Detection of swine influenza virus in nasal specimens by reverse transcription-loop-mediated isothermal amplification (RT-LAMP).

Detection of swine influenza virus (SIV) in commercial swine herds is important for understanding the infection status of the herd and for controlling disease. Current molecular diagnostics require that specimens be submitted to a laboratory which provides results to the growers after some time which is generally too late to intercede in disease control. Moreover, current diagnostic assays are time-consuming, typically costly, and require skilled technical expertise. We have instituted a reverse transcription loop-mediated isothermal amplification (RT-LAMP) diagnostic assay based on conserved regions of the SIV matrix (M) gene and H1N1 hemagglutinin (HA) sequences. The RT-LAMP assay was optimized to use both colorimetric and fluorescent endpoints and was validated. The M and HA RT-LAMP assays have a limit-of-detection (LOD) sensitive to 11 and 8-log-fold dilutions of viral RNA, respectively, and are capable of discriminating between H1 and H3 strains of SIV. Additionally, the RT-LAMP assay was optimized for direct amplification of SIV from field samples without the need for viral RNA isolation. The direct RT-LAMP detected >86 % of qRT-PCR validated SIV samples, and >66 % of negative samples when spiked with viral RNA or SIV. The diagnostic RT-LAMP assay is a rapid, sensitive, specific, and cost-effective method for the detection of SIV in herds substantially aiding diagnosis and surveillance.

Molecular epidemiology and glycomics of swine influenza viruses circulating in commercial swine farms in the southeastern and midwest United States.

Tracking the genetic diversity and spread of swine influenza viruses (SIVs) in commercial swine farms is central for control and to reduce the potential emergence of SIV reassortants. We analyzed the diversity of SIVs in nasal washes or oral fluids from commercial swine farms in North Carolina using influenza M qRT-PCR and hemagglutinin (HA) and neuraminidase (NA) subtyping. We found a predominance of H1 HAs and N2 NAs in the samples examined. The majority of the H1 HAs could be further classified into gamma and delta subclusters. We also identified HAs of the H1 alpha cluster, and those of human novel pandemic origin. Glycan binding profiles from a representative subset of these viruses revealed broad α2,6 sialylated glycan recognition, though some strains exhibited the ability to bind to α2,3 sialic acid. These data show that SIV surveillance can aid our understanding of viral transmission dynamics and help uncover the diversity at the human-swine interface.