Detection of Avian Influenza Virus Subtype H5 Using NASBA

Nucleic acid sequence-based amplification with electrochemiluminescent detection (NASBA/ECL) of avian influenza virus subtype H5 was developed,comparing with virus isolation in embryonated chicken eggs. The NASBA technique described here can detect three isolates of infuenza A subtype H5 and do not detect infuenza A subtype H1,3,6,9, other avian viruses and allantoic fluid of SPF chicken, showing rapidness and high specificity for identificating infuenza A subtype H5 viruses. Dilution of a known virus was used to determine the limit of sensitivity for both NASBA and classic virus isolation. The NASBA/ECL method was equivalent in sensitivity to virus isolation in eggs,with limit of 10+{-1.5} ELD-{50} dose. Virus was isolated from anal swabs, blood and tissues of chickens artificially infected with highly pathogenic avian influenza A/Chicken/Hong Kong/1000/97 (H5N1). NASBA/ECL could detect viral nucleic acid in anal swabs from the day following artificial infection until death. Conversely, Nucleic acid molecule