Viro plenary/practicals Flashcards
(226 cards)
1
Q
Two forms of excistence
A
Virion and Vegetative virus
2
Q
Virion
A
- Inert particles transferring genetic information
- Size: 20-300 nm
- High variety in shape
- Content: NA (DNA or RNA), protein, +/- lipid
3
Q
Vegetative virus
A
The infected cell
4
Q
Reproduction of viruses
A
- Cannot divide as bacterias do
- Multiplication 10^3-10^6 vision/host cell
- -> makes the cell a virus factory
- No energy production
- No protein synthesis enzymes
- No (or not complete) NA replicating enzymes
5
Q
Occurence
A
- Whidespread
- Bacteria (phages), fungi, plants, animals, human hosts
- Multitude of viruses: in 1 ml shallow (0-300) seawater even 8 million vision particle can be found
- Viruses represent about 10% of the biomass
6
Q
Significance
A
- Pathogens of domesticated and wild animals
- Human pathogens
- -> Emerging new viruses
- -> The simplest living creatures
- -> Many viruses are not pathogenic
7
Q
Useful virus?
A
- Not in natural symbiosis
- Placenta development
- Biotechnology, gene therapy (vector)
- Viral proteins - potential medicines
8
Q
The origin of viruses
A
- Most of eukaryotic viruses have probably been evolved from procaryotes (becteriophages)
- They do not fit into the phylogenetic three of living creatures
9
Q
Cell degeneration (theory)
A
The parasite cell lost its independence. Outside of the cell only carries the information.
- Complicated viruses
10
Q
Runaway cell components (with genetic information)
A
- mRNA –> +ssRNA virus
- Chromosome fragment –> dsDNA virus
11
Q
Concentration and purification of viruses, aim
A
Virus analytical investigations
12
Q
Concentration and purification of viruses, prerequisite
A
Microbiologially clean cultures
- Virus isolate –> plaque purification –> virus strain
- -> propagation of genetically identical viruses in large scale
13
Q
Release of viruses from infected cell - Mechanical method
A
Freezing-thawing x3
14
Q
Release of viruses from infected cell - Sonication
A
Heat generation - virus protein may damage
15
Q
Release of viruses from infected cell - Detergents
A
For NA investigations
16
Q
Rough purification - Centrifugation
A
Sedimentation of cells, cell debris
17
Q
Rough purification - Filtration
A
Removal of particles larger than viruses, 450 nm filter pore size
18
Q
Concentration - Precipitation
A
- (NH4)2SO4, PEG 6000, ethanol
- Resolve in buffer
19
Q
Concentration - Adsorption
A
- Al(OH)3, Ca3(PO4)2
- Non-specific chromatography
20
Q
Concentration - Ultrafiltration
A
- Hydrostatic pressure
- Pore sizes are smaller than the dm of viruses
21
Q
Concentration - Dialysis
A
- Osmotic pressure
Through a semipermeable membrane
22
Q
Concentration - Pelletisation
A
- Virion sedimentation at 25k-200k x g
- In ultracentrifuge
- Even 1000 x concentration
23
Q
Conc and purification methods - Affinity chromotography
A
- Virus speific AB are bound to the chromatography column matrix
- Adsorption of viruses
- Rinsing
Elution with buffer (pH change)
24
Q
Conc and purification methods - density gradient ultracentrifugation
A
- Sedimentation of viruses in dense solutions
25
Stokes Law (1-2)
Sedimentation rate is proportional with the size of the particle and with the difference between the density of the particle and the density of its environment
26
Conc and purification methods
- Affinity chromatography
| - Density gradient ultracentrifugation
27
Rate zonal technique
based on sedimentation rate in sequential gradients, which are less dens than the viruses (Stoke 1)
28
Isopicninc technique
based on the density differences
| - Virus will sink as deep as their buoyant density is equal to the environment´s density
29
Conc and purification methods - preparative
For purification
30
Conc and purification methods - analytical
For measuring buoyant density
31
Virion buoyant density
NA/protein/lipid ratio (incomplete virions are lighter)
32
Nucleic acid hybridization
```
dsDNA heating = double helix splits
and cooling down = the completeary threads rejoin
Sample + probe
Heating - cooling
Washing (removal of unbound probe)
Autoradiography or substrate
```
33
dsDNA heating
double helix splits
34
What is a Probe?
Labeled oligosaccharide (DNA or mRNA) complementary to the viral genome
35
What is Nucleic acid hybridisation used for?
