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J Clin Microbiol. Jan 2013; 51(1): 46–54.
PMCID: PMC3536225
Neutralizing DNA Aptamers against Swine Influenza H3N2 Viruses
Manoosak Wongphatcharachai,abc Ping Wang,c Shinichiro Enomoto,c Richard J. Webby,e Marie R. Gramer,c Alongkorn Amonsin,ab and Srinand Sreevatsancorresponding authorcd
aEmerging and Re-Emerging Infectious Diseases in Animals, Research Unit, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand
bDepartment of Veterinary Public Health, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand
cDepartment of Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota, USA
dDepartment of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota, USA
eInfectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee, USA
corresponding authorCorresponding author.
Address correspondence to Srinand Sreevatsan, sreev001/at/umn.edu.
Received August 8, 2012; Revisions requested September 10, 2012; Accepted October 9, 2012.
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 (Kd) 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.
Influenza is an acute contagious disease caused by viruses belonging to the family Orthomyxoviridae. This family of viruses contains five genera: influenza viruses A, B, and C, thogotovirus, and isavirus (1). All influenza pandemics in human have been caused by genus influenza virus A. Moreover, influenza A viruses (IAVs) have been reported to infect a wide range of animal species including humans (26). Based on the antigenic properties of hemagglutinin (HA) and neuraminidase (NA) genes, IAVs can be divided into 17 HA and 9 NA subtypes (7).
IAVs of swine were first isolated from pigs in 1930 and identified as H1N1 (8). IAV is not only an important cause of respiratory disease for the swine industry throughout the world but also represents an important public health concern. Since human, avian, and swine IAVs can all replicate in pigs to various levels, swine has been implicated as a species that can generate novel viruses with wide host ranges (911). Pigs also can be infected with many subtypes of IAVs; however, three major subtypes (H1N1, H1N2, and H3N2) are endemic in swine populations globally (6, 10, 12).
Since 1998, triple reassortant IAV H3N2 were identified in North America and then have been circulating in swine herds worldwide (13, 14). Phylogenetic analysis of HA gene of H3N2 IAV from swine in North America describe their origin as coming from distinct human-to-swine transmission events and resulting in three genetic and antigenic clusters, namely, clusters I, II, and III (13, 15). Cluster III viruses were the most common in North America in the early 21st century. By 2005, cluster IV viruses that emerged as possible vaccine escape variants of cluster III have since become dominant in pig populations (1618).
In 2011, novel reassortant H3N2 variant (H3N2v) IAVs containing internal genes of the 2009 pandemic H1N1 have been reported in pigs (19). In the same year, the Centers for Disease Control and Prevention (CDC) reported two pediatric cases of H3N2v. This virus has been identified as “novel reassortant” because their genomes contain seven gene segments of triple reassortant H3N2 circulating in North America swine since 1998 and matrix gene of the 2009 pandemic H1N1 (20).
Current diagnostic tools for IAVs rely on isolation of virus, followed by serotyping via hemagglutination or genome sequencing, both of which can be expensive and time-consuming (21). Thus, novel subtype-specific ligands and methods are needed for rapid testing and subtyping of IAVs. Although antibodies have been made for a wide range of applications and have become the indispensable agents for most diagnostic tests, they have inherent limitations. The process of antibody production starts in animals or cell cultures that are demanding and costly to maintain. Frozen stocks of antibodies might lose their activity over time for no apparent reason. The performance of the same antibody may have batch-to-batch variation. Moreover, antibodies are sensitive to high temperature and irreversible after denaturation; thus, they have a limited shelf-lives and are not suitable for transport at ambient temperatures (22).
Aptamers are short, unique, single-stranded nucleic acid ligands that can bind specifically to their target molecules. They can be completely selected and characterized from complex synthetic libraries by an in vitro process called “systematic evolution of ligands by exponential enrichment” (SELEX) (23, 24). This process starts with random oligonucleotides flanked with primer-binding regions for PCR amplification.
In the present study, high-affinity DNA aptamers against recombinant HA protein from H3 cluster IV IAV of swine were selected and characterized. Several studies of aptamers for IAVs have been reported (2528). However, these studies did not demonstrate that these aptamers could be applied for heterologous viral subtype. This is the first report of DNA aptamers for IAVs demonstrating homologous and heterologous viral subtype specificity. Furthermore, our study shows that selected candidates bind directly to virus, suggesting that these ligands can be translated into a rapid testing device for both laboratory and field use.
Randomized single-stranded DNA library, aptamers, and primers.
The single-stranded DNA library (WAP40) consisting of randomized 40-mer DNA sequence flanked by constant primer-binding regions, primers (sense strand [WP18] and antisense strand [WP20]), and aptamers were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). Sequences of primers and DNA library are shown in Table 1.
Table 1
Table 1
Sequences of primers and DNA library used in this study
Recombinant His6-tagged H3 hemagglutinin protein.
Recombinant His6-tagged H3 hemagglutinin protein from an H3N2 strain A/swine/Minnesota/SG-00235/2007 (swH3) was cloned and expressed in a baculovirus system in our laboratory. To verify that the recombinant swH3 protein was a functional protein, we performed a hemagglutination (HA) test parallel with the corresponding swH3 virus, and the results were similar. The recombinant swH3 protein was used as a template for the selection and characterization of the DNA aptamers. To test the specificity of aptamers, the reference recombinant HA proteins in different subtypes of IAVs obtained from Biodefense and Emerging Infections Research (BEI) resources were used. Detailed descriptions of the recombinant HA proteins used in the present study are provided in Table 2.
Table 2
Table 2
Detailed descriptions of the recombinant HA proteins used in this study
Preparation of recombinant swH3 protein and Ni-NTA magnetic agarose bead matrix.
Approximately 100 μl of Ni-NTA magnetic agarose beads (Qiagen, Hilden, Germany) was separated from solution by using magnetic apparatus. The beads were washed three times with 500 μl of 50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, 0.005% Tween 20 (pH 8.0) (washing buffer) and then conjugated with recombinant swH3 protein in 500 μl of 50 mM NaH2PO4, 300 mM NaCl, and 20 mM imidazole (pH 8.0) (binding buffer) at 4°C with gentle shaking. After 48 h of incubation, protein-bead matrix was washed three times with 500 μl of washing buffer. After washing, the protein-bead matrix was stored at 4°C and used within a day. In subsequent iterations of SELEX, the recombinant swH3 protein was gradually reduced from 30 to 0.4 μg.
Negative selection.
Negative selection was performed in every iteration of SELEX to remove any nonspecific candidates that bind to the Ni-NTA magnetic agarose beads. The aptamer library was denatured at 95°C for 10 min and then incubated at room temperature for 10 min prior use. Three sets of ~100 μl of Ni-NTA beads were prepared by separating them from solution by using a magnetic separation apparatus and three washes with 500-μl portions of washing buffer. The denatured aptamer library was added to Ni-NTA beads in 500 μl of 50 mM NaH2PO4–50 mM NaCl–20 mM imidazole pH 8.0 (interaction buffer), followed by incubation at room temperature for 30 min in a rotisserie shaker. After the third negative selection, the unbound library was subjected to positive SELEX against H3.
SELEX.
The aptamer library was conjugated with protein-bead matrix in 500 μl of interaction buffer, followed by incubation at room temperature using a rotisserie shaker. After 1 h of incubation, the unbound library or poor binders were removed by 11 wash steps with 1× phosphate-buffered saline (PBS) containing 0.05% Tween 20 (1× PBST). Protein-bound aptamers were recovered with 100 μl of nuclease-free water and then subjected to PCR. PCR was performed to amplify all protein-bound aptamer candidates. Briefly, 3 μl of protein-bound aptamers were mixed with 5 pmol of forward primer (WP18) and biotinylated reverse primer (Bio-WP20), 2× HotStarTaq DNA polymerase (Qiagen), and nuclease-free water (final volume, 50 μl). PCR was performed using the following conditions: 95°C for 15 min, followed by 15 cycles of 95°C for 30 s, 63°C for 30 s, and 72°C for 30 s, and finally 72°C for 7 min. Amplicons from PCR were then used as a template for asymmetric-touchdown PCR to enrich for the sense strand to be applied back in the next round of SELEX. Briefly, 2 μl of amplicons was mixed with 30 pmol of WP18 and 1.2 pmol of Bio-WP20 (forward primer/reverse primer ratio of 25:1). Touchdown PCR conditions used were 95°C 15 min, followed by 9 cycles of 95°C for 15 s, 72°C for 15 s (gradually decreasing by 1°C each cycle), and 72°C for 15 s, followed by 11 cycles of 95°C for 15 s, 63°C for 15 s, and 72°C for 15 s, and a final extension at 72°C for 3 min. Approximately 400 μl of amplicons from asymmetric-touchdown PCR was purified by MiniElute PCR purification kit (Qiagen) and then eluted with 60 μl of nuclease-free water. Subsequent rounds of SELEX were performed only with the sense strand aptamers. To remove the antisense strand, PCR amplicons were purified by heat denaturing and quick chilling on ice and then mixture with Dynal M280 streptavidin super-paramagnetic beads (Invitrogen/Dynal AS, Oslo, Norway) for 15 min. The SELEX process was repeated 15 times. The binding affinity and the specificity of aptamer candidates were investigated by chemiluminescent electromobility shift analysis (LightShift chemiluminescent EMSA kit; Pierce, Rockford, IL) before cloning and sequencing.
Counter-SELEX to remove any His tag binding candidates.
During the last two rounds of SELEX (i.e., rounds 14 and 15), His6-tagged recombinant nucleoprotein (rNP) from an H1N1 virus, A/swine/Minnesota/07002083/2007 (swH1), was used for counter-SELEX to remove any nonspecific candidates that bound to the His6-tagged portion of the protein. A protein-bead matrix was prepared according to the same procedures used for the recombinant swH3 protein. The aptamer pool was incubated with rNP at room temperature for 1 h, and then the supernatant was collected using a magnetic apparatus. The supernatant containing aptamer candidates was then submitted to positive SELEX.
Cloning and sequencing of the aptamer candidates.
PCR amplicons containing the enriched aptamer pools of rounds 8 and 15 were purified by using a MiniElute PCR purification kit (Qiagen). The purified amplicons were cloned into TA vector (pCR2.1-TOPO) and then transformed into chemically competent TOP10 Escherichia coli cell by heat shock at 42°C according to the manufacturer's recommendations (TOPO TA cloning kit; Invitrogen, Carlsbad, CA). Transformed E. coli cells were plated on Luria-Bertani agar containing ampicillin (50 μg/ml) and X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside). White colonies of E. coli cells were chosen and amplified by PCR with M13 primers. PCR amplicons were then submitted to the Biomedical Genomics Center at the University of Minnesota (BMGC; St. Paul, MN) for sequencing using the Sanger sequencing method (ABI Prism 3730xl DNA analyzer). The DNA sequences were analyzed, and a phylogenetic tree was prepared to identify redundancy in the selected pool using MEGA4 (29). Selected aptamer sequences were further analyzed for secondary structure prediction using the Mfold web server (30).
EMSA.
Selected aptamer sequences were synthesized and labeled with biotin (5′), while amplicons containing enriched aptamer pool were amplified by PCR with 5′-biotin-labeled forward primer (Bio-WP18). Aptamer-protein binding assay was analyzed by using a LightShift chemiluminescent electrophoretic mobility shift assay (EMSA) kit (Pierce) in accordance with the manufacturer's instructions with a few modifications. Briefly, selected aptamers (20 fmol) were added to recombinant swH3 protein in 1× binding buffer and nuclease-free water (final volume, 20 μl), followed by incubation at room temperature for 30 min. The samples were loaded onto an 8% native polyacrylamide gel (Bio-Rad, Hercules, CA) in 0.5× Tris-borate-EDTA buffer. Electrophoresis was performed at 80 V for 60 min, and then the samples were electrotransferred to a positively charged nylon membrane (Biodyne B