Viral diagnostics for animal diseases

I.SophiaP-1699VBM

substances or devices which aid in the diagnosis of viral infections

Classification of viral diagnostic tests

(i) direct virus detection

(ii) viral antigen detection

(iii) virus isolation

(iv) viral antibody detection.

Viral diagnostics

Electron microscopy, immunoelectron microscopy

Histopathology

Fluorescent antibody technique – direct, indirect

Immunohistochemistry

ELISA

Immunochromatography

Latex agglutination test

Direct identification of viruses

Electron microscopy

ELECTRON MICROSCOPY

Rapid diagnosis

No need of additional probes Disadvantage

Procurement and maintenance of the instrument – expensive

Require skilled & well trained personnel for operation.

Sensitivity is comparatively low – require at least 10 6 viral particles for + ve diagnosis.

Single specimen examined at a time.

Applications: Rotavirus from faecal samples, rabies virus from

brain, norwalk virus from faecal samples

Employs viral antibody

Increases the sensitivity & specificity of EM

Two methods (i) classical IEM (ii) solidphase IEM

classical IEM: Requires prior incubation of the clinical sample with virus specific antibody .

Solidphase IEM: solid support (copper grid) is coated with specific antibody which captures viruses from the clinical sample.

Immunoelectron microscopy (IEM)

Helps in diagnosis of viral inclusion bodies

Viral inclusion bodies: Aggregates of viral nucleocapsids in the cytoplasm or nucleus.

Can be detected by H&E staining

Applications

Negri bodies in rabies virus

Guarnieri bodies – vaccinia virus

Bollinger bodies - fowlpox

Histopathology

Use of two specific antibodies – nanoparticle conjugated antibody, antibody fixed in chromatography paper.

The sample dropped on sample pad – forms complex with the antibody. When moves along the membrane pad- sandwiched between two antibodies- production of colour.

diagnosis of avian influenza, dengue etc.

IMMUNOCHROMATOGRAPHY

Particulate antigen +virus specific antibody - crosslinking of polyvalent antigens – agglutination.

Visibility can be improved by coating the latex beads with virus specific antibodies (sensitized latex beads).

Sensitized latex beads + clinical sample (containing viruses) visible aggregates.

Applications: Avian influenza Rota virus FMD (Sugimura et al.,2000)

Latex agglutination test

Fluorescence antibody technique (FAT)

Fluorochromes like fluorescent isothiocyanate and rhodamine isothiocyanate. Are used.

Viral antigens can be detected from smears from clinical samples, frozen tissue sections and also from formalin fixed tissue samples.

Direct Fluorescence antibody technique The virus specific antibodies are directly tagged with fluorescent dyes.

Indirect fluorescence antibody technique (IFAT)second antibody known as anti-species immunoglobulin (Igs) are used.

Advantage: Available easily commercially than the virus specific antibodies More sensitivity than direct FAT. Better signal amplification

Immunohistochemistry

Principle Employs antibodies conjugated with enzymes like horseradish peroxidase or

alkaline phosphatase.

When suitable substrates are used, colour reaction takes place in case of positive sample which can be viewed through light microscope.

Both direct and indirect immunoperoxidase tests are used.

Advantages The procedure can be applied for formalin fixed tissues as well.

Comparative study of site of localization of viruses and the tissue damage is possible

Sandwich ELISA

The viral antigen is sandwiched between two antibodies namely capture and detection antibodies. For the assay both the antibodies should target different epitopes of the antigen. This is followed by addition of enzyme conjugated anti-species immunoglobulin. On adding suitable chromogen-substrate colour reaction develops.

AdvantagesAssay is quantitative, amount of viral antigen can be detectedAssay has high sensitivity and specificityMore samples can be tested at the same time

DisadvantagesNeed ELISA reader for result interpretation; not possible under field conditionsThe method is time consuming and labourious.Applications: Used in diagnosis of PPR, Bluetongue, FMD etc

Immunoblotting

Applications: useful in differentiation of infected and vaccinated animals in FMD using recombinant proteins (Fu et al., 2011).

Complement fixation test

Principle The ability of the complement system to fix the antigen- antibody complex

forms the basis of complement fixation test. Sheep RBCs and its corresponding antibody acts as indicator system. When the antigen reacts with specific antisera, antigen antibody complex will

be formed and the complement will be unavailable for the indicator system. Then there will be no haemolysis and the test is positive.

If the antiserum is not specific, then the complement is free to fix the indicator system resulting in haemolysis.

Sensitive assay like ELISA have replaced it. Simple; easy to perform.

Applications Complement fixation test is prescribed by the OIE for diagnosis of equine

diseases like African horse sickness, equine encephalomyelitis (eastern, western, Venezuelan), vesicular stomatitis.