Classification of viruses, relatedness and diagnostics
36
DNA microarray technique
- DNA samples are bound to glass slides or membrane filters = "DNA chip"
- Hybridization with fluorescently labeled probes
- Laser scanning
- Computer analysis (identification and "typing" of viruses)
37
Polymerase Chain Reaction (PCR)
- Template+ primers + free deoxy-nucleotides + thermo-resistant polymerase (Taq)
- Heating and cooling in cycles (two copies of the fragment)
NB 72 degrees to find the prim. enzyme site
38
Polymerase Chain reaction detect
amplification of specific DNA fragments
39
Polymerase Chain reaction detection
Agarose-gel electrophoresis, NA hybridisation etc
40
Real-Time PCR detect
the amplification, can also be used for identification
41
Real-Time PCR
- Fluoerscent labeling
- Laser detection of amplification products, computer analysis
- Quantification
42
What is sequencing?
determination of the nucleotide sequence
43
What can you detect by sequencing?
the complete in formation about the nucleic acid
44
Sanger´s method (sequencing)
- Polymerization
- Template + primer + deoxy-nucleotides + labeled dideoxy-nucleotides (chain termination) + polymerase enzyme
=Polymerization of the complementary thread
45
Gel typ in Sequencing by Sanger´s reaction
Gel-electrophoresis
46
Oligonucleotide fingerprint technique investigate
RNA viruses -> relation and epidemiology investigations
47
Oligonucleotide fingerprint technique
- digestion with ribonuclease A or T1 ribonuclease
| - Twodimensional PAGE
48
Reverse transcriptase investigate
RNA viruses
49
Reverse transcriptase
- RNA dependant DNA polymerase --> transcription to dsDNA
| - Use of DNA investigation methods
50
Reverse transcriptase
- RNA dependant DNA polymerase --> transcription to dsDNA
| - Use of DNA investigation methods
51
Advantages of indirect virus demonstration
isolation of the causative agent within short time (NA/Ag detection)
52
Disadvantages of indirect virus demonstration
can be performed only for few days expensive
53
Indirect virus demonstration
detect ion of antibodies
54
Serological advantages of indirect virus demonstration
higher chance of demonstration (longer persistence of antibodies), and it is cheaper
55
Serological disadvantages of indirect virus demonstration
cannot differentiate maternal AB, Vaccines and seroconversion
- need to get age and vaccines history of the animal!!
56
Aim of indirect virus demonstration
1. Survey viruses present in the stock, random sampling
| 2. Individual diagnosis, identification of the causative agent, paired sera (serum titer increase)
57
Sample 1 and 2 in indirect virus neutralisation test
Sample 1: about the 3rd-4th day of the infection
| Sample 2: 10-14 days after the first
58
Indirect virus demonstration methods
- VN --> all cytopathogen viruses and non cytopathogen viruses
- HAI --> Haemagglutination viruses
- ELISA --> marker vaccines
- AGID
- CF
59
Virus neutralisation test (VN) principle
- detection of AB
- indirect method
in the presence of blocking antibodies reacting with anti-receptors of viruses the virus is not able to adsorb the cells.
Serotype, species specific
60
Virus neutralisation test, VN
- Indirect method
- Detection of antibodies
- Blood or body fluids
- Sample 1 and 2
- Increase of AB in sample 2 proves immune response
- Indicates the animals protection as well
61
Serum neutralization titre
the highest dilution of the serum where there is 50% CPE (the amount of blocking AB was sufficient yet)
62
Constant virus varying serum dilution method (VN)
| - steps
AB detection + determination of AB-level in the blood
1. Inactivation of heat-sensitive blocking factors at 56* for 1 hour
2. Serial twofold dilutions from the sera
3. Mixing each serum dilutions with a constant amount and volume of the virus suspension
4. incubation at 37* for 1 hour (the AB neutralise the viruses)
5. inoculation of at least 2 cell-cultures with each serum dillution
6. incubation at 37* for several days
7. investigate sample for CPEs
63
Validity of the virus neutralisation test, VN
- a virus between 30-300 TCID50
| - actual titer of the known positive serum did not change more than one dilution level
64
Constant serum varying dilution method (VN)
| - steps
Neutralization index calculation, for virus identification
1. Two serial tenfold dilution of the given virus suspension
2. adding negative serum
3. adding positive serum
4. incubation at 37* for 1 h
5. incubation of the cell-cultures from each dilution
6. incubation at 37* for several days
7. CPE
65
Evaluation of the constant serum varying dilution method
Nautralization index = virus+negative serum titre/virus + positive serum titre
66
Rapid fluorescent focus inhibition/fluorescent AB VN test
e. g rabies
1. VN (20 or 48h incubation, virus conc=FFD50/TCIS50)
2. adding of fluorescent labelled virus-specific AB: residual virus titre shows the AB content of the serum sample
67
Indirect immunofluorescent test (ilFA)
1. VN
| 2. adding of fluorescent labelled host Ig-specific AB: the fluorescence shows the AB content of the serum sample
68
Plaque reduction test
fewer plaques compared to the control viruses
69
Plaque reduction titer of the serum
The highest dilution of the serum where in 50% of the cell cultures the plaque formation is inhibited
70
Haemagglutionation test (HAI)
quantitative detection of the serum, only for haemagglutionation viruses!!!
