# Porcine Sample Type Characteristics Associated with Sequencing and Isolation of Influenza A Virus

## TL;DR

This study identifies which pig sample types are best for detecting and isolating influenza A virus, helping improve surveillance and diagnostic testing strategies.

## Contribution

The study provides specific Ct value thresholds for successful virus sequencing and isolation across different sample types in pigs.

## Key findings

- Nasal swabs and lung tissue had the highest probability of successful influenza A virus sequencing and isolation.
- Oral fluids can be effective for gene sequencing but require lower Ct values for success.
- Ct thresholds for 95% success varied by sample type and gene, such as 27.8 for HA in nasal swabs and 25.7 for HA in lung tissue.

## Abstract

Influenza A virus is frequently detected in pigs worldwide, and surveillance through diagnostic testing is important for understanding virus circulation on farms. This study assessed different sample types and the probability of successfully identifying and growing the virus in the veterinary diagnostic laboratory. Scientists analyzed thousands of samples submitted to a diagnostic laboratory between 2018 and 2024. Nasal swabs and lung tissue samples had the best results, with a high chance of successfully identifying and isolating the virus when present in sufficient quantities. Oral fluids, which are easier and less stressful to collect from pigs, also worked well, but virus levels had to be much higher. These findings help veterinarians and researchers choose the best sample types for influenza A virus surveillance and understand how likely they are to obtain useful results from testing based on the amount of virus present in the sample. This can better inform veterinarians before requesting diagnostic tests, such as sequencing and virus isolation, leading to more effective monitoring in pigs, which is important for animal health and for preventing possible spread to humans.

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 reverse transcription real-time polymerase chain reaction (RT-rtPCR) cycle threshold (Ct) values. The Ct values corresponding to 95%, 75%, and 50% probabilities of sequencing or virus isolation success were determined for each sample type. For antemortem samples, a 95% probability of success for HA Sanger sequencing on nasal swabs exhibited a Ct value of 27.8 from 1046 samples and 23.6 for NA sequencing based on 66 nasal swabs. Using oral fluids, HA and NA Sanger sequencing success was at Ct values of 27.3 from 3446 samples and 22.1 from 137 samples, respectively. For postmortem samples, lung tissue had the highest number of sequences for the HA and NA, with Ct values of 25.7 and 21.5, respectively. For a 95% probability of successful virus isolation, nasal swabs demonstrated a Ct value of 21.1 from 647 samples, while lungs had a Ct value of 18.7 from 5892 samples. This study determined that nasal swabs and lung tissue had the highest probability of IAV gene sequencing 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 gene sequencing when using lower IAV RT-rtPCR Ct values, i.e., ≤27.3. These results provide practical expectations for successful IAV HA and NA gene sequencing and virus isolation at 95%, 75%, and 50% probabilities based on sample type and RT-rtPCR Ct values to improve diagnostic testing strategies in swine populations.