Single-step multiplex reverse transcription polymerase chain reaction assay for detection and differentiation of the 2009 H1N1 Influenza A virus pandemic in Thai swine populations

Abstract

A recently emerged H1N1 Influenza A virus (pandemic H1N1 (pH1N1)) with a Swine influenza virus (SIV) genetic background spread globally from human-to-human causing the first influenza virus pandemic of the 21st century. In a short period, reverse zoonotic cases in pigs followed by a widespread of the virus in the pig population were documented. The implementation of effective control strategies, rapid diagnosis, and differentiation of such virus from endemically circulating SIV in the various swine populations of the world is needed. To address the problem, a multiplex reverse transcription polymerase chain reaction assay utilizing a combination of the PB1, H1, and N1 primers that can rapidly and simultaneously subtype and screen for the presence of pH1N1 virus infection in Thai pigs was developed. The assay had 100% specificity and did not amplify genetic material from other subtypes of SIV, seasonal H1N1 human influenza (huH1N1) virus, highly pathogenic influenza H5N1 virus, and other important swine respiratory viral pathogens. The assay was able to both detect and subtype pH1N1 virus as low as 0.1-50% tissue culture infective doses/ml (TCID(50)/ml). The assay was used to screen 175 clinical samples obtained from SIV suspected cases, of which 6 samples were pH1N1 positive and were confirmed through virus isolation and whole genome sequencing. The results of the study suggested that the assay would be useful for the rapid diagnosis of pH1N1 in suspected Thai swineherds, where genetics of the endemically circulating SIV differ from the strains circulating in North American and European herds.