- To investigate the AB content after infection with heamagglutination viruses
- If the serum contains AB, the haemagglutination will be prevented
71
Haemagglutionation inhibition
exhausting of the sera at 37* in 1 h
72
Haemagglutionation test (HAI), steps
1. serial twofold dilution of the serum sample
2. 4-8 HAU of virus to each serum dilutions
3. incubation in 1 h at a virus-specifictemperature
4. addition of washed electrocytes
73
Serum hemagglutination inhibition titre
the highest dilution where there is no HA (enough AB to block the HA proteins)
74
Validity of the HAI
- a virus between 4-8 HAU
| - actual titre of the known positive serum did not change more than on dilution level since the previous test
75
Antibody detecting ELISA plate is labelled with
viral antigen
76
Competitive ELISA advantages
"multispecies" kits, discriminate ELISA-s
77
compeditive ELISA
AB in the serum binds/no AB
Secunder AB: peroxidase labelled, specific to viral antigen
Binding/no binding (competition)
Binding = color change = negative test
No binding= no color change = positive test
78
Indirect ELISA
AB in the sera binds / no binding
Secunder AB: peroxidase labelled, host Ig-specific
Binding = color change = positive test
No binding= no color change = negative test
79
Aim of titration of viruses
"Standardization" of virus suspension, amount of infected viruses, titration, quantification
- Diagnostics (VNT, HAI)
- Vaccine production
- Experimental animal infection (challenge)
80
Methods of titration of viruses
- Physical assays
| - Biological assays
81
Physical assays
direct particle counting
| EM, haemagglutnation
82
Biological assays
determination of the infective titre (endpoint method), plaque assay, focus assay, pock formation etc
- incomplete/defective particles are not detected
- purification may damage virions
- succesful infection may also depend on the cell metabolic stage
83
Endpoint dilution (infective titre)
- method
- detects
- serial tenfold dilution of the virus suspension inoculation of the cell-cultures with each dilution incubation
- in vitro propagation and identification
84
Endpoint titre is the reciprocal of
the highest dilution virus that infects 50% of the host system. This endpoint dilution contains one 50% Tissue Culture Infecting Dose (TCID50) of virus
(The amount usually used is 30-300 TCID50 per unit volume)
85
What does the endpoint dilution contain at the endpoint titre
one 50% Tissue Culture Infecting Dose (TCID50) of virus
86
Infective titre
CPE in 50% of the inoculated cell-cultures
87
1 Tissue Culture Infective Dose
TCDI50/ml
88
Titration of embryonate eggs
determination of EID50 (pock assay)
89
Titration of experimental animals
determination of LD50
90
Calculation
Spearman-Kärber method
91
Spearman-Kärber method
ID50= x + 1/2d -dr1
92
Plaque counting (infective titre)
- steps
- semisolid maintenance
- serial tenfold dilution of the certain virus suspension - - -- inoculation of the cell cultures from each dilution
- adsorption in 1 h at 37*
Agar or CMC
- incubation characteristic to the virus
- local CPE in the inoculated cell-cultures
- staining with vital stain
- -> gentian purple
- -> janus green
- -> evans blue
93
Evaluation of plaque counting
The concentration of the virus suspension is given in plaque forming units = PFU/ml
94
Plaque counting method
At the inoculated cell cultures containing less than 10 plaques/inculated cell-cultures we take the average number (mean of the plaques), multiply it with the negative reciproc of the dilution level = infective titre of the virus suspension
95
Haemagglutinating titer´s determination
- serial twofold dilutions of the virus suspension
| - washed erythrocytes (1%) of proper species incubation at proper temp for the virus
96
Haemagglutination titer
The highest dilution of the virus suspension, in which we can observe hemagglutination
97
Haemagglutination spectrum
Different in the different haemaggl. viruses
98
"Obligatory detection on laboratory"
- notifiable diseases --> Foot and mouth disease, African swine fever, Newcastle disease etc
- suspected zoonosis --> rabies etc
- Eradication programs
- Official certifications of free status, specified pathogen free herd
99
The accompanying letter should have
information regarding the location and contact details of the submitter and premises sampled, description of the case associated to epidemiological information etc
100
