The effect of sample type, temperature and RNAlater on the stability of avian influenza virus RNA.

As a result of continuing worldwide outbreaks of highly pathogenic avian influenza (HPAI) caused by the Asian lineage of H5N1, surveillance of targeted avian species in selected regions has been implemented. In these wild bird surveys, the use of real-time reverse transcription (rRT)-PCR has proved to be an invaluable tool as a frontline screening assay for the detection of avian influenza virus (AIV) RNA. However, verification of HPAI diagnosis, particularly in a primary outbreak situation, requires confirmation by a national, community or world reference laboratory. This may necessitate freezing and thawing of samples, sub-sampling and transportation to the reference laboratory. The deleterious effects of such handling on the infectivity of virus and the yield of viral RNA have been observed. The objective of this study was to investigate the effects of freezing and thawing, time, sample type and transportation on the yield of AIV RNA. Additionally, the effect of the RNA stabilisation agent, RNAlater was investigated. It was demonstrated that the quality of AIV RNA in faecal homogenate was markedly reduced by freezing and thawing, but that treatment with RNAlater protected the viral RNA from deterioration. When using RNAlater even low titre AIV samples were protected from the detrimental effects of time and transportation conditions.