Virology
See also: Special requirements for testing of stock for overseas export
Virological examinations involve demonstration of a pathogenic virus or detection of antibody to virus. Findings must be interpreted in the light of history, clinical findings, lesions, etc.
It is not possible to screen for a wide range of viruses. Submitters should forward specimens to be tested for specific viruses. If there is any doubt about the availability of a test, the laboratory should be contacted for advice.
Diagnosis of viral disease
The diagnosis of a viral disease can be based on Histopathology, Virus isolation, Virus Serology, or Virus or Virus Antigen Detection.
Histopathology
Cytological changes can indicate a viral aetiology.
Virus isolation
Cultivation and identification of virus grown in tissue culture or eggs inoculated with specimen.
NB. Failure to cultivate the virus does not rule out a viral aetiology.
Cultivation of a virus does not necessarily mean it caused the disease process.
Viral serology
Detection and quantitation of virus specific antibody in the infected animal.
Presence of antibody can be a result of clinical disease, unapparent infection, passive immunity or vaccination.
Tests variously demonstrate group specific antibody, type specific antibody or a cross reaction.
Viral serology can be applied with the following limitations:
Single Serum
- Useful only to eliminate a diagnostic possibility. Presence of antibody in the acute phase may eliminate the diagnostic possibility; absence of antibody in the convalescent phase eliminates the diagnostic possibility.
- Accurate interpretation often difficult because time of collection may be critical. A negative test on serum < 3 weeks after a suspected viral condition leaves the possibility that the animal was infected but had not yet produced detectable antibody.
Paired Sera
- A change from negative to positive antibody status is known as seroconversion and indicates infection.
- A four-fold rise in antibody titre between acute and convalescent samples can also indicate infection by the specific pathogen.
However both the above may also be due to cross reaction or booster immunisation. They may also indicate stress induced reactivation of latent infection.
The relationship between virus and antibody varies between diseases. In some, virus does not occur in animals with detectable antibody, e.g., Akabane, Ephemeral Fever whereas in others, virus and antibody can be present in the one animal, e.g., EIA, IBR, CAE, EBL and occasionally Pestivirus.
NSW Department of Primary Industries offers the following types of viral serology tests:
- Agar Gel Immunodiffusion Test (AGID)
- Virus Neutralisation Test (VNT)
- Haemagglutination Inhibition (HI) Test
- Enzyme Linked Immunosorbent Assay (ELISA)
Agar Gel Immunodiffusion (AGID) Test
AGID involves diffusion of viral antigen and antibody towards each other through a gel. When they combine, they precipitate in the gel. This produces a visible line where the concentrations of antigens and antibody are balanced. An excess of antigen or antibody can alter the location and appearance of the precipitin line.
Each test sample for viral antibody is tested against a known viral antigen to examine the relationship of any precipitin line formed with an adjacent standard reference line formed between known positive serum and virus antigen. This reference line is optimised to give a strong visible precipitin line centrally located between the antigen and positive serum wells (termed a 3 reaction - see below).
For viral serology, a specific precipitin line formed by the tested serum sample is recorded as 1, 2, 3, or > 3 to describe its relative position to the serum well and the antigen well:
| Description/Position of Precipitin Line | Result |
|---|---|
| Turn on end of reference line | 1 |
| Line, closer to serum well | 2 |
| Line, midway between serum well and antigen well | 3 |
| Line, closer to antigen well | >3 |
A 1, 2, 3 or >3 antibody reaction is a positive test for antibody, and indicates that an animal has been infected with the specific virus (or a related virus).
Strength of viral antibody levels in the test generally does not reflect the severity and stage of infection with any certainty.
Virus Neutralisation Test (VNT)
Serum is usually titrated in two fold serial dilutions beginning at 1/4 or 1/10. The virus neutralising antibody titre is the reciprocal of the serum dilution that will neutralise a standard amount of virus (usually 100TCID50).
Two serial dilutions rise (i.e. 4-fold) in titre in the convalescent serum sample compared to the acute serum sample is considered significant. This occasionally occurs as a result of technical or statistical variation, so greater differences can be accepted with greater confidence.
Haemagglutination Inhibition (HI) Test
HI tests are used to detect antibodies to viruses which agglutinate erythrocytes. HI tests depend on antibody binding a fixed amount of antigen, preventing it from causing haemagglutination. Sera is tested in two-fold dilutions, and results expressed as a titre (negative, 2, 4, 8, .......) etc. More accurate measurement of flock status can be gained by repeat sampling of the flock to determine if a rise in titre (e.g. a rise in titre of two dilutions or more is arbitrarily considered significant) has occurred.
Enzyme linked immunosorbent assay (ELISA)
ELISA results can express the detection of antibody in a qualitative (+ or -) or a quantitative way. The three common means of expressing a quantitative result are absorbance (optical density), ELISA ratio and ELISA value (unit).
Absorbance (Optical Density)
The amount of antibody in a sample is proportional to the absorbance (optical density, OD) given by it. The OD may be standardised against one or more serum controls on the ELISA plate.
ELISA ratio
The ELISA ratio indicates the strength of the sample OD compared to that of a negative serum control on the same ELISA plate. (E/R = OD test/OD neg control).
ELISA value or ELISA units
The sample OD is fitted to a curve determined by the performance of control positive and negative samples, and the result given as an ELISA value (e.g 0 - 100). This is often a measurement of how the sample OD compares with that of a high positive control (taken as a value of 100).