Type of submitted sample material
- Swabs - nasal, pharyngeal, faecal etc
- Blood - EDTA, heparinised, coagulated, serum
- Organs - liver, spleen, lung, kidney, heart, intestines, brain, placenta ec
101
Direct methods (samples)
cadavers, organs, tissues, secretions etc
- for investigating the pathogen itself
- usually in early/acute phase
102
Indirect methods (samples)
coagulated blood or serum, milk, liquor, ventricle and organ secretion
- for AB investigation
- late/chronic phase, for immune response investigation
103
Respiratory signs ante-mortem sampling and post-mortem sampling
- Swabs - nasal, pharyngeal, EDTA-blodd
| - Lung and mediastinal lymph nodes (liver, spleen, kidney)
104
Gastro-enteric signs ante-mortem sampling and post-mortem sampling
- Faeces or faecal swabs (EDTA-blood)
| - Parts of intestine, mesenterical lymph nodes (lung, liver, kidney, spleen)
105
Neurological signs ante-mortem sampling and post-mortem sampling
- EDTA-blood; conjuctival- and nasal swabs (liquor cerebrospinalis)
- Parts of brain and spinal cord (lung, liver, spleen, kidney)
106
Skin/mucosal diseases ante-mortem sampling and post-mortem sampling
- Pieces (biopsy, skin scrapings) of affected skin, papillomas, sarcoids, vesicular wall, vesicular fluid (both cases)
107
Minimum AB titer increase to prove infection
4x is considered significant
108
Direct virus demonstration
The whole virus or its components (proteins, NA) can be investigated by virus isolation or more specific test; HA, ELISA, IF, protein detection and NA detection (PCR, NA hybridisation)
109
Indirect virus demonstration
Serological methods by which the AB from the blood or body fluids of the infected animals can be detected - ELISA, VN, indirect IF, HAI
110
Propagation of viruses (Direct virus demonstration) aim
detection of viruses - virology diagnostics
| - Large scale production of viruses - virus analytical studies, vaccine production
111
Why is living cells needed for the cultivation of viruses
Because viruses are obligatory cell parasites
112
Propagation of viruses in cell (tissue) cultures
In vitro maintenance and propagation of living cells
113
Production of primary monolayer culture (steps)
- Need cells that are the most susceptible for virus infections and able to divide actively (organs of origin)
- The organ must be processed within a few hours
- Separate the cells by the use of trypsin-EDTA
- The cells and the trypsin containing supernatant are separated by centrifugation
- Then the supernatant is removed by pipetting and the sedimented cells are resuspended in culturing medium
114
The culturing medium is
an optimal environment for the cells created by its components.
- It is isotonic, isoionic and isoosmotic and nutritive
- It usually contains foetal calf serum (FCS, from colostrum-free calves) which is a protein source and provides mediators og cellular division
- -> in growth medium 5-10%, in maintenance medium 2%
- The culturing medium must be supplemented with antibiotics and antimycotics in order to prevent the bacterial and fungal infection of the cell culture
115
Health of cell culture
Sick cell = more black, break the light more
| Healthy cell= can hardly see the cell
116
"Contact inhibition"
The cells will divide until there is contact with between the neighbouring cells cytoplasmic membrane
117
Primary cell cultures
Monolayer, produced directly from organs
118
Subculturing process (passage)
Cells are removed from the flask sf using trypsin. Resuspension in fresh growth medium, then the cell culture is divided into more flasks = more room for division. The cell is incubated and divides until the contact inhibition happens. At the end of the process this is a secondary cell culture
119
Advantages of secondary cell cultures
more homogenous, smoother cells, fresh culture with dividing activity, increase the amount of cells
120
Disadvantages of secondary cell culture
less susceptible to some viral infections
121
Permanent cell lines
Diploid cultures are formed by cell cloning
122
Advantages of permanent cell lines
- Standard, homogenous cells
| - Cells can be stored frozen for years and revitalised if needed
123
Disadvantages of permanent cell lines
- Less susceptible to some viral infections
| - Virus cannot choose among different cells
124
Aneuploid cell cultures
are made of tumor cells having high mitotic activity, usually primordial cell type. Less susceptible to som viral infections.