Haemagglutination inhibition tests

Principle Certain viruses namely paramyxoviruses and orthomyxoviruse have the ability to bind to sialic acid residues in RBCs. This property is called haemagglutination. If specific antibody and viruses are mixed prior to the addition of RBCs haemagglutination is inhibited. HI test is used for serotyping and also for measuring antibody titre.

Applications OIE recommends this test for diagnosis of viral infections like blue tongue,

avian laryngeo tracheitis, avian influenza, marek’s disease, infectious bursal disease, enzootic bovine leucosis, equine infectious anaemia (Coggin’s test), myxomatosis and caprine arthritis encephalitis disease.

Complement fixation test

-ve

+ve

Virus neutralization test

Principle When a serum sample to be tested is specific for the virus, there will be

antigen-antibody complex formation. The virus particles are unavailable to cause cytopathic effect in cell culture which can be visualized microscopically or by staining with vital dyes.

Applications Prescribed test by OIE for diagnosis of pseudorabies, rabies, Riftvalley fever,

vesicular disease and IBRT-IPV.

Plaque reduction neutralization test

Principle The ability of virus specific antibody to inhibit the plaque forming property of viruses by 50% when plated in semisolid media is the basic principle involved.

ApplicationsPRNT is recommended by the OIE for diagnosis of eastern equine encephalitis and Venezulean equine encephalitis disease.

Disadvantages:The interpretation of results requires at least 3-5 days which is very slow when compared to EIA and RIA.Both the tests involve use of cell culture where false results due to contamination of cell culture are possible.

Agar gel immunodiffusion test

Principle It is basically a precipitation test in which soluble antigen reacts with its

homologous antibody in the presence of electrolytes. At the optimum proportion, an insoluble antigen-antibody precipitate will be formed.

Applications OIE recommends this test for diagnosis of viral infections like blue tongue,

avian laryngeo tracheitis, avian influenza, marek’s disease, infectious bursal disease, enzootic bovine leucosis, equine infectious anaemia (Coggin’s test), myxomatosis and caprine arthritis encephalitis disease.

Counter immunoelectrophoresis

PRINCIPLE Counter immunoelectrophoresis is the immunodiffusion modified by electrophoresis to drive antigen and antibody towards each other. The specimen to be tested is placed in the cathode side, and the antiserum is placed in the anode. At neutral or alkaline pH, antigens are negatively charged and hence migrate towards anode in barbital or veronal buffer.

Applications: Counter immunoelectrophoresis is used in diagnosis of blue tongue virus, orf virus and pox viral diseases

Radio immunoassay

Principle It is the competitive binding assay which binding of known amount of radiolabeled substrate and antibody. When the test sample is added, the radiolabeled substrate gets displaced by it resulting in free radiolabelled substrate in the solution. Finally, the radioactivity of free substrate in the solution is measured which is proportional to the amount of unknown antigen bound to antibody.The assay is more sensitive and specific compared to the above methods.Disadvantage:Assay needs sophisticated instruments like gamma counter.Radioactive compounds are hazardous to human health ie. Carcinogenic. Therefore, it is not routinely used for viral diagnosis.

Indirect ELISA

PRINCIPLE The viral antigen is immobilized in a solid support followed by addition of

serially diluted antibodies. The antibodies in turn are captured by enzyme conjugated anti-species immunoglobulin.

Addition of chromogen / substrate facilitates the colour reaction which can be measured calorimetrically.

Currently, use of recombinant viral proteins as antigens reduce the risk of handling of dangerous organisms and made the assay sensitive and specific.

AdvantagesSimplicity; easy to performRapid than neutralization assaysSafer: Use of enzymes instead of radiolabels Require ELISA reader for result interpretation

Nucleic acid based diagnostics

PCR

RFLP

Microarray

Hybridization techniques

RFLP

The nucleotide signatures of each species are unique. This fact is explored in

the technique where endonucleases recognizing restriction sites are used to cut

the genome at various sites for each organism. The genome fragments are

resolved by running in agarose gel electrophoresis. The characteristic nucleotide

base pair length corresponds to each organism.

PCR

Real time PCR

MICROARRAY

Nanobiosensor

There are various types of nanobiosensors based on various principles namely, electrochemical biosensors, voltametric and amperometric sensors, impedance sensors, optical fiber based sensors, surface plasmon resonance based biosensors, quartz crystal microbalance and atomic force microscopy based nanobiosensors.

Nanobiosensors have been designed for diagnosis of dreadful human viral diseases like HIV, hepatitis B, Hepatitis C, Ebola virus etc. Nanobiosensors have also been designed for diagnosis of animal diseases like avian influenza virus, infectious bovine rhinotracheitis, rabies, bovine leukemia virus.