Test methods

The following test methods are carried out in our laboratory:

Test methods

Virus isolation in cell culture
The detection of viruses after replication in cell culture is a common method in virological laboratory diagnostics. For this purpose, around 20 different cell lines of different origins (animal species, organ origin) are routinely kept in our laboratory. For the detection of viral pathogens, the samples are processed (production of suspensions from organ samples, preparation of cells from coagulated blood samples or swab samples) and then used to inoculate test cultures. The selection of the cell lines used depends on the origin of the test material and the diagnostic question. The multiplication of viruses in culture cells often causes a cytopathic effect (cpe), i.e. a morphological change in cells and/or cell layer. If a cpe is observed, the virus is further characterized, e.g. by immunofluorescence, PCR or electron microscopy. Several "blind" passages may be necessary until a cpe occurs. Some viruses multiply in cell culture without cpe, e.g. non-cytopathogenic (ncp) representatives of the virus of bovine viral diarrhea (BVDV). Ncp BVD viruses are routinely detected by immunofluorescence using the test cultures.
Advantages: a wide range of viral pathogens can be detected depending on the susceptibility spectrum of the cells used.
Disadvantages: high expenditure on equipment and long process duration. For some viruses a cell culture system is not available .

(Immune) Electron microscopy
Viro Vet Diagnostik has its own electron microscope, which is mainly used in virological infection diagnostics.
Representatives of various virus families can be distinguished using an electron microscope. Negative contrast is a quick method for the display and morphological characterization of virus particles in suspension. Among other things, a virus infection can be detected directly from sample material. In addition, virus particles can be identified after replication in cell cultures. A prerequisite for a successful virus detection is a high number of virus particles in the material to be examined.
Very small virus particles (e.g. Parvoviridae) are even in an electrin microscope difficult to detect. Immune electron microscopy can help to faciliate the diagnosis. Virus particles are clotted using (gold) labelled antiviral antibodies and therefore easier to see.
Advantages: "catch-all-method", can find several pathogens at once
Disadvantages: high doses of virus needed, high expenditure on equipment.
Indications for the use of electron microscopic (EM) virus diagnostics include viral intestinal diseases and virus-induced skin and mucous membrane changes. Application examples for EM diagnostics are listed in the following table:

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Indirect immunofluorescence assay
Indirect immunofluorescence can be used to detect antibodies against virus antigens from patient sera. Test cells infected with a known virus isolate are incubated with the serum in question and the binding of antibodies from the serum is detected using a fluorochrome-labeled secondary antibody. Indirect immunofluorescence is used e.g. to detect antibodies against the feline coronavirus (FCoV, including pathogen of feline infectious peritonitis (FIP)), against the Borna disease virus (BoDV) of mammals and the avian Bornavirus (mainly PaBV).
Advantages: fast process with high sensitivity.
Disadvantages: high equipment expenditure, unspecific reactions possible (depending on the test material and the quality of the antibodies).