- only vaccines that will be inactivated later are allowed to propagate in tumor cells
125
Virus propagation (steps)
- sample processing
- inoculation when the possibly virus-containing sample-supernatant is added to the maintenance fluid of the cell culture
126
Steps of Organ sample
1. Using sterilized scissors and forceps, cut approx 1 g organ into 1-2 mm pieces
2. Transfer the organ pieces into a ceramic mortar, add approx 0,5 g sterile quartz sand
3. Homogenize the organ pieces
4. Add 9 ml sterile PBS to dilute
5. Mix the suspension and transfer approx 1 ml into a sterile micro centrifuge tube
6. Centrifuge the sample at 5000 x g for 5 min
127
Steps of Swap sample
1. Shake the swab in the tube with PBS using vortex to wash the sample out of the swab
2. Squeeze the swab into the tube with the help of sterilised forceps
3. Transfer about 1 ml of the suspension into a sterile microcentrifuge tube
4. Centrifuge the sample at 5000 x g for 5 min
128
Steps of faeces sample
1. Dilute 0,1 g sample with 1 ml sterile PBS
2. Homogenize the sample using vortex
3. Centrifuge the sample at 5000 x g for 5 min
129
When is absorption method used for the inculation of sample supernatant into the cell culture?
when the sample is toxic for the cell culture
| - e.g dead animals can contain toxic substances that can cause non-specific effects (CPE) and harm the cell culture
130
Absorption method steps
1. Check the cell culture in inverted microscope
2. Remove all of the culturing medium using micropipette
3. Inoculate 200 microL sample supernatant onto the cells
4. Incubate for 30-60 min at room temperature
5. Washing step 1: Rinse the sf of the cells with 750 PBS
6. Washing step 2: repeat step 5
7. Add 2250 microL maintenance medium into the cells
8. Place the cells into the incubator
131
Suspension method steps
1. Check the cell culture in inverted microscope
2. Remove all of the culturing medium using micropipette
3. Washing step 1: Rinse the sf of the cells with 750 PBS
4. Washing step 2: Rinse the sf of the cells with 750 microL trypsin-versene solution
5. Add 200 micrL trypsin to the cells
6. incubate for 5 min in room temp
7. Check the effect if the trypsin by microscope
8. Add 200 micrL growth medium and suspend the cells by using micropipette
9. Transfer 200mcL cell suspension into an empty, sterile culturing dish (negative control)
10. Add 1500 mcL growth medium to the cells
11. Add 200 mcL inoculum to the cells (except to the negative control)
12. Place the cell culture into incubator
132
Virus specific clinical signs in the embryonic egg
- egg necropsy
- dwarfism
- disortion
- death of the embryo
133
Haemagglutination test can be performed on embryonic eggs by
using the allantoic fluid
134
Inoculation of embryonated egg into the allantoic sac, steps
1. Candling - check and mark positions
2. Disinfection
3. Measure 200 mcL into a syringe
4. Pierce the cell with the needle, over the air sac
5. Push the needle into the allantoic sac
6. Inoculate the sample into the egg
7. Remove the needle and cover the hole with tape
8. Place the egg into incubator
(Should be candled every day to check)
135
Virus titration, aim
determination of the virus-content in a virus-suspension
136
Reasons for titration of viruses
- Serology --> standard amount
- Vaccine production --> determination of the antigen conc of vaccines
- Pathogenicity tests: vaccine testing
137
Virus titration methods
- Electron microscopic investigation --> counting
- Determination of the infective titre in cell culture --> appearance of CPEs in the different dilutions
- Determination of haemagglutination titre by HA
138
Examples of viruses with sf proteins (haemagglutins)
Orthomyxoviridae, Paramyxoviridae, Coronaviridae, Parvoviridae
139
Haemagglutination test detects
haemagglutination viruses
140
Haemagglutination, negative test
All viruses are NOT haemaggl viruses = RBC just sediment (little, clear, red dot)
141
What are CPEs
alterations in the morphology of cells due to virus infection, therefore mainly observed in cell cultures
142
CPEs are usually a result of degenerative processes
- direct damage of virus cells
- toxic effect of adsorption
- virus proteins inhibit cellular translation
- proteins inhibit cellular NA transcription and replication
- cytoskeleton depolymerisation
143
Main types of CPEs
- Inclusion body formation
- Cell rounding
- Syncytium-formation
- Lumpy cell nucleus
- Cell vacuolisation
- Cytolysis
- Haemadsorption
144
Inclusion body formation
Inclusion bodies appear at the site of assembly of nucleocapsid. Observed in stained cells, have homogenous staining, surrounded by a narrow stripe ("halo) due to shrinkage after fixation
145
Intranuclear inclusion bodies are caused by
DNA viruses replication in the nucleus
| Cowdry-A, Cowdry-B types can differentiate them (staining: basophil)
146
Intracytoplasmic inclusion bodies are caused by
```
RNA viruses (staining: eosinophil)
- Negri bodies or Guernireri bodies
```
147
Cell rounding
one of the most frequent CPEs
| - Change of the shape of the healthy cell because of cytoskeleton depolymerisation and loss of electrolytes
148
Syncytium-formation is caused by
enveloped viruses. Fusal proteins on the sf of the virus are primary for viral penetration. Membrane tunnels are formed bw cells = intracellular spread of viruses is possible. Giant cells with many nuclei are formed (polykaryotes/syncytia)
= virus can hide from AB
149
Lumpy cell nucleus
consequence of chromatin conglomeration, rearrangement or changed refraction. Characteristic for EHV-1 in the liver of foals and ASFV in lymphatic system
150
Cell vaculisation
Vacoules are formed in the cells. Can occur in the nucleus or in the cytoplasm
151
Cytolysis occurs because of
damage of the cell membrane or caused by lysosomal enzymes. The cell can explode
152
Haemadsorption
Between the infected cell and the RBC.