Percent inhibition
The results of blocking or competitive ELISAs are expressed as the reduction in OD (as a %) relative to the OD given by a reference positive (often monoclonal) antibody.
ELISA ODs, ratios or values may be placed into categories (negative/inconclusive/positive) determined by previous experience of the performance of the test.
Virus or virus antigen detection
The following types of test are available for the direct detection of virus or viral antigen:
- Electron microscopy (EM)
- Agar Gel Immunodiffusion (AGID) Test
- Enzyme linked Immunosorbent Assay (ELISA)
- Latex Agglutination Test
- Haemagglutination (HA) Test
Electron microscopy (EM)
Allows detection and identification of virus particles on a morphological basis e.g. Rotavirus, Poxvirus.
Agar Gel Immunodiffusion (AGID) Test
Antigen detection is useful where virus infected tissues contain soluble virus antigens that visibly precipitate with specific antibody, eg:
- Parvovirus in mummified pig foetuses
- Pestivirus in gut scrapings of cases of mucosal disease
- Rotavirus in faeces.
In such cases, the sample is tested against a known positive serum and the relationship of any precipitin line formed with a standard reference line, originating from an adjacent positive antigen, is examined.
A test sample giving a line of similar position and intensity, which is continuous through a turn of identity with the reference line is classed as +++ for viral antigen.
The results are recorded in terms of relative strength (+, ++, +++, or XS) against the reference line.
Enzyme linked Immunosorbent Assay (ELISA)
Can be used to detect either virus antigen or intact virus particles. Results can be expressed qualitatively (+ or ) or quantitatively (OD's or ELISA ratios or ELISA values). eg:
- Pestivirus detection in the Pestivirus antigen capture ELISA (PACE).
Latex Agglutination Test
Can be used to detect viral antigen or virus particles. Results are only qualitative (+ or ), eg:
- Rotavirus detection
Haemagglutination (HA) Test
Can be used to screen for haemagglutinating viruses, eg:
- Newcastle Disease
- Avian Influenza
- Egg Drop Syndrome
On poultry tissue or allantoic fluid from eggs. Results can be given as a titre derived from a doubling dilution of the sample (e.g. 2, 4, 8, ...etc).
To determine which virus is responsible, HA positive samples can be tested in the presence of specific antibody in a direct (or reverse) HI test. The results are qualitative (+ or - ).
Standard Containers and Equipment
Blood vacuum tubes, silicone coated
For serum samples and blood clots (for isolation of, or detection of antigen to, some viruses). Please do not use serum separator tubes.
Blood vacuum tubes with heparin or EDTA
For blood for virus isolation (of mainly the arboviruses) or preparation of buffy coat samples for antigen detection.
Sterile bottles for samples of tissues, organs
Sterile 5 ml vials: For serum and fluid samples.
Swabs and PBGS bottles: For discharges, etc. Plain sterile swabs are used and immediately placed in small bottles of phosphate buffered gelatin saline (PBGS). These are available from the Regional Veterinary Laboratories.
Sterile instruments
An adequate range of sterile scissors and tissue forceps should be available.
The containers and equipment listed are suitable for the routine collection of tissues for virus examination. Containers and equipment for special requirements will normally be supplied by the laboratory, after prior arrangements have been made.
Collection of Specimens
Specimens for virus isolation must be collected by aseptic techniques, using sterile instruments and sterile containers. Avoid contamination from other tissues as well as that from extraneous sources. Submissions for virus isolation should be accompanied by serum from the affected animal(s) whenever possible.
Blood
This should be collected using the appropriate blood vacuum tube, avoiding contamination of the sample. A separate needle should be used for each animal.
Avoid contamination of the outside of the tube by blood, soil and faeces, as this creates difficulties in handling the specimens within the laboratory.
Serum
If blood samples cannot arrive at the laboratory within 2 days, the serum should be poured off aseptically into a sterile 5 ml vial. The clot should also be submitted as it may be required for virus isolation or antigen detection.
Tissues and organs
These must be collected aseptically, using separate sterile instruments and containers for each tissue or organ. Contamination between tissues must also be avoided.
Portions of tissue (no greater than 2 cm x 2 cm x 2 cm) should be taken.
Scabs
Scabs and underlying tissue should be submitted in a sterile bottle.
Swabs
Swabs from excretions, exudates, mucosal surfaces and orifices should be taken carefully, avoiding contamination from other sites.
Each swab should then be transferred to PBGS at room temperature, then chilled (but never frozen) for transport to the laboratory.
Faeces
At least 10 g of fresh faeces should be collected into a clean jar.
Storage and Despatch of Specimens
All virological specimens should be chilled prior to and during transport. If more than 48 hours is to elapse between collection and receipt at the laboratory, specimens except blood samples should generally be frozen.
For isolation of Arboviruses (especially Bluetongue) and Herpesviruses (e.g. IBR), specimens should never be frozen. These viruses have extremely poor survival at 20°C with just a single freeze, and tissues for their isolation must be kept chilled.
All specimens should be clearly labelled and sent in a leak-proof container. Check that screw caps are tight, especially after freezing.
All samples should be packed in insulated containers with sufficient icebricks to ensure that they are still cold when received at the laboratory. However, care should be taken to prevent direct contact between coolant bricks and specimens, which may otherwise become frozen.