ICVs were cultured in Madin-Darby Canine Kidney (MDCK) cells [13]. The ICVs used to evaluate analytical performance included three historical ICV strains—C/Aomori/1974 (KA176 lineage), C/Kansas/2/1979 (AI181 lineage) and C/Yamagata/10/1981 (Taylor/47 lineage) —and three recent ICV strains— C/Minnesota/29/2015 (SP82 lineage), C/Minnesota/05/2014 (KA176 lineage) and C/Minnesota/1/2016 (KA176 lineage).

IAV and IBVs used in this study were cultured in either MDCK cells or embryonated chicken eggs (ECE) [14]. The IAV and IBV virus titers were measured by tissue culture infectious dose-50 % (TCID₅₀/ml) or egg infectious dose-50 % (EID₅₀/ml) [15].

Viral RNA was extracted from 100 μL of virus isolates or clinical specimens and eluted in 100 μL elution buffer using the MagNA Pure Compact RNA isolation kit on a MagNA Pure Compact instrument (Roche Applied Science) according to manufacturer’s instructions. While the MagNA Pure Compact was recently sunset from manufacturing, we have shown comparable performance between this instrument and other similar extractors, such as the Qiagen EZ1 (data not shown).

We designed the InfC rRT-PCR assay based on nucleotide sequence data available from the National Center for Biological Information (NCBI) GenBank database and the Global Initiative on Sharing All Influenza Data (GISAID). The oligonucleotide primers and probe were designed to target a highly conserved region of the matrix (M) gene to allow universal detection of ICVs (Table 1). Specifically, we used PrimerExpress 3.0.1 software (Applied Biosystems, Foster City, USA) to design the primers/probes to have annealing temperatures of approximately 60 °C and 70 °C, respectively. We confirmed primer/probe specificity through comparisons with sequence data from NCBI and GISAID databases.

We evaluated two quencher chemistries for Taqman^® hydrolysis probes, namely, Blackhole Quencher^™ (BHQ^™) and the ZEN^™ Double-Quenched Probe. Both probe types were labeled at the 5′-end with the reporter molecule, 6-carboxyfluorescein (FAM). The BHQ^™ probe contained a Black Hole Quencher 1 (BHQ-1) at the modified “T” residue, and a 3’-end Spacer 6 (Sp6, Biosearch Technologies, Inc., Novato, CA) (Table 1). Meanwhile, the ZEN^™ Double-Quenched Probe contained a ZEN Quencher between base residues 9 and 10, and an Iowa Black FQ quencher at the 3′-end (Integrated DNA Technologies, Coralville, IA). The CDC Biotechnology Core Facility (CDC, Atlanta, GA) synthesized all primers and dual-labeled Taqman^® hydrolysis probes.

A modified probe was also designed with a degenerate base “Y” at nucleotide position 9.

We optimized reaction parameters for the InfC rRT-PCR assay using the Invitrogen SuperScript^™III Platinum^® One-Step quantitative RT-PCR Kits (Life Technologies) on the Applied Biosystems^™ (AB) 7500 Fast Dx Real-Time PCR systems. A thermal gradient analysis of InfC rRT-PCR assay reactions indicated comparable performance at annealing temperature range from 50 °C to 62.5 °C (Table S1, Figure S1). Consequently, we set the final reaction annealing temperature at 55 °C (Table S1, Figure S1).

Reaction conditions for rRT-PCR were based upon U.S. Food and Drug Administration (FDA)–approved CDC Human Influenza Virus Real-Time RT-PCR Detection and Characterization Panel (CDC Flu rRT-PCR Dx Panel): Total reaction volume for the rRT-PCR reactions performed was 25 μl, with primer and probe reaction concentrations at 0.8 μM and 0.2 μM, respectively. The thermocycling rRT-PCR conditions were as follows: 50 °C for 30 min, Taq activation at 95 °C for 2 min and 45 cycles of 95 °C for 15 s and 55 °C for 30 s [16,17].

The analytical sensitivity and specificity of the InfC rRT-PCR assay were evaluated by using the ZEN^™ probe.

Analytical sensitivity of the InfC assay was determined on 3 historical strains, 3 recent strains, and a synthetic RNA sequence derived from the M gene of C/Minnesota/01/2016 (KA176 lineage) virus (Armored RNA Quant^® CDC-9, AsuraGen, Inc.). The analytical specificity was evaluated using 14 IAVs, 6 IBVs, and 15 non-influenza viruses that commonly infect the human respiratory tract via the nasopharynx.

Since 2008, we have supported a number of ICV detection and surveillance programs, including one with the Minnesota Department of Health. In 2017, we received 39 specimens collected from Minnesota between December 2014 and February 2016 that tested positive for InfC virus using the CDC InfC rRT-PCR assay. Of these, 32 specimens were collected from patients who were admitted with severe acute respiratory illness (ARI), while seven were from outpatients with influenza-like illness (ILI) or ARI [18].

We tested the 39 suspect ICV specimens, which were all collected from the upper or lower respiratory tract of patients, along with 30 additional respiratory specimens from unrelated patients received by CDC for testing using the InfC rRT-PCR assay (ZEN^™ probe) and the CDC Flu rRT-PCR Dx Panel-Influenza A/B Typing Kit. We further sequenced the HEF genes of ICV-positive samples according to CDC’s protocol to confirm the rRT-PCR result [18].

The InfC rRT-PCR assay was developed using the ABI 7500 Fast Dx Real-time PCR system for the qualitative detection and characterization of ICV RNA in respiratory specimens from patients presenting with ILI and/or ARI. Based on the thermal gradient analysis, we set the annealing temperature at 55 °C, which is identical to current IAV and IBV diagnostic assays of the CDC Flu rRT-PCR Dx Panel [16,17] and accommodates potential nucleotide mismatches in the primer/probe regions due to virus genetic variability.

We measured reaction efficiencies of the InfC assay by testing ten-fold dilution series of viral RNA extracted from two historical ICV strains, C/Kansas/2/1979 (AI181 lineage) and C/Yamagata/10/1981 (YA2681 lineage). Overall, we observed 98.9–100 % efficiency (Fig. 1). Furthermore, both hydrolysis probe types— ZEN^™ and BHQ^™ — yielded comparable Ct values for C/Minnesota/29/2015 (SP82 lineage) tested using both quencher chemistries (Table 2)

To determine the analytical sensitivity of the ICV assay, we extracted viral RNA from three historical ICV strains—C/Aomori/1974 (KA176 lineage), C/Yamagata/10/1981 (Taylor47 lineage) and C/Kansas/2/1979 (AI181 lineage)—and three recent ICV strains—C/Minnesota/29/2015 (SP82 lineage), C/Minnesota/05/2014 (KA176 lineage) and C/ Minnesota/1/2016 (KA176 lineage)—then tested a 10-fold dilution series according to the optimized RT-PCR parameters. All six viral RNAs tested positive for ICV by the InfC rRT-PCR assay, even at 10⁻⁷ dilution (Fig. 2 and Table S2).

We also tested a synthetic RNA derived from ICV, C/Minnesota/1/2026(KA176 lineage) (Armored RNA Quant^® CDC-9, AsuraGen, Inc.) to determine the minimum RNA copy number which the InfC rRT-PCR assay could consistently detect. Overall, we determined the limit of detection (LoD) at 5 copies of RNA per reaction (Table 3).

To demonstrate that the ICV assay retained exclusivity of IAVs, IBVs, and other common respiratory pathogens, we tested several of these pathogens on the InfC assay to confirm lack of positivity. In total, we tested six each of seasonal IAV or IBV subtypes or lineages, including the 2009 pandemic influenza A(H1N1) (H1N1pdm09) and influenza A (H3N2) subtypes. We also tested two avian influenza viruses; one each of A(H5N1) and A(H7N9) viruses (Table 4). All IAVs and IBVs tested negative by the InfC rRT-PCR assay. Likewise, all 15 non-influenza viral pathogens also tested negative (Table S3).

Thirty-nine ICV suspected specimens that had tested positive with the CDC InfC rRT-PCR assay from the Minnesota Department of Health were subjected to a confirmatory InfC rRT-PCR test in 2017 by the CDC’s Influenza Division. All 39 specimens also tested positive with the InfC rRT-PCR assay performed at CDC. The specimens were also tested with the CDC Flu rRT-PCR Dx Panel-Influenza A/B Subtyping Kit. All specimens tested positive for the Human Rnase P (RP) assay indicating the specimens were of good quality. Thirty-eight of the 39 specimens tested negative by the universal-influenza A and B (InfA and InfB) assays, and one specimen tested positive for InfA (Table 5).

We also evaluated the clinical performance of the InfC rRT-PCR assay by testing an additional 30 residual clinical respiratory specimens previously tested for influenza, including 10 specimens positive for IAVs, 10 specimens positive for IBVs and 10 influenza negative specimens (Non influenza); the results were in 100 % agreement with the expected result for each specimen (Table 5).

The HEF gene genetic sequence analysis provided additional confirmation of the InfC rRT-PCR positive result. Thirteen of the 39 HEF genes from the positive samples were SP82 lineage, while 26 were KA176 lineage.

To assess potential mismatches between ICV M gene sequences and the primers and probes designed, we performed in silico analysis that compared the primers and probe against the ICV M gene sequences that had been deposited in GISAID between 2000 and 2024. Within the forward primer region, we found only one virus with a mismatch. Within the reverse primer region, 6.3 % (21/332) and 1.8 %(6/332) viruses had one or two mismatches, respectively. Within the probe region, 29.0 % (98/338) viruses had one mismatch and 3.6 % viruses (12/338) had two mismatches. Additionally, we analyzed the probe region sequences for a cytosine to thymine change at nucleotide position 9; in total, 24.0 % (81/338) viruses contained this change, including 9 viruses with two mismatches. As a result, within the modified probe, 6.8 % (26/338) and 0.9 (3/338) viruses had only one or two mismatches, respectively (Table 6).