A viral protein, haemagglutinin, incorporates the cytoplasmic membrane of the infected cell = it is able to adsorb the erythrocytes on the surface of the monolayer
- Paramyxoviruses!
153
Type of focal CPE
Plaque
154
What happens in plaque
the CPE spreads from cells to neighbouring cells concentrically - in the centre CPE is stronger
- Syncytia are also seen as plaques
155
When is plaque-formation useful?
In case og virus purification, when subsequent passages of viruses taken from singular plaques are propagated, by which establishment of virus strains are possible
156
What is plaque counting used for in virus quantification and plaque reduction?
to determine the quantity of virus particles or AB content of the sample respectively
157
What supplement/test is used in non-cytophatogenic viruses?
Immunoperoxidase assay (IPA) or immunofluorescence (IF) test
158
Immunoperoxidase assays is when
virus-specific ABs labelled with peroxidase enzyme is added to the sf of the cell culture. The ABs will bind to these cells, and after adding a substrate of the enzyme, a color change will reveal the precense of virus
159
Immunofluorescence test
the virus-specific AB is labelled with fluorescence staining, and after it binds to infected cells, the cell culture is investigated using fluorescence microscope (yellow/green-ish indicate virus in the cell)
160
Test/assays that can be used for investigation of non-cytqpatogenic viruses:
- immunoperoxidase assay
- immunofluorescence test
- artificial infection of susceptible test animals
- Electromicroscope (EM) investigation
- Immuno-electromicroscope (IEM) investigation
- Complemet-fixation test
- Agar-Gel precipitation (AGP)
- Agar-Gel Immune Diffusion (AGID)
- Counter current immunoelectrophoresis (ccIEF)
- ELISA
- HA
- polymerase chain reaction
161
Microscopic investigations to identify viruses
- CPE
- Tissue specificity
- Type of pocks
- Haemadsorption
162
EM investigations to identify viruses
look at the dimension, size, shape of the virion, symmetry, surface etc
163
Enveloped/non-enveloped virus
If the virus loses its infectivity after chloroform treatment, it is enveloped
164
DNA/RNA virus
Halogenated deoxy-uridine is added to the growth media. It will inhibit the replication in DNA, but will not affect RNA
165
Single/double stranded NA
Stain with acridin-orange staining, then the single strand will show reddish-orange fluorescence while double stranded will be greenish-yellow.
166
Suitable methods to investigate group-specific antigens
- AGP
- IPA
- IF
- IEM
- ccIEF
- ELISA
- RIA
- HA
167
Suitable methods to investigate the determination of the serotype
- VN
| - HAI
168
Suitable methods to investigate the determination of subtype
- clinical signs
- investigation of NA
- investigation with monoclonal ABs
169
What is PCR used for?
to amplify a single or a few copies of a DNA, e.g in investigation of NA (direct method)
170
Advantages of using PCR
- fast method
- specific
- sensitive
- robust
171
Disadvantages of using PCR
- targeted investigation (can only detect certain viruses)
- needs specific equipment
- false negative and/or positive results can occur
172
Purification of the NA
Proteinase K- lysis of proteins, followed by purification with chromotography using NA adsorbing matrices
173
Chromotography method in PCR
- virus suspension is mixed with Proteinase K containing buffer
- after short inc. time ethanol is added for the precipitation of the NA
- during the centrigugation the NA is bound the the filter it is going through, while the digested protein molecules and the fluid goes through (discharged)
- after washing (washing solution + centrifuge), the filter can be cleaned of the molecules besides the NA
- elusion buffer is added to the filter, which release the NA from the filter and work as a restoration buffer
- the purified NA suspension can be stored at -80*
174
PCR (thermal cycling)
cycles of repeated heating and cooling of the reaction mixture. Necessary first to physically separate the two strands in a DNA double helix (high temp), then in low temp each strand act as template in DNA synthesis by the DNA polymerase to selectively amplify the target DNA
- In optimal conditions the amount of DNA target is doubled in each cycle
- Very sensitive method=a small amount of NA can be detected
175
Why do we say that the PCR is selective?
