Effect of stabilizers on the detection of swine influenza A virus (swIAV) in spiked oral fluids over time

Aggregated samples such as oral fluids (OFs) display an animal friendly and time and cost-efficient sample type for swine Influenza A virus (swIAV) monitoring. However, further molecular and biological characterization of swIAV is of particular significance. The reportedly inferior suitability of aggregated samples for subtyping of swIAV presents a major drawback compared to nasal swabs, still considered the most appropriate sample type for this purpose (Garrido-Mantilla et al. BMC Vet Res 15(1):61, 2019). In addition, the viral load in the original sample, storage conditions and characteristics of different swIAV strains might further compromise the eligibility of aggregated samples for molecular detection and subtyping. Therefore, the present study aimed to evaluate the suitability of stabilizing media to minimize the degradation of viral RNA and thus increase the detection and subtyping rate of swIAV by RT-qPCR in spiked OFs under different conditions (virus strain, storage temperature and viral load in the original sample) over a time span of 14 days.

The use of stabilizing media in spiked OFs resulted in a significant higher probability to detect swIAV RNA compared to OFs without stabilizers (OR = 46.1, p < 0.001). In addition, swIAV degradation over time was significantly reduced in samples suspended with stabilizer (OR = 5.80, p < 0.001), in samples stored at 4 °C (OR = 2.53, p < 0.001) and in samples spiked with the avian derived H1N2 subtype (OR = 2.26, p < 0.01). No significant differences in swIAV RNA detection and degradation of swIAV RNA in spiked OFs over time were observed between the three different stabilizing media.

Addition of stabilizers and storage of samples under cooled conditions significantly improved detection and subtyping of swIAV in spiked OFs.