One important public health lesson learned from the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic has been the importance of time to action and preparedness for detection of novel viruses with pandemic potential. A recent publication in the Proceedings of the National Academy of Science (PNAS) by Sun H. and colleagues described the predominant genotype “G4” Eurasian (EA) avian-like H1N1 virus that has been spreading among pigs in China since 2016, which has H1N1 pandemic 2009 (H1N1pdm09) and triple-reassortant (TR)-derived internal genes (1). It is well established that efficient human-to-human transmission is a critical feature of pandemic influenza viruses (2,3). The authors determined that the emergent novel G4 reassortment EA H1N1 virus has high infectivity and transmissibility potential via direct contact and respiratory droplets amongst ferrets, suggesting ability to infect humans as the previous pandemic H1 in 2009 (4,5). Additionally, the reported serological surveillance amongst swine workers with occupational exposure showed 10.4% were positive for G4 swine EA H1N1 with younger patients 18-35 years old with higher rates of seropositivity. These data caught the attention of the US Center for Disease Control and Prevention which has already determined that their current Influenza Virus Real-time RT-PCR Influenza A (H1/H3/H1N1pdm09) Subtyping panel would detect this swine influenza virus (SIV) (6). This would yield a positive result for the subtype H1N1, which would indicate that the H1N1 subtype assay shows cross-reactivity to swine variants of the H1 genotype.
The overlap of viral etiologies of Influenza Like Illness (ILI) during the influenza season has increased the attention to syndromic diagnostics with multiplex capability, as well as the need to increase ability to detect and distinguish new variants. The QIAstat-Dx® Respiratory SARS-CoV-2 Panel offers a multiplex real time RT-PCR platform with 22 targets (CE-IVD) or 21 targets (FDA EUA) as one of the currently available respiratory syndromic solutions (7).
One distinguishing feature of the QIAstat-Dx® Respiratory SARS-CoV-2 panel is the double target approach to Influenza A detection. Specifically, this detection is based on a generic assay designed to detect any Influenza A strain, as well as 3 specific assays to discriminate between the three main Influenza A Virus (IAV) subtypes infecting humans: seasonal IAV H1, seasonal IAV H3, and IAV H1N1pdm09. The dual target approach enhances the detection of the IAV genotypes affecting humans, while allowing the detection of novel strains by detecting Influenza A targets without subtype (ruling out H1, H3 and H1N1pdm09). The mentioned generic Influenza A design targets the Matrix gene as the one with highest homology among IAV.
After performing an in-silico analysis of the G4 EA H1N1 strains defined in Sun H. et al. (1) against the 4 Influenza A assays included in the QIAstat-Dx® Respiratory SARS-CoV-2 Panel, we generated the hypothesis that upon testing with the QIAstat-Dx® Respiratory SARS-CoV-2 Panel, the G4 EA H1N1pdm09-like lineage would be detected as generic Influenza A positive with no subtype discrimination given the specificity of the human H1 and H1N1pdm09 subtype assays on the panel. We sought to test this hypothesis in-vitro to confirm that the QIAstat-Dx® Respiratory SARS-CoV-2 Panel would be able to detect this new G4 swine EA H1N1 strain providing the expected level of specificity.