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.

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.

Genetic variations of nucleoprotein gene of influenza A viruses isolated from swine in Thailand.

BACKGROUND: Influenza A virus causes severe disease in both humans and animals and thus, has a considerably impact on economy and public health. In this study, the genetic variations of the nucleoprotein (NP) gene of influenza viruses recovered from swine in Thailand were determined. RESULTS: Twelve influenza A virus specimens were isolated from Thai swine. All samples were subjected to nucleotide sequencing of the complete NP gene. Phylogenetic analysis was conducted by comparing the NP gene of swine influenza viruses with that of seasonal and pandemic human viruses and highly pathogenic avian viruses from Thailand (n = 77). Phylogenetic analysis showed that the NP gene from different host species clustered in distinct host specific lineages. The NP gene of swine influenza viruses clustered in either Eurasian swine or Classical swine lineages. Genetic analysis of the NP gene suggested that swine influenza viruses circulating in Thailand display 4 amino acids unique to Eurasian and Classical swine lineages. In addition, the result showed 1 and 5 amino acids unique to avian and human lineages, respectively. Furthermore, nucleotide substitution rates showed that the NP gene is highly conserved especially in avian influenza viruses. CONCLUSION: The NP gene sequence of influenza A in Thailand is highly conserved within host-specific lineages and shows amino acids potentially unique to distinct NP lineages. This information can be used to investigate potential interspecies transmission of influenza A viruses. In addition, the genetic variations of the NP gene will be useful for monitoring the viruses and preparing effective prevention and control strategies for potentially pandemic influenza outbreaks.