# Impact of Viral Co-Detection on the Within-Host Viral Diversity of Influenza Patients

## TL;DR

This study explores how the presence of multiple viruses in influenza patients affects the genetic diversity of the influenza virus within the host.

## Contribution

The study is the first to investigate the impact of viral co-detection on the within-host genetic diversity of influenza viruses.

## Key findings

- Most influenza A samples had fewer than five intra-host single-nucleotide variants (iSNVs) at low frequencies.
- Age groups of elderly individuals and school-aged children were significantly associated with higher iSNV counts.
- Viral co-detections had a minor or negligible impact on influenza virus evolution based on the findings.

## Abstract

Numerous studies have documented the evidence of virus–virus interactions at the population, host, and cellular levels. However, the impact of these interactions on the within-host diversity of influenza viral populations remains unexplored. Our study identified 13 respiratory viral pathogens from the nasopharyngeal swab samples (NPSs) of influenza-like-illness (ILI) patients during the 2012/13 influenza season using multiplex RT-PCR. Subsequent next-generation sequencing (NGS) of RT-PCR-confirmed influenza A infections revealed all samples as subtype A/H3N2. Out of the 2305 samples tested, 538 (23.3%) were positive for the influenza A virus (IAV), while rhinovirus (RV) and adenoviruses (Adv) were detected in 264 (11.5%) and 44 (1.9%) samples, respectively. Among these, the co-detection of more than one virus was observed in ninety-six samples, and five samples showed co-detections involving more than two viruses. The most frequent viral co-detection was IAV–RV, identified in 48 out of the 96 co-detection cases. Of the total samples, 150 were processed for whole-genome sequencing (WGS), and 132 met the criteria for intra-host single-nucleotide variant (iSNV) calling. Across the genome, 397 unique iSNVs were identified, with most samples containing fewer than five iSNVs at frequencies below 10%. Seven samples had no detectable iSNVs. Notably, the majority of iSNVs (86%) were unique and rarely shared across samples. We conducted a negative binomial regression analysis to examine factors associated with the number of iSNVs detected within hosts. Two age groups—elderly individuals (>64 years old) and school-aged children (6–18 years old)—were significantly associated with higher iSNV counts, with incidence rate ratios (IRR) of 1.80 (95% confidence interval [CI]: 1.09–3.06) and 1.38 (95% CI: 1.01–1.90), respectively. Our findings suggest a minor or negligible contribution of these viral co-detections to the evolution of influenza viruses. However, the data available in this study may not be exhaustive, warranting further, more in-depth investigations to conclusively determine the impact of virus–virus interactions on influenza virus genetic diversity.