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Title: 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

Author
item THONTIRAVONG, AUNYARATANA - Chulalongkorn University
item TANTILERTCHAROEN, RACHOD - Chulalongkorn University
item TUANUDOM, RANIDA - Chulalongkorn University
item SRETA, DONREUTHAI - Chulalongkorn University
item THANAWONGNUWECH, ROONGROJE - Chulalongkorn University
item AMONSIN, ALONGKORN - Chulalongkorn University
item ORAVEERAKUL, KANISAK - Chulalongkorn University
item Kitikoon, Pravina

Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/29/2011
Publication Date: 9/1/2011
Citation: Thontiravong, A., Tantilertcharoen, R., Tuanudom, R., Sreta, D., Thanawongnuwech, R., Amonsin, A., Oraveerakul, K., Kitikoon, P. 2011. 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. Journal of Veterinary Diagnostic Investigation. 23(5):1017-1021.

Interpretive Summary: The introduction of the novel Influenza A H1N1 virus (pH1N1) from human into the pig population calls for a rapid and effective assay to detect and discriminate pH1N1 from the various endemic swine H1N1 (sH1N1) viruses currently circulating in pigs. Because the genetic background of sH1N1 viruses in Thai pigs differ from the ones circulating in North American and European pigs presently available reverse transcription-polymerase chain reaction (RT-PCR) based assays can not be used. This study developed a multiplex RT-PCR test that can simultaneously subtype and screen for the presence of pH1N1 virus infection in Thai pigs. The assay was shown to be accurate and can be used to effectively screen nasal swab samples for the presence of pH1N1 virus. This test will benefit the diagostic laboratory and Thai swine practioners and veterinarians as it can provide rapid pH1N1 test results that will contribute to the disease control management in a timely manner.

Technical Abstract: A recently emerged H1N1 Influenza A virus (pandemic 1 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 wide spread 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 human influenza H1N1 (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 (TCID50/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 swine herds, where genetics of the endemically circulating SIV differ from the strains circulating in North American and European herds.