Systematic Veterinary Virology

LABORATORY DIAGNOSIS

Isolation and identification

• Clinical materials
• i. Faeces from affected animals or from dead animals is the material of choice.
• ii. Loops of affected small intestine, ligated at each end to retain the contents are also the preferred specimens for isolation of virus.
• Isolation systems: TGEV does not grow well in cell culture. However, they grow in 3–4-day-old primary or secondary pig kidney monolayers and low passage porcine cultures (such as thyroid or testis).
• The sample material is homogenised in cell culture medium or phosphate buffered saline (PBS), pH 7.2, containing antibiotics.
• This homogenised sample is allowed to stand out off direct sunlight for 30 minutes at room temperature.
• The suspension is then sonicated and clarified by low-speed centrifugation.
• The supernatant fluid is mixed with an equal volume of heat-inactivated bovine serum in order to reduce the cytotoxic effect of the material and it is then used to inoculate susceptible tissue cultures.
• The cytopathic effect (CPE) may be observed after 3–7 days, characterised by cell rounding, enlargement and formation of syncytia. Wild-type TGEV (virus obtained from an outbreak in field is referred as wild type) does not grow readily in tissue culture, so several subpassages may be necessary before distinctive CPE become apparent.
• Antigen identification tests: Since CPEs are not produced immediately and require number of blind passages, following antigen identification tests are performed.
• Immunofluorescence
• Reversed passive hemagglutination
• Enzyme-linked immunosorbent assays (ELISAs)
• Radioimmunoassay (RIA)
• DNA probes
• Electron microscopy
• RT-PCR
• Identification of antigen directly in tissue sections/clinical materials
• Fluorescent antibody test: Used to identify viral antigens in sections of small intestine
• Double antibody sandwich ELISA: This test is based in the capture of the viral antigen from the faecal sample by three MAb, two specific for the S protein (site A and D) and one for the nucleoprotein N.
• In situ hybridisation (ISH) and RT-PCR: This technique is used for the direct detection of TGEV in clinical samples and for differentiation from PRCV.