Virus - PCR - PRRS diagnostics

Virus detection techniques provide a useful indicator of disease, although some are used primarily as research tools because they are labour-intensive, expensive and slow. One such example is virus isolation, which uses specific cell lines to amplify viruses from tissue samples through a process of infection and replication. Another is electron microscopy, which can play an important role in characterising new strains not recognised by specific molecular or antibody approaches. Newer, rapid and less costly techniques, described below, are now widely used, being more practical for routine and timely diagnosis.

Reverse transcriptase polymerase chain reaction
Reverse transcriptase polymerase chain reaction (RT-PCR) is a rapid, highly sensitive and specific method used to detect PRRSv in a range of tissues (including serum, semen, oral fluids, lung, foetuses, lymph nodes, spleen and tonsils), and also in environmental samples. The process involves extraction of viral RNA from the biological sample, conversion to DNA by reverse transcriptase, amplification by PCR, and detection of amplified DNA.



A drawback of RT-PCR is that the window for detection of virus after infection differs both between tissues sampled and between animals. Infection of both young animals and animals naïve to PRRSv leads to higher and more long-lasting detectable virus loads than for mature animals. Furthermore, the high sensitivity of RT-PCR can result in false positive results due to contamination, whereas the specificity of primers may result in false negative results for some genetically diverse viruses. As these data can impact significantly on herd management decisions, secondary testing of samples using alternative strategies is advisable where appropriate. RT-PCR is also unable to differentiate between wild-type and attenuated virus, an issue particularly relevant when assessing PRRSv in environmental samples.

Immunohistochemistry
Immunohistochemistry (IHC) is used in conjunction with microscopy to detect PRRSv in tissue from a number of sites, including lung, lymph nodes, thymus, tonsil, spleen and kidney. Typically, tissues are first fixed with formalin (although fresh tissues can also be used), and then labelled with monoclonal antibodies targeting the viral nucleocapsid. These are visualised using a tagged secondary antibody. While IHC has high specificity (100%), it shows only moderate sensitivity (67%). This can, however, be improved by processing of tissues within 48 hours of fixation. Sensitivity is highest at early stages of infection, but progressively decreases during later stages, such as more than  28 days post infection (DPI), and so is not recommended after 90 DPI. While IHC is effective in identifying vertically transmitted virus in piglets, it may not be effective for some genetically diverse isolates.



Fluorescent antibody staining
Fluorescent antibody (FA) staining, typically used for evaluation of BAL fluid or fresh/frozen lung tissue, is seldom used today. In this technique, samples are labelled directly with fluorescent antibodies to visualise nucleocapsids. This process is quicker and cheaper than IHC for measuring PRRSv, but, as with IHC, may not detect genetically diverse isolates and is not recommended in the later stages of infection. Furthermore, false positives can be an issue due to poor specificity of the primary antibody or high background staining. In both cases, positive tests are ideally confirmed using RT-PCR (or virus isolation).
 


Field test strips
Field test strips are a relatively new development in PRRSv diagnostics and, although not yet widely used, have the potential to provide rapid and convenient PRRSv detection without the need for specialised expertise or equipment. Field test strips detect virus in serum or tissue homogenates by immunochromatography. Samples are mixed with gold-labelled monoclonal antibodies targeting specific PRRSv proteins, forming protein-antibody complexes which are captured on the strips. Captured complexes are then detected using a labelled secondary antibody. In Chinese studies, test strips have demonstrated a sensitivity of over 93% and specificity of over 96%, although current data are available only on a limited range of viruses.