DETECTION OF ROSS-RIVER-VIRUS IN CLINICAL-SAMPLES USING A NESTED REVERSE TRANSCRIPTION-POLYMERASE CHAIN-REACTION

Background: Ross River virus (RRV) is a mosquito borne alphavirus that has been found in Australia, Papua New Guinea and the Pacific Islands. It is the aetiological agent of epidemic polyarthritis, a debilitating illness whose symptoms are arthritis, arthralgia, lethargy, rash and fever which may persist for weeks or months. Diagnosis is made on a serological basis, but in many cases is presumptive rather than definite. Objectives: To apply the polymerase chain reaction (PCR) to detection of RRV in human sera to assess its suitability for application in disease diagnosis. Study design: Sensitivity of the nested RT-PCR assay was determined by detection of virus of known titre diluted in uninfected serum. Clinical serum samples from patients serologically diagnosed of having RRV infection were tested by nested RT-PCR to assess its diagnostic value. Results: Sensitivity of the nested RT-PCR assay was determined to be detection of 0.01 PFU of virus stock in 100 mu l serum. Clinical samples tested showed that 10 of 26 (38%) serum samples with low or negative (non-diagnostic) virus-specific antibody titres were PCR-positive, whereas all 22 specimens with high antibody titres were PCR-negative. PCR positivity was unaffected by repeated freezing and thawing of samples. Conclusions: While PCR cannot replace serology as a means of RRV diagnosis, it may be useful in conjunction with serological testing, particularly for forming definitive diagnoses in those samples with low (inconclusive) antibody titres. It is faster and more sensitive than virus isolation by tissue culture, and could also prove useful in investigations of disease pathogenesis.