Flu Pandemic Preparedness: Detection of the Potentially Pandemic G4 EA Avian-Like H1N1 Strains Using QIAstat-Dx Respiratory SARS-CoV-2 Panel

A recent publication by Sun H. and colleagues in the Proceedings of the National Academy of Science (PNAS) has drown global attention to the predominant genotype “G4” Eurasian (EA) avian-like H1N1 Inuenza A virus that has been spreading among pigs in China since 2016, which is predicted to have a signicant pandemic potential. Since pigs are hosts for the generation of pandemic Inuenza A viruses (IAVs), surveillance and preparedness are critical to prevent pandemics. In this regards, one distinguishing feature of the QIAstat-Dx ® Respiratory SARS-CoV-2 panel is the double target approach Inuenza A detection of seasonal strains affecting humans, by amplication of a generic Inuenza A assay plus one of the specic assays discriminating H3, H1 and H1N1pdm09 subtypes. The generic Inuenza A assay is designed to amplify any IAV, a key feature for preparedness. Here we report an initial in-silico analysis that predicts that the G4 EA avian-like H1N1 strains tested in the QIAstat-Dx ® Respiratory SARS-CoV-2 Panel would be detected yielding a positive result for the generic Inuenza A assay and negative results for the seasonal H3, H1 and H1N1pdm09 assays. This prediction was conrmed in-vitro using dsDNA fragments mixed to mimic the genomes of the different reported G4 EA IAV strains. In conclusion, the QIAstat-Dx ® Respiratory SARS-CoV-2 Panel could be a useful tool to differentially diagnose zoonotic strains from the seasonal Inuenza A strains commonly affecting humans.


Introduction
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 e cient human-to-human transmission is a critical feature of pandemic in uenza 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 In uenza Virus Real-time RT-PCR In uenza A (H1/H3/H1N1pdm09) Subtyping panel would detect this swine in uenza 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 In uenza Like Illness (ILI) during the in uenza 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 In uenza A detection. Speci cally, this detection is based on a generic assay designed to detect any In uenza A strain, as well as 3 speci c assays to discriminate between the three main In uenza 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 H1N1pdm09like lineage would be detected as generic In uenza A positive with no subtype discrimination given the speci city of the human H1 and H1N1pdm09 subtype assays on the panel. We sought to test this hypothesis in-vitro to con rm 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 speci city.

Results
Bioinformatic analysis showed high homology between the generic In uenza A assay (no subtype) included in the QIAstat-Dx ® Respiratory SARS-CoV-2 Panel and all screened 29 G4 strains. Mismatches were low in frequency and located in no-critical positions in the oligonucleotide sequences, rendering a positive ampli cation prediction by the generic In uenza A assay when testing the described any of the described G4 swine strains.
On the contrary, detection by the In uenza A H1 subtypes assays (H1 and H1N1pdm09) was as predicted negative. Several mismatches were found in both seasonal H1 and H1N1pdm09 RT-qPCR assays, predicting no ampli cation by RT-PCR.
Further laboratory testing of simulated G4 strains corroborated the in-silico prediction ( Table 1). All 13 sample combinations covering the complete genetic variability observed among the 29 G4 strains resulted in In uenza A positive with no subtype discrimination. None of the subtype assays presented a cross-reaction with this new G4 genetic cluster. Those results demonstrate the good speci city of the QIAstat-Dx ® Respiratory SARS-CoV-2 Panel with the ability to discriminate only human-speci c IAV H1 strains. The presence of EA capsid genes (HA and NA genes) of the swine G4 lineage (1) facilitate the discrimination from human H1 or H1N1pdm09 subtypes.

Discussion
Although there are currently no reports of the In uenza G4 EA H1N1pdm09-like viruses spreading from person-to-person, it is important to remain vigilant of potentially infective In uenza strains from other species and be prepared to take action in the event of a public health threat (2,4,5). To cover the possibility of a spillover from other species, the overall detection strategy of In uenza A in the QIAstat-Dx ® Respiratory SARS-CoV-2 Panel is to speci cally detect and differentiate the human seasonal H1, the human seasonal H3 and the H1N1pdm09 subtypes while providing a generic In uenza A-no subtype assay. This detection strategy using a generic In uenza A assay ful ls the double function of con rmation of the human-speci c subtypes detection while also allowing the potential detection of nonhuman IAVs. In this regards, the generic In uenza A assay in the QIAstat-Dx ® Respiratory SARS-CoV-2 Panel is predicted to detect the recorded zoonotic variants such as avian IAVs subtypes A(H5N1),

A(H7N9), and A(H9N2) and swine IAVs subtypes A(H1N1), A(H1N2) and A(H3N2) (8).
Here we con rm experimentally that the newly emergent swine In uenza A G4 EA H1N1pdm09-like viruses reported by Sun H. and colleagues (1) would be detected by the QIAstat-Dx ® Respiratory SARS-CoV-2 Panel yielding a positive generic In uenza A result and negative result for speci c human H3, H1 and H1N1pdm09 subtypes. This contrasts with the report by the CDC of the detection of this potentially pandemic emergent swine G4 strain as seasonal H1N1 positive result using the In uenza Virus Real-time RT-PCR In uenza A (H1/H3/H1N1pdm09) Subtyping panel (6).
The results we report supports the overall In uenza A detection strategy of the QIAstat-Dx ® Respiratory SARS-CoV-2 Panel allowing human-speci c In uenza A subtype detection as well as non-human IAVs. In conclusion, in the event that the G4 swine EA IAV adaptation causes human-to-human transmission, the QIAstat-Dx ® Respiratory SARS-CoV-2 Panel could be a useful tool to differentially diagnose zoonotic strains from the seasonal IAVs commonly affecting humans. The output alignment was curated manually.

Materials And Methods
A speci city assessment on the criticality of the mismatches between the viral sequences and the oligonucleotide sequences was made to establish a prediction on the potential ampli cation of the targeted regions of the G4 EA H1N1pdm09-like viral genomes. Every screened primer set (general In uenza A detection; and H1, H3 and H1N1pdm09 subtypes) was considered unspeci c for G4 EA lineage if: 1) three or more mismatches were found among any oligonucleotide sequence and the strain gene sequences or 2) variations were placed in the three last nucleotides of the 3'-end or in the 5'-end of the probe affecting the PCR ampli cation (9,10,11). This approach has been used previously to characterize inclusivity of speci c RT-qPCR assays for viral detection (12).
In addition, laboratory testing was performed to con rm in-silico prediction. A total of 22 gBlocks (dsDNA fragments of 500 bp size) were designed to cover all the genetic variability observed in the sequence alignments among the 29 G4 EA strains described in Sun H. et al. (1), corresponding to the different In uenza A genome regions targeted by the QIAstat-Dx ® Respiratory SARS-CoV-2 Panel. Those 22 gBlocks were mixed accordingly in 13 unique combinations (1E+05 copies/mL concentration) in order to simulate all the genetic variability of the viral genomic regions targeted by the QIAstat-Dx ® Respiratory SARS-CoV-2 Panel. Finally, two analytical samples were also included in the analysis as controls corresponding to a known seasonal H1 and H1N1pdm09 human strains (