Neutralizing DNA aptamers against swine influenza H3N2 viruses

Abstract

Triple reassortant influenza A viruses (IAVs) of swine, particularly the North American H3N2 subtype, circulate in swine herds and may reassort and result in the emergence of novel zoonotic strains. Current diagnostic tools rely on isolation of the viruses, followed by serotyping by hemagglutination or genome sequencing, both of which can be expensive and time-consuming. Thus, novel subtype-specific ligands and methods are needed for rapid testing and subtyping of IAVs in the field. To address this need, we selected DNA aptamers against the recombinant HA protein from swine IAV H3 cluster IV using systematic evolution of ligands by exponential enrichment (SELEX). Four candidate aptamers (HA68, HA7, HA2a, and HA2b) were identified and characterized. The dissociation constants (K(d)) of aptamers HA68, HA7, HA2a, and HA2b against recombinant H3 protein were 7.1, 22.3, 16.0, and 3.7 nM, respectively. The binding site of HA68 to H3 was identified to be between nucleotide residues 8 and 40. All aptamers inhibited H3 hemagglutination. HA68 was highly specific to all four lineages within the North American H3N2 subtype. Further, the other three aptamers specifically identified live viruses belonging to the phylogenetic clusters I, II/III, and IV especially the virus that closely related to the recent H3N2 variant (H3N2v). Aptamer HA68 was also able to bind and detect H3N2v isolated from recent human cases. In conclusion, we provide subtype-specific aptamers against H3N2 IAVs of swine that can now be used in rapid detection and typing protocols for field applications.

Fig 1

Phylogenetic clusters of IAV H3 subtypes used for aptamer specificity testing. The relative phylogenetic positions of HA sequences of archived IAVs from North American swine (blue) used for aptamer dot blot assay are shown. Isolates were obtained from the UMDLV. The H3 IAV used for SELEX is indicated in pink and belongs to cluster IV.

Fig 2

DNA sequences from enriched aptamer pool of the 15th iteration of SELEX were aligned by using CLUSTAL W to identify redundancy in the selected sequence motifs. The sequence similarity was compared, and five sequences representative of redundant clusters were chosen for further characterizations (red arrows).

Fig 3

Aptamer dot blot assay performed to test the specificity of the four candidate aptamers. Sample 1, A/swine/Minnesota/SG235/2007 (H3N2); 2, A/Solomon Islands/3/2006 (H1N1); 3, A/Singapore/1/1957 (H2N2); 4, A/Uruguay/716/2007 (H3N2); 5, A/Hong Kong/156/1997 (H5N1); 6, A/bar-headed goose/Qinghai/1A/2005 (H5N1); 7, A/teal/Hong Kong/W312/1997 (H6N1); 8, A/Canada/rv444/2004 (H7N3); 9, A/Netherlands/219/2003 (H7N7); 10, A/Hong Kong/1073/1999; 11, NP protein of A/swine/MN/07002083/2007 (H1N1). Detailed descriptions of each sample are shown in Table 2. Abbreviations: a, avian; h, human; s, swine.

Fig 4

Two selected aptamer candidates, HA68 and HA7, were screened by using EMSA.

Fig 5

An HI test was performed to show the neutralization activity and specificity of selected aptamers to the HA protein of A/swine/Minnesota/SG-00235/2007 (H3N2).

Fig 6

The dissociation constant (K_d) of each DNA aptamer was used to describe the strength of the binding affinity between aptamer and recombinant swH3 protein. The K_d was calculated from the dot blot chemiluminescence intensity based on a nonlinear regression equation.

Fig 7

The aptamer binding site of HA68 was identified by a DNase I footprinting assay. (Upper right panel) FAM-HA68 was reacted with recombinant swH3 protein (blue peaks) or recombinant human H1 protein (pink peaks). (Lower right panel) Nucleotide sequence chromatogram. (Left panel) Secondary structure prediction of HA68 and putative binding sites.

Fig 8

An aptamer dot blot assay was performed to detect archived IAV isolates from North American swine in different phylogenetic lineages of H3 (samples 1 to 7 and sample 9). SwH1N2 IAV (sample 8), AIV H4N8 (sample 10), and 10% fetal bovine serum (FBS) in MEM (sample 11) were used as negative controls. The numbers on the left side of the figure are the virus titers (in HA U/50 μl). Abbreviations: a, avian; s, swine; Dig, digoxigenin labeled.