Indirect immunofluorescence Borna disease virus
ELISA (Enzyme-linked immunosorbent assay)
VVD uses a competitive ELISA for the detection of antibodies in sheep and goat sera against CAEV/MVV. The wells of ELISA test plates are coated with inactivated CAEV antigen, which specifically binds antibodies. Once serum antibodies are bound they block the binding of a second,  enzyme (HRP)-labeled CAEV specific monoclonal antibody. The enzyme of bound monoclonal antibody would react with a substrate added to the well and this reaction would lead to a visible change in colour. Previously bound serum antibodies block this clour change. Thus in such a competitive test the color intensity is reciprocally related to amount of antibodes in the respective sample (serum / plasma or milk), is measured using a photometer and compared with the corresponding positive and negative controls.
Advantage: fast method with high sensitivity.
Disadvantage: high complexity of equioment, unnspecific reactions possible(depending on sample material and antibody quality).
Immunoblot
We use the immunoblot for the detection of antiviral antibodies against feline immunodeficiency virus (FIV). Proteins of a defined virus preparation are separated according to their size with the aid of SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins are then transferred (blotted) to a nitrocellulose membrane. This membrane is then incubated with the test serum. After washing an enzyme-labelled species-specific antibody is added and further incubated.  Following final washing steps the membrane is inoculated with a substrate that is converted by the enzyme linked to the secondary antibody which leads to chemiluminescence.
Advantage: Because of its high specificity, the test is recommended as a confirmatory test for positive rapid test results.
Disadvantage: Time and  material consuming.
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Polymerase Chain Reaction (PCR)/ quantitative real time PCR (qPCR)
In the polymerase chain reaction (PCR), certain nucleic acid segments, for example from viruses, are multiplicated, separated through gel electrophoresis and finally made visible by staining.
The respective steps of a PCR test are as follows: double-stranded DNA is first  denatured at 94 ° C, then annealing (attachment of the primers) takes place at a temperature of 50-60 ° C and in the last step, a synthesis of the defined new DNA segment is carried out with the help of deoxyribonucleotides and a thermostable polymerase at 72 ° C. These 3 reaction steps are carried out one after the other until a measurable amount of the DNA fragment has been created through the exponential replication (about 30 cycles).
Primers are short, single-stranded sections of DNA that are complementary to the beginning and end of the sequence to be amplified. They are specific for each PCR and have to be chosen in order to create the PCR product of interest.
For RNA viruses, a reverse transcription (RT) step must precede the PCR. In this step RNA is transcribed into DNA so that the PCR steps can follow.
Virus-containing secretions / excretions (puncture, swabs, EDTA blood, liquor, urine, feces) and organ material are suitable as sample material.
The quantitative real-time PCR is based on the emission of light signals during the process of nucleic acid amplification. The amount of light signals correlates with the amount of amplified DNA and is related to a standard dilution series.
Advantages: very small amounts of DNA can be detected, high sensitivity, quick results, detection of viruses that cannot be grown on cell culture.
Disadvantages: high expenditure on equipment, possible false negative results due to non-fitting primers.
Immunofluorescence assay
Fluorochrome labelled antibodies can be used to detect viral antigen in cell preparations (e.g. of nasal swabs) or cell cultures (after virus isolation). The staining can be direct (antiviral antibody is fluorochrome labelled) or indirect ( a second fluorochrome labelled antibody binds to the antiviral antibody). In both cases a positive fluorescence signal shows the presence of the viral antigen.
Advantages: rapid assay, high sensitivity
Disadvantages: high equipment expenditure, unspecific reactions possible (depending on the test material and the quality of the antibodies).
Serum neutralization test
Virus neutralization can be used to detect and quantify neutralizing antibodies in serum samples. Therefore cytopathogenic virus strains are used that lead to visible cytopathic effect. If non cytopathogenic viruses are used (e.g. rabies virus) the cpe has to be made visible by immunofluorescence staining.
For this assay sera are diluted and incubated with a defined amount of serum for a certain time period. After addition of an indicator system (culture cells) the test is incubated for some days (usually 3-5 days, for some viruses 8 days can be necessary).
Using statistics the neutralization titer of the respective serum is calculated. A statement of the course of infection can only be made if a paired serum sample is tested. This assay can't differentiate between antibodies due to a vaccination and such due to a natural infection (even if a marker vaccine is used). In most cases a titer can't be related to a certain protectivity against a virus.
Advantages: high sensitivity and specificity.
Diasadvantages: time, material and staff consuming method; assay difficult to be standardized between laboratories due to different virus strains and cell culture systems used.
Fluorescent Antibody Virus Neutralisation (FAVN) assay
FAVN assay is a serum neutralization assay and serves to the determination of antibodies against rabies virus. Test sera are diluted and incubated with a constant amount of test virus, finally cells susceptible for the virus are added. NEutralizing antibodies in the test sera prevent a cytopathic effect in the used indicator cells. Virus reproduction is detected by direct immunofluorescence staining as rabies virus is a non cytopathogenic virus. Neutralization titer is calculated using the Spearman–Kärber method and indicated as international units (IU) compared to a determinated standard. This assay is used for vaccine efficiency control or as a prescribed test in accordance to travel purposes with dogs, cats and ferrets. More information regarding the latter you find here.
Immunodiffusion assay (Coggins assay)
Soluble viral antigen as well as antiviral antibodies are diffusing in an agar gel. A positive reaction always results in visible immune complexes, thin white precipitin lines (in the so called  zone of equivalence). This test can be used for either antigen or antibody detection. The "Coggins" assay is the prescribed method for the detection of antibodies against equine infectious viremia virus (EIAV). Test sera are applied alternating with positive control sera, the result can be seen after 48 hours.
Positive Coggins test

 
Hemagglutination inhibition assay (HI or HAI)
Some viruses (e.g. Influenzavirus) are able to agglutinate erythrocytes of certain animal species (e.g. chicken erythrocytes), so called hemagglutination. By pre-incubation of virus and antiviral antibodies a hemagglutination van be prevented, this is called hemagglutination inhibition.
In this assay test sera are diluted and incubated with a defined amount of test virus, finally erythrocytes are added. During the following incubation time non inactivated virus can agglutinate the erythrocytes. Agglutination doesn't happen in the presence of antiviral antibodies. Non agglutinated erythrocytes sediment quickly and are visible as red "buttons" at the bottom of the well.
 
left : complete HI, erythrocytes non agglutinated, visible "red button"; right: erythrocytes agglutinated resultung in a red "layer"