Because that the use of short primers, single-stranded oligodeoxynucleotide molecules, complementary to the (viral) DNA region targeted for amplification
176
Why is only NA complimentary to the primer amplified during PCR?
Because primer design is based on the genome sequence of the target virus. The primers are necessary for the polymerase enzyme as an initiator of the polymerisation
177
PCR thermal cycling
High temp: separate the two strands in double stranded helix
| Low temp: Strands act like template in DNA synthesis. DNA polymerase amplify the targeted DNA
178
Basic PCR setup
- DNA template
- Two primers (short segment of sense and anti-sense strands)
- Heat resistant polymerase enzyme
- Deoxynucleoside triphosphates
- Buffer solution
- Naase-free water
(In case of RNA template RNase inhibitor is added)
179
Positive control for PCR
Contains NA from a previously identified, specific viral NA
- If the positive control contains negative results it should be repeated = the first result cannot be accepted
- Positive control is also used for the identification of the DNA produced in the sample tube
180
Agarose Gel Electrophoreses (PCR) positive control
Comparing size of the DNA product of the positive control and the DNA product amplified in the sample tube
Similar size= most likely similar viral NA
Different size= not virus specific DNA var amplified in the sample
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PCR procedure (short)
1. Initiation step
2. Denaturation step (temp)
3. Annealing step (temp)
4. Extension/elongation step (temp)
5. Final elongation
6. Final hold
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Initiation step (PCR)
single temp. step at high temperature. Heating the reaction to a temp of 94-96* for 1-15 min (heat activation of DNA polymerase)
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Denaturation step (PCR)
94-98* for 20-60 seconds = unwinding of the DNA template helix (disrupting H-bonds). Separating of the complementary strands
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Annealing step (PCR)
50-65* for 20-60 sec. Allowing the annealing of the primers to the dingle-stranded DNS template. Stable DNA-DNA H-bonds are formed only when the primer sequence closely matches the template sequence
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Extension/elongation step (PCR)
temp depends on the DNA polymerase used. DNA polymerase binds to the primer-template hybris and synthesises a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5´ to 3´ direction.
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Final elongation (PCR)
70-74* for 5-15 min, to ensure that any remaining single-stranded DNA is fully extended
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Final hold (PCR)
4* for short-time storage of the reaction mixture
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PCR program, DNA
- Activation of polymerase
- Separation of DNA strains
- Primer annealing
- Extension
- Final extension
- Storage
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PCR program, RNA
- RT
- Denaturation of RTase, activation of polymerase
- Activation of polymerase
- Separation of DNA strains
- Primer annealing
- Extension
- Final extension
- Storage
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VN advantages
AB present longer in blood
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VN disadvantages
Cannot differentiate maternally v.s vaccine derived AB, or seraconversion. Maternal immunity and immunisation should be taken into considerations
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How many antibody-titre proves immune response (seroconversion) against a certain virus in VN?
At leat 4 (2x dilutions)
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How do vi detect the neutralisation in the VN?
By the lack of reactions such as CPE, plaque formation or disease in animals.
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What kind of result will the determination of the neutralising antibodies give (VN)?
serotype-specific result
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Constant virus varying serum dilution method (steps)
(is used for AB detection and determination of AB-level of the blood sample)
1. Preparation of the samples - inactivate heat-blocking factors
2. Serial twofols dilutions
3. Mixing in a constant volume of the virus suspension
4. Incubation 1 h at 37*, AB neutralize viruses
5. Inoculation of at least 2 cell-cultures with each serum dilution
6. incubation at 37* for days
investigate for CPE
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Parts of the 96- well plastic plate
- Serum part, serum samples are investigated
- Virus control part, virus content of the virus suspension is checked
- Cell control part, negative control
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Cell control (negative control)
- only the cell culture and cell-culturing medium
To check the health of the cells
Positive control: If CPE can be seen. Cannot use the cell culture. Can be toxins, bacterial/fungal infection (environment, aging)
Negative control: No CPE = healthy cells
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Virus control
- Cell culture + Virus(diluted) + Cell culturing medium
Gives us quantitative info about the AB content.
Accurate: known standard amount of viral antigen react with AB (have to determine the amount of infective virion particles in the virus suspension)
- Titrate, serial tenfold dill
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Serum (assay) wells
- Cell culture + virus(const) + Sera (diluted) + Cell culturing media
- Titration of the neutralising content of the serum, serial twofold dilutions.
- Positive control are also diluted
- Each dilution is mixed with an equal volume of virus suspension containing the same amount of virus
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Agarose Gel electrophoresis detect
To check if the PCR-virus specific amplification product is positive for the tested virus, AGE is used for size separation and visualisation of the PCR product-
- Separates the DNA molecules on electric current
- Move negative charged molecules through the agarose matrix
- Compare the size and weight to the positive control
(DNA in PCR can be identified)
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Agarose Gel electrophoresis, steps
1. Casting the gel
2. Loading the PCR products
3. Electrophoresis
4. Evaluation
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AGE - casting the gel
- Electrophoresis buffer, TAE, TBE
- Agarose powder, synthetic polysaccharide, large pores
- Intercalating dye (mixed with the gel)
- DNA virtualised, UV-light
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AGE - Loading the PCR products
- Put the solid gel into electrophoresis tank filled with TAE and TBE (to provide ions and keep constant pH)
- Mixed with loading buffer which contains dense compound. Increase density = DNA at the bottom
- Colored dyes;
- -> Large fragment: xylene cyan (light blue)
- -> Smaller fragment: bromophenol blue (dark blue)
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AGE - ELectrophoresis
- Negative charged DNA migrate from negative to positive pole
- Speed determined by its size
- Horizontally
- 10 V/cm electric current
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AGE - Evaluation
- View the gel with UV-transmitter
- Stained with intercalating dye, the DNA fragments appear as fluorescent bands in the gel
- Size of DNA can be compared to a molecule weight marker
Positive PCR sample: DNA size= size of the prod of the positive control
Negative PCR sample: < or > 100 bp size of fragment
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HAI
- Quantitative investigation of the serum
- -> to investigate the AB content after infection with haemagglutinating viruses
- -> If the serum contains AB, haemagglutination will be prevented
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Haemagglutinating inhibition titre
The highest dilution of the serum where the amount og AB is still sufficient to prevent the haemagglutination
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Assay steps in HAI
1. inactivation of the sera at 37* for 1 h
2. twofold serial dilution
3. each dilution is mixed with a same volume of the virus suspension
4. incubation at 37* for 1 h
5. the same volume of 1% RBC suspension is added to each well
6. incubation at room temp
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Exhausting in HAI
In order to eliminate the non specific, possibly haemagglutination AB, the serum is mixed with 10-25% RBC or 10% kaolin, which is eliminated by later centrifugation
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Each well of the serum /assay) wells contain
- Cell culture
- Cell culturing media
- Virus (constant)
- Sera (diluted)
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Each well of the virus control contain
- Cell culture
- Cell culturing media
- Virus (10-fold dilution)
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Each well of the cell control conatins
- Cell culture
| - Cell culturing media
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Why do we inactivate the sera in the first assay- step of HAI-test
In order to eliminate the non-specific, possibly haemagglutination ABs
- mix serum with 10-25% RBC or 10% kaolin, later eliminated by centrifugation
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Pandemic/panzootic disease
when the epidemic spreads throughout the world
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Endemic/enzootic disease
is the disease that permanently exists in a region or animal herd
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Epidemic/epizootic disease
outbreaks of the disease attacks many animals at around the same time, and might spread through one or several herds
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Sporadic disease
a disease that occurs infrequently and irregularly, and involves individual animals or small groups
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Indirect methods
Presence or increase level of AB
- VN
- HAI
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Direct methods
Presence of virus
- virus isolation
- HA
- PCR
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Paired sera investigate...
the difference of AB level between serum 1 and 2
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Virus neutralisation titre of the serum
That dilution of which we see CPE in half of the infected cell culture
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Virus endpoint titre
Highest dilution of virus that infects 50% of the host system, TCID50
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Serum (endpoint) titration
Contains one AB unit and is the reciprocal of the highest dilution of the antiserum protecting against the virus
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Serum titration
The serum is titrated against the homologous virus of the AB expected to be present in the serum. Serial twofold dilutions
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Virus control
to check the virus-content of the virus suspension.
- Serial tenfold dilutions
- Cell suspension was added to the well
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Cell control
used as negative control to check the health of the cell culture
- If CPEs appear = failed
