Impact of influenza virus escape‐mutations on influenza detection by the rapid influenza diagnostic test

Abstract

During the influenza pandemic of 2009-2010, rapid influenza diagnostic tests (RIDTs) were used to detect influenza viral infections because they are quick and simple to use. However, retrospective studies showed that RIDTs performed poorly when used to diagnose pandemic viral infections. Determining how amino acid sequence changes in pandemic or epidemic influenza viral antigens impact clinical value of RIDTs has not been possible, because the viral epitopes recognized by RIDTs have been not mapped. In this study, the effect of escape-variations or mutations in influenza viral antigens upon the sensitivity and specificity of an RIDT was investigated by characterizing the immunological properties of the antibodies used in the RIDT. Escape-mutants were generated by cultivating A/Korea/01/2009 in the presence of an excess of the same antibodies used in the RIDT. Escape-mutants not recognized by the RIDT were selected. Epitopes recognized by the RIDT were mapped by comparing the sequence and immunological analysis of the escape-variants and wild-type isolates. The RIDT antibodies recognized epitopes on the Sa antigenic site and in the F subdomain in hemagglutinin. Variants bearing mutations in these epitopes were not detected by the RIDT. The frequency of escape-variants emerging since the 2009-2010 pandemic was calculated as 1.27% using in silico surveillance of influenza sequence databases. These results suggest that mapping the relevant epitopes of RIDTs and making such information available to clinics would be helpful for determining whether RIDTs match newly emergent strains and subtypes prior to retrospective re-evaluation of the RIDTs using clinical specimens.

Immunological characterization of monoclonal antibodies used in rapid influenza diagnostic test for detection of the 2009 pandemic influenza A(H1N1)pdm09 infection

“…Rudneva et al (2012) also generated H1N1pdm09 specific mAbs (5F7 and 6A3), and it was demonstrated that escape-mutants generated by immune pressure of the mAbs have HA G172E substitution. As previously mentioned, immunological pressure by our mAb-D383 also generated HA G172E escape-mutation on the HA of the H1N1pdm09 virus (Yi et al, 2013). Reversegenetics experiments showed that a single G172E HA on the Sa site of HA was sufficient to escape from sera isolated from ferrets infected with an H1N1pdm09 strain (A/California/ 07/2009).…”

“…4B) compared with those of 1918 H1N1 and 2009 H1N1 viral HAs. Also, the amino acid sequences corresponding to the residues of the HA close-up views shown In the blue Sa antigenic sites, G172E, P176Q, and K180E mutation sites that were elicited as escape-mutation sites to the mAb-D383 in our previous study (Yi et al, 2013) are highlighted in yellow. In the purple F' subdomains, the G56E mutation site that was elicited as an escape-mutation site from mAb-I38 is marked in green.…”

“…Blue-colored spots in background indicate the nucleus of MDCK cells stained by DAPI and green colored cells indicate FITC-labeled antimouse IgG. The influenza strains depicted in the left side of the figure are escape-mutant viruses (mI38 and mD383) and a non-escape mutant virus (KR01) generated in our previous study (Yi et al, 2013). The mI38 and mD383 contain G56E and G172E/P176Q escape-mutations from mAb-I38 and mAb-D383 respectively on their HAs.…”

“…3). In addition, the amino acid residues [the escape-mutation sites: G172, P176, and K180 for mAb-D383; G56 for mAb-I38 (Yi et al, 2013)] recognized by mAb-D383 and -I38 were also mapped directly to the HA structures of H1N1 viral strains ( Fig. 4A and B).…”

“…The escape-mutant viruses used in this study were generated in our previous study (Yi et al, 2013). Briefly explaining the generation of escape mutants, an H1N1pdm09 strain (A/ Korea/01/2009) was incubated with an excess amount (more than 1 mg/ml in which both mAb-I38 and mAb-D383 were able to neutralize A/Korea/01/2009, data not shown) of the indicated mAbs (mAb-D383 and -I38) for 1 h at 20°C, and the mixture was inoculated into 10-day-old embryonated chicken eggs and incubated for 48 h at 37°C.…”