An optimized cell-based assay to assess influenza virus replication by measuring neuraminidase activity and its applications for virological surveillance

Abstract

Year-round virological characterization of circulating epidemic influenza viruses is conducted worldwide to detect the emergence of viruses that may escape pre-existing immunity or acquire resistance to antivirals. High throughput phenotypic assays are needed to complement the sequence-based analysis of circulating viruses and improve pandemic preparedness. The recent entry of a polymerase inhibitor, baloxavir, into the global market further highlighted this need. Here, we optimized a cell-based assay that considerably streamlines antiviral and antigenic testing by replacing lengthy immunostaining and imaging procedures used in current assay with measuring the enzymatic activity of nascent neuraminidase (NA) molecules expressed on the surface of virus-infected cells. For convenience, this new assay was named IRINA (Influenza Replication Inhibition Neuraminidase-based Assay). IRINA was successfully validated to assess inhibitory activity of baloxavir on virus replication by testing a large set (>150) of influenza A and B viruses, including drug resistant strains and viruses collected during 2017-2022. To test its versatility, IRINA was utilized to evaluate neutralization activity of a broadly reactive human anti-HA monoclonal antibody, FI6, and post-infection ferret antisera, as well as the inhibition of NA enzyme activity by NA inhibitors. Performance of IRINA was tested in parallel using respective conventional assays. IRINA offers an attractive alternative to current phenotypic assays, while maintaining reproducibility and high throughput capacity. Additionally, the improved turnaround time may prove to be advantageous when conducting time sensitive studies, such as investigating a new virus outbreak. This assay can meet the needs of surveillance laboratories by providing a streamlined and cost-effective approach for virus characterization.

Keywords: Antiviral resistance; Baloxavir; HINT; Influenza; NA inhibitor; Neuraminidase.

Fig. 1.

Linear relationship between infected cell population (ICP) and the NA activity of infected cells. Three viruses, A/Illinois/08/2018 (A), A/Louisiana/50/2017 (B), and B/North Carolina/25/2018 (C) representing A(H1N1)pdm09, A(H3N2), and B/Victoria lineage, respectively, were serially diluted to determine the relationship of ICP and NA activity (expressed in RFU) of infected cells. Plotted data is representative of three independent experiments. Dashed grey lines indicate cut-offs for acceptable ICP range. Black dots represent values used for best-fit trendline (black dotted line) with 95% confidence. The correlation coefficients (r) between ICP and RFU were calculated by the Pearson correlation test (p = < 0.0001). RFU: relative fluorescence units.

Fig. 2.

Baloxavir susceptibility testing of reference and surveillance viruses using IRINA and HINT. (A) Pair of A(H3N2) viruses (A/Louisiana/50/2017-PA-I38 and A/Louisiana/49/2017-PA-I38M) tested in a minimum of six independent experiments. Virus inoculum was determined by NA activity for IRINA, and ICP for HINT. EC₅₀ values were calculated using RFU for IRINA, and ICP for HINT and shown as box and whiskers plots, where whiskers stretch to the minimum and maximum EC₅₀ values. Median EC₅₀ and corresponding fold increase values are indicated. (B, C) Results for surveillance viruses (n = 96): A (H1N1)pdm09 and A(H3N2) subtypes (B), and B/Victoria and B/Yamagata lineages (C). IRINA and HINT were conducted separately, each in at least two independent experiments. Median EC₅₀ values are indicated, and standard deviations are shown as error bars. (A–C) Unpaired student’s t-test was used for statistical comparison of EC₅₀ values determined using IRINA vs. HINT. * indicates statistically significant difference (P < 0.05).