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. 2013 Apr 15;13:34.
doi: 10.1186/1472-6750-13-34.

Modular Glycosphere Assays for High-Throughput Functional Characterization of Influenza Viruses

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Free PMC article

Modular Glycosphere Assays for High-Throughput Functional Characterization of Influenza Viruses

Sven N Hobbie et al. BMC Biotechnol. .
Free PMC article

Abstract

Background: The ongoing global efforts to control influenza epidemics and pandemics require high-throughput technologies to detect, quantify, and functionally characterize viral isolates. The 2009 influenza pandemic as well as the recent in-vitro selection of highly transmissible H5N1 variants have only increased existing concerns about emerging influenza strains with significantly enhanced human-to-human transmissibility. High-affinity binding of the virus hemagglutinin to human receptor glycans is a highly sensitive and stringent indicator of host adaptation and virus transmissibility. The surveillance of receptor-binding characteristics can therefore provide a strong additional indicator for the relative hazard imposed by circulating and newly emerging influenza strains.

Results: Streptavidin-coated microspheres were coated with selected biotinylated glycans to mimic either human or avian influenza host-cell receptors. Such glycospheres were used to selectively capture influenza virus of diverse subtypes from a variety of samples. Bound virus was then detected by fluorescently labelled antibodies and analyzed by quantitative flow cytometry. Recombinant hemagglutinin, inactivated virus, and influenza virions were captured and analyzed with regards to receptor specificity over a wide range of analyte concentration. High-throughput analyses of influenza virus produced dose-response curves that allow for functional assessment of relative receptor affinity and thus transmissibility.

Conclusions: Modular glycosphere assays for high-throughput functional characterization of influenza viruses introduce an important tool to augment the surveillance of clinical and veterinarian influenza isolates with regards to receptor specificity, host adaptation, and virus transmissibility.

Figures

Figure 1
Figure 1
Illustration of an influenza glycosphere assay.
Figure 2
Figure 2
Binding specificity of the representative receptor motifs used in the glycosphere assay. A, Recombinantly expressed hemagglutinin of the avian-adapted influenza strains A/Chicken/PA/2004 (CkPA04, H2N2) and A/Vietnam/1203/2004 (Viet04, H5N1) binds quantitatively and selectively to its cognate avian glycan domain. B, Hemagglutinin of the human pandemic strains A/Albany/6/58 (Alb58, H2N2) and A/California/04/2009 (Ca04, H1N1) binds with high specificity to only its cognate human glycan domain.
Figure 3
Figure 3
Quantitative receptor binding analysis of a representative panel of influenza viruses. Influenza A subtypes H1N1, H3N2, as well as influenza B showed a selective and dose-dependent binding to glycospheres coated with the human receptor motif LSTc (red). H5 strains of both human and avian origin demonstrated highly selective binding to the avian receptor motif LSTa (blue). SM15, A/Singapore/SM15/09; Bb07, A/Brisbane/59/07; NC99, A/New Caledonia/20/99; SM19, A/Singapore/SM19/09; WY03, A/Wyoming/03/03; HK68, A/Hong Kong/8/68; TK05, A/turkey/Turkey/1/05; VN04, A/Vietnam/1194/04; SG97, A/duck/Singapore/97; Bb08, B/Brisbane/60/08; Tw62, B/Taiwan/2/62; Lee40, B/Lee/40. Full details of influenza strains used in this study are provided in Additional file 4.
Figure 4
Figure 4
Assay performance characteristics. A, Assay reproducibility as determined by three independent receptor binding assays performed in different weeks with independently prepared reagents and non-identical virus stocks of A/Fort Monmouth/1/1947(H1N1). B, Limit of detection as determined by comparing the fluorescent signals of increasing viral titers of A/Hong Kong/8/1968(H3N2) with the no-virus control (NV). Statistical significance was determined by an unpaired t-test and categorized as not significant (ns), p < 0.05 (*), p < 0.01 (**), or p < 0.001 (***). C, Assay robustness as determined by comparing the signal intensity of identical viral titers in different assay media. A/Fort Monmouth/1/1947(H1N1) was well detected over a range of viral titers when serially diluted in phosphate buffered saline (PBS), virus growth medium (MEM), or Universal Transport Medium (UTM).

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