Porcine sample type characteristics associated with sequencing and isolation of Influenza A virus

Authors: MORAES, DANIEL - Iowa State University; OSEMEKE, ONYEKACHUKWU - Iowa State University; ZELLER, MICHAEL - Iowa State University; Baker, Amy; SILVA, GUSTAVO - Iowa State University; TREVISAN, GIOVANI - Iowa State University; LINHARES, DANIEL - Iowa State University; GAUGER, PHILLIP - Iowa State University

Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/14/2025
Publication Date: N/A
Citation: N/A

Interpretive Summary: Influenza A virus (IAV) is a respiratory pathogen affecting swine worldwide and is a public health concern since IAV can be shared between humans and pigs. Diagnostic testing and sequencing at veterinary diagnostic laboratories are necessary for applying appropriate intervention strategies to reduce the impact of disease and potential for spreading. Testing for IAV in swine largely relies on the expense of the producer and veterinarian consultation of the appropriate tests and number of animals. Thus, profitability may influence the timely diagnosis and characterization of IAV on swine farms. The objective of this study was to evaluate the probability of success for IAV hemagglutinin (HA) and neuraminidase (NA) Sanger sequencing and virus isolation in Madin-Darby Canine Kidney (MDCK) cells across different porcine sample types submitted to the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) from 2018 to 2024. These results provide practical expectations for successful IAV HA and NA gene sequencing and virus isolation based on sample type and screening criteria to improve diagnostic testing strategies in swine populations. This information will enable veterinarians to provide cost-effective testing strategies for their producer clients.

Technical Abstract: Understanding how sample type may influence the probability of Influenza A virus (IAV) sequencing and isolation success can help improve the use of diagnostic tests and refine surveillance strategies in swine populations. The objective of this study was to evaluate the probability of success for IAV hemagglutinin (HA) and neuraminidase (NA) Sanger sequencing and virus isolation in Madin-Darby Canine Kidney (MDCK) cells across different porcine sample types submitted to the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) from 2018 to 2024. Antemortem and postmortem sample types were selected and analyzed based on the reverse transcription real-time polymerase chain reaction (RT-rtPCR) cycle threshold (Ct) value. The Ct values corresponding to a 95% probability of sequencing or virus isolation success were determined for each sample type. For antemortem samples, nasal swabs had a Ct value of 27.8 for HA Sanger sequencing from 1,046 samples and 23.6 for NA sequencing based on 66 samples. Oral fluids had a Ct value of 27.3 from 3,446 samples and a Ct value of 22.1 for NA Sanger sequencing from 137 samples. For postmortem samples, lung tissue had the highest number of samples sequenced for the HA and NA, with Ct values of 25.7 and 21.5, respectively. For a 95% probability of detection in virus isolation, nasal swabs had a Ct value of 21.1 from 647 samples, while lungs had a Ct value of 18.7 from 5,892 samples. This study identified nasal swabs and lung tissue as the samples with the highest probability of IAV sequencing by the Sanger technique and virus isolation success, while oral fluids, a common swine diagnostic sample type that is easy to collect and welfare-friendly, can be effective for sequencing when lower Ct values, i.e., 27.3, are detected in the sample. These results provide practical expectations for successful IAV HA and NA gene sequencing and virus isolation based on sample type and RT-rtPCR Ct values to help improve diagnostic testing strategies in swine populations.