickettsia, chlamydia
  • 5-7 days old embryo
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40
Q

Characteristics for the Allantoic cavity, Amniotic cavity (embryo)?

A
  • Orthomyxo-, paramyxo-, coronaviruses

- 9-12 days old embryo

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41
Q

Characteristic for Chorio-allantoic membrane (CAM)?

A
  • Pox-, herpesviruses

- 10-13 days old embryo

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42
Q

characteristic for Intravenous inoculation?

A
  • Orbiviruses (i.e Bluetongue virus)

- 16-17 days old embryo

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43
Q

Incubation temp for inoculation of embryonated eggs

A

33-37 *C

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44
Q

Control after inoculation of embryonated eggs to check?

A
  • Tranillumination
    Death within 24h – usually non-specific
  • Egg necropsy (after 4-5days)
    Dwarfism, distorsion, death
    CAM: pock (size, inflammation, haemorrhage, necrosis)
  • Haemagglutination test on the allantoic fluid
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45
Q

If there is no CPE of the embryonated egg:

A
  • Blind-passage (increase of virus titer and CPE

- Auxiliary examinations: EM, HA, IF, IP

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46
Q

Final steps of inoculation of embryonated egg:

A

isolate –> plaque -isolation –> purification –> virus strain

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47
Q

Daily examination to check:

embryonated egg

A
  • Sample collection: allantoic fluid, CAM, embryo

* Auxillary examinations: HA, EM, histopathology

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48
Q

Aim for concentration and purification of viruses:

A

virus analytical invesstigations

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49
Q

Prerequisite or concentration and purification of viruses:

A

Microbiologically clean cultures
- Virus isolate –> plaque purification –> virus strain –> propagation of genetically identical viruses in larger scale

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50
Q

Name different methods for release of viruses from infected cells:

A

• Mechanical method: freezing-thawing (3x)
• Sonication (heat generation – virus protein may damage)(ultrasound)
• Detergents (for nucleic acid investigations)
–> Opens the cell membrane. Virus can be dead or alive.

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51
Q

Rough purification can be done by?

A

• Centrifugation
- sedimentation of cells, cell debris (3000-5000 x g)
- the virus stays in the supernatant, NOT in the sediment
• Filtration
- Removal of particles larger than viruses (bacteria, yeasts, moulds, cell components)
- 450nm filter pore size

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52
Q

concentration methods depends on?

A

The physico-chemical resistance of viruses

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53
Q

Why do we use Precipitation?

A
  • When we don’t need the suspected virus, only the Nucleic Acids etc.
  • (NH4)2SO4, PEG 6000, ethanol
  • Resolve in buffer
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54
Q

Methods for concentration:

A
  • Precipitation
  • Adsorption
  • Ultrafiltration
  • Dialysis
  • Pelletisation
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55
Q

Why do we use Adsorption, and what do we use:

A
  • Can not separate the virus and smaller particles.
  • Al(OH)3, Ca3(PO4)2
  • Non-specific chromatography
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56
Q

Why do we use ultrafiltration, and what do we use

A
  • Hydrostatic pressure

- Pore size are smaller than the diameter of viruses

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57
Q

Why do we use Dialysis, and what do we use

A
  • Osmotic pressure

- Thorugh a semi permeable membrane

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58
Q

Why do we use Pelletisation, and what do we use

A
  • Virion sedimentation at 25 000-200 000 x g
  • In ultracentrifuge (mitochondria and other organells can also be analysed)
  • Even 1000 x concentration
59
Q

What are contaminating elements that also should be removed?

A

elements similar to viruses in size (eg mitochondria, chromosomes, ribosomes)

60
Q

Concentration and purification methods:

A
  • Affinity chromatography
  • Density gradient ultracentrifugation
  • Rate Zonal technique
  • Isotopic technique
61
Q

Affinity chromatography method:

A
  • Virus-specific antibodies are bound to the chromatography column matrix
  • Adsorption of viruses
  • Rinsing
  • Elution with buffer (pH change)
  • Filter that binds antibody specific pathogens:
62
Q

What is the function of filter that binds antibody specific pathogens?
(Affinity chromatography method)

A
  • Virus suspension go through the filter.
  • The virus binds specifically.
  • Mitochondria etc. only stays on the filter.
  • With the buffer, you remove all the cell organelles except the virus itself
63
Q

What is the principle of Stoke´s Law in Density gradient ultracentrifugation?

A

The virus particles density divides until it has the same density as the density of the tube.

64
Q

What is used for sedimentation of viruses in dense solutions?

A

CsCl, Cs2SO4, sucrose, metrizamide

65
Q

What is Rate Zonal technique based on?

A

on sedimentation rate in sequential gradients, which are less dense than the viruses (Stoke 1)

66
Q

What is Isotopic technique based on?

A

based on density differences.

- Viruses will sink as deep as their buoyant density is equal to the environemnt´s density (Stoke2)

67
Q

Preprative ultracentrifugation is used for?

A

purification

68
Q

Analytical ultracentrifugation is used for?

A

measuring buoyant density

69
Q

What is virion buoyant density?

A

nucleic acid/protein/lipid ratio (incomplete virions are lighter)

70
Q

dialysis is used for?

A

collect and purify virus-specific zones

71
Q

Physico-chemical test for virus identification:

A
  • Electron-microscopic investigation
  • Chloroform resistance
  • Halogenic uridin derivates
  • Acridin-orange staining
72
Q

When is Electron microscopic investigation used?

A

virus identification
–> morphological recognizable virions (complete/incomplete)
Advantage in case of viruses not replicating in cell-cultures

73
Q

Methods of electron microscopic investigation:

A
  1. Ultra thin section
  2. Negative contrast method
  3. Immune-electron microscopic method
74
Q

Explain the method of Ultra thin sections in Electron-microscopic investigation:

A
  1. Ultra thin section (from the organs, from the predilection sites)
    - fixation (glutaraldehyde)
    - Embedding (durcupan resin)
    - Tungsten- or urnanium salts treatment (electron absorbent)
75
Q

Explain the method of Negative contrast method in Electron-microscopic investigation:

A
  1. Negative contrast method (diluted sample e.g: fecal)
    - Treatment with contrast material (phosphotungsten acid)
    - Drying onto the grid (contrast material does not bind)
76
Q

Explain the method of Immune-electron microscopic method in Electron-microscopic investigation:

A
  1. Immune-electron microscopic method (diluted samples)
    - centrifugation
    - Immune serum added to the sample –> precipitation
    - Centrifugation –> precipitated virus in the sediment
77
Q

How to investigate serotye

A

species –> antigen investigation, serological methods (VN)

78
Q

Subtype, variants:

- Alteration in genotype:

A

NA examination: REA, sequencing

79
Q

Subtype, variants:

- Alteration structure protein:

A

MAB, PAGE

80
Q

what is needed for Virus antigen detection:

A

with specific antibodies (virus identification too)

  • Incomplete virions, non-matures proteins are demonstrated
  • Proper serum
81
Q

What is characteristic for the proper serum needed for Virus antigen detection?

A
  • Hyperimmune
  • Monospecific (polyclonal)
  • Monoclonal
82
Q

What test can be used for Virus Antigen Detection?

A
  • Immunofluorescence (IF) test
  • Immunoperoxidase (IPA, PLA) test
  • Complement fication (CF) test
  • Agar Gel Immune electro-foresis (CIEF) test
  • Radio Immune Assay (RIA) test
  • Enzyme linked Immunosrobent Assay (ELISA) test
83
Q

When can you use IF test for virus antigen detection:

A

Immunofluorescence (IF) test: antigen intracellularly

  • Direct: rabies, classical swine fever
  • Indirect: 10x more sensitive
  • Specificity  sensitivity optimation
84
Q

When can you use IPA, PLA test for virus antigen detection:

A

Immunoperoxidase (IPA, PLA) test: antigen intracellularly

  • Histology slide/cell culture
  • Spec, antibody labelled with peroxidase enzyme
  • Substrate digestion  colour change
85
Q

When can you use CF test for virus antigen detection:

A 
Complement fixation (CF) test: antigen released off the cells
- The hemolysin lyses the sheep RBCs in the presence of complement (FMD)
86
Q

When can you use AGID test for virus antigen detection:

A

Agar Gel Immune Diffusion test (AGID):

- Adeno, bluetongue, EHD

87
Q

When can you use CIEF test for virus antigen detection:

A

Counter current immune electro-forezis (CIEF):

- Rota, parvo, adeno

88
Q

When can you use ELISA test for virus antigen detection:

A

Enzyme Linked Immunosorbant Assay (ELISA):

  • Direct/indirect: antigen/antibody detection
  • Competitive: discriminating/multispecies antobody detection
  • Sandwich (FMD antigen detection)
89
Q

Nucleic acid hybridization

- Theory: heating/cooling down

A
  • dsDNA heating a–> the double helix splits

- Cooling down: the complementary threads rejoin

90
Q

What is the function of the probe in Nucleic acid hybridization?

A

Probe: labels oligonucleotide (DNA or mRNA) Complementary to the viral genome Labelling: isotope (32P) or enzyme

91
Q

Southern Blot:

A
  1. Agarose gel lectrophoresis
  2. Nitrocellulose/nylon filter
  3. Hybridization
    - Electricity first horizontally, then vertically
92
Q

DNA microarray technique:

A

• DNA samples are bound to glass slides or membrane filters –> “DNA chip “
• Hybridization with fluorescently labelled probes
• Laser scanning
• Computer analysis
–> Identification, “typing” of viruses
–> Comparison of virus strains, genetic relationships

93
Q

What are the different Nucleic acid investigation methods:

A
  • Nucleic acid hybridization
  • Southern blot
  • DNA microarray technique
  • Polymerase chain reaction
  • Real time PCR
94
Q

Polymerase chain reaction is used for:

A

Amplification of specific DNA fragments

95
Q

What is needed in PCR?

A

Template + primers + free deoxy-nucleotides + thermos-resistant polymerase
(Taq)

96
Q

How PCR works:

A

• Template + primers + free deoxy-nucleotides + thermos-resistant polymerase
(Taq)
• Heating and cooling in cycles (n)
• Polymerase enzyme; moves at 72°C (important number), makes a new
complementary strand –> double amount of nucleic acid
• RNA: reverse transcription
- ssDNA stay ss –> reverse transcription
• Compare sequence strains to see if it is a zoonosis, mutation or epidemic virus
ex.
–> 2n copies of the fragment
• Detection: agarose-gel electrophoresis, NA hybridization etc.

97
Q

What is the function of real-time PCR?

A

(detect amplification of NA during the reaction)
• Fluorescent labelling
• Laser detection of amplification products, computer analysis
• Quantification only

98
Q

What is sequencing

A

determination of the nucleotide sequence

99
Q

What is Sangers method?

A

A type of Sequencing
Sangers method: polymerization
- Template + primer + deoxy nucleotides + labelled dideoxy-nucleotides + polymerase
enzyme –> polymerization of the complementary thread

100
Q

What is needed for sequencing:

A
  • The complete information about the nucleic acid
  • Sangers method
  • Dideoxy- nucleotide: chain termination
  • Polyacrylamide gel-electrophoresis
101
Q

What is Next generation sequencing methods:

A

• High efficacy – 20 million base read within a few hours
• Fragmentation of the DNA sample, binding to carrier surface
• Non-specific amplification
• Separation into wells of a microplate
• Sequencing reactions, detection of signal generated by the incorporating nucleotides
• Automated processing of the reads:
Alignments of repeatedly determined, overlapping sequences, consensus sequence generation

102
Q

Name some Next generation sequencing methods:

A
  • Oligonucleotide fingerprint technique

- Reverse transcriptase conversion

103
Q

What is Oligonucleotide fingerprint technique?

A
  • investigation of RNA viruses
  • Digestion with ribonuclease A, or T1 rubonuclease
  • Two dimensional PAGE –> relation, epidemiology investigation
104
Q

What is Reverse transcriptase conversion?

A
  • Investigation of RNA viruses
  • RNA dependent RNA polymerase –> transcription to dsDNA
  • Use of DNA investigation methods.
105
Q

Aim of titration of viruses:

A

“standardization” of the virus suspension –> amount of infective viruses –> titration, quantification

106
Q

Methods for titration of viruses:

A
  • Physical assays: direct particle counting – EM, Haemagglutination
  • Biological assays: determination of the infective titre (endpoint method), plaque assay, focus assay, pock formation etc.
107
Q

When is End point dilution - infective titer used?

A

in vitro propagation and identification

108
Q

Method of End point dilution - infective titer:

A
  • Serial tenfold dilution of the virus suspension
  • Inoculation of the cell-cultures with each dilution
  • Incubation, the period is characteristic to the virus
109
Q

Diagnostics with Titration of viruses:

A

Virus neutralization test, Haemagglutination inhibition test

110
Q

What is infective titer?

A

CPE in 50% of the inoculated cell-cultures

111
Q

What is characteristic to the virus suspension?

A

The highest dilution of the virus, which cause CPE in 50% of the inoculated cell-cultures

112
Q

What does TCID50/ml mean?

A

The highest dilution of the virus, which cause CPE in 50% of the inoculated cell-cultures, is characteristic to the virus suspension
–> Its virus concentration unit is; 1 Tissue Culture Infective DOSE TCID50/ml

113
Q

What does EID50 mean?

A
  • Titration in embryonated eggs – determination of EID50
114
Q

What does LD50 mean?

A
  • Titration in experimental animals – determination of LD50
115
Q

Methods of calculation:

A

• Reed-Muench method: very first, most commonly used. We are looking for
concentration of original virus suspension.
• Spearman-Karber method: easier, no need of lot of numbers, only the data of
dilution which is the next higher dilution with 100% response (CPE).
- A difficult equation, but also looking for the original concentration of the virus suspension.

116
Q

Method of Plaque counting - infective titer:

A

• Both stained and unstained
• Serial tenfold dilution of the certain virus suspension
• Inoculation of the cell cultures from each dilution
• Adsorption: 1 hour at 37 degrees
• Covering with semisolid maintenance: agar or CMC
- Incubation characteristic to the virus
- Local CPE in the inoculated cell-cultures
- Staining with vital stain; gentian purple, Evans blue, Janus green

117
Q

How do you calculate the Infective titre in plaque counting?

A

at the inoculated cell-cultures containing less than 10 plaques/inoculated cell-cultures, we take the average number (mean) of the plaques, multiply it with the negative reciprocal of the dilution levelàthis gives the infective titre

Evaluation: The concentration of the virus suspension is given in plaque forming units = PFU/ml

118
Q

Lattice formation =

A

no infection – only RBC + virions (or hemagglutinating proteins of
virions)

119
Q

Method of Haemagglutinating tire determination:

A

• Lattice formation, no infection – only RBC + virions (or hemagglutinating proteins of
virions)
• Often species specific
• On micro-haemagglutination plates (Takatsy plates)
• Serial twofold dilution of the virus suspension
• Washed erythrocytes (1%) of proper species
• Incubation –> at proper temperature for the virus

120
Q

Haemagglutinating tire =

A

The highest dilution of the virus suspension, in which we can observe hemagglutination

121
Q

Virus demonstration Advantage/disadvantage:

A
  • Advantage: isolation of the causative agent within short time (in case of NA/aG detection).
  • Disadvantage: can be performed only for few days, and it is expensive.
122
Q

Serological methods Advantage/disadvantage:

A
  • Advantage: higher chance of demonstration (longer) persistence of antibodies), and it is cheaper. Antibodies present for a much longer time –> higher chance of detection.
  • Disadvantage: cannot differentiate maternal, vaccine and seroconversion antibodies
  • -> see the age and vaccination history in accompanying letter
123
Q

Method of Virus neutralization:

A

• Mix virus with antibodies –> cant infect. Then see CPE on cell. Answer over 2 –>
enough antibodies to neutralize virus
• Heat inactivation of the sera at 56 degrees in 30 min. –> non-specific antibodies are
heat sensitive

124
Q

Principle of Virus neutralization:

A

In the presence of blocking antibodies reacting with antireceptors of
viruses, the virus is not able to adsorb to the cell
- Serotype (species) specific

125
Q

Method of Serial twofold dilution from sera:

A
  • 100 TCID50 (or EID50 or PFU50) viruses
  • Incubation 1 hour at 37 degrees –> antibodies neutralize the viruses - Inoculation of the cell-cultures with each dilution
  • Incubation at 37 degrees for several days
  • -> CPE
126
Q

What is Serum neutralizing titer:

A

the highest dilution of the serum where there is 50% CPE

127
Q

Method of Rapid fluorescent focus inhibition/fluorescent antibody virus neutralization test:

A

• RFFIT/FAVNT – ex. Rabies

  1. Virus neutralization – 20 or 48 hours, virus concentration: FFD50/TCID50
  2. Addition of fluorescent labelled virus-specific antibody: residual virus titer shows the antibody content of the serum sample
128
Q

Method of

Indirect immunofluorescent test (iIFA):

A
  1. Virus neutralization test
  2. Addition of fluorescent labelled host IG-specific antibody: the fluorescence shows the antibody content of the serum sample
129
Q

method of Constant serum varying virus dilution method:

A

• Neutralization index calculation –> for virus indentification

  • Two serial tenfold dilution of the given virus suspension - Adding negative serum
  • Positive serum
  • Incubation at 37 degrees for 1 hour
  • Inoculation of the cell-cultures from each dilution - Incubation at 37 degrees for several days
  • -> CPE
130
Q

Plaque reduction test is used to:

A
  • To see if it is enough antibodies in blood

* Fewer plaques compared to the control viruses

131
Q

What is the plaque reduction titre of the serum:

A
  • The highest dilution of the serum where in 50% of the cell cultures the plaque formation is inhibited (reduction % is characteristic to the given virus)
132
Q

Method of Hemagglutination inhibition (HAI):

A

• Which sample does not agglutinate? Opposite of hemagglutination test
• Exhausting the sera 37 degrees for 60 minutes – eliminates the non-specific
hemagglutinating activity: 10-25% RBC or with 10% caolin
• Only for hemagglutinating viruses
- Serial twofold dilution of the serum sample
- 4-8 HAU (standard virus suspension) of virus to each serum dilutions - Incubation for 1 hour at a temperature characteristic to the virus
- Addition of washed erythrocytes (1%)

133
Q

What is the Serum hemagglutination inhibition titer?

A

the highest dilution where there is no hemagglutination (enough antibodies to block the HA proteins)

134
Q

Validity of the HAI test:

A
  • A virus between 4-8 HAU
  • Actual titer of the known positive serum did not change
    reduction test
  • More than one dilution level since the previous test
135
Q

Antibody detecting can be done by:

A

ELISA (cELISA, iELISA)

136
Q

Advantage of cELISA?

A

Competitive ELISA
Advantage:
- “multispecies” kits, discriminative ELISAs (ex. IBR)
- antisbody in the serum binds / no antibody
- Secunder antibody: peroxidase labelled, specific to viral antigen, binding/no binding – competition

137
Q

What is true in the case of Binding in cELISA?

A

colour change–sample is negative

138
Q

What is true in the case of No Binding in cELISA?

A

No colour change–sample is positive

139
Q

Advantage of iELISA?

A

Indirect ELISA

  • Antibody in the serum binds/no binding
  • Secunder antibody: peroxidase labelled, host Ig-specific
140
Q

What is true in the case of Binding in iELISA?

A

colour change–serum sample is positive

141
Q

What is true in the case of No Binding in iELISA?

A

No colour change–serum sample is negative

142
Q

What is true for AGID test?

positive/negative test

A

Agarose gel immune diffusion test (AGID)
o Positive- precipitation occurs
o Negative – nothing happens

143
Q

What is true for CF, iCF test?

positive/negative test

A

(Indirect) complement fixation test (CF, iCF)
o Positive – sedimenting RBC
o Negative–hemolysis

Virology (25 decks)

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