Neutralizing DNA Aptamers against Swine Influenza H3N2 Viruses

Abstract

e 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.

Oligonucleotide aptamers: promising and powerful diagnostic and therapeutic tools for infectious diseases

“…Thus far, more than 10 aptamers against HA have been developed for diagnostic purposes. [128][129][130][131][132][133][134][135][136][137][138][139] RNA aptamer P30-10-16 binds to HA (human influenza A virus subtype H3N2) with more than 15-fold stronger affinity as compared with a conventional anti-HA monoclonal antibody. 130 Bai et al developed a portable SPR aptasensor (an aptamer-based biosensor) based on the specific DNA aptamer for the rapid detection of avian influenza virus (AIV) H5N1.…”

Cross-Protection of Influenza A Virus Infection by a DNA Aptamer Targeting the PA Endonuclease Domain

“…SELEX procedure. The selection of aptamer relied on a nickel nitrilotriacetic acid (Ni-NTA) magnetic agarose bead (Qiagen), as described previously (40). Purified PA or PA N (30 g) was first immobilized on the magnetic bead to form a protein-bead matrix.…”

Aptasensors for viral diagnostics

“…Recently, a whole range of virus-specific aptamers were generated against vaccinia virus [36,40], dengue virus [41], severe acute respiratory syndrome (SARS) [42][43][44][45], hepatitis C [46][47][48][49][50][51], human immunodeficiency virus (HIV) [52][53][54][55], apple stem pitting virus [56][57][58], bovine viral diarrhea virus [39], norovirus [34,59], rabies virus [60], hepatitis B [61], Ebola [62] and influenza [31,33,35,[63][64][65][66][67][68][69][70][71][72][73][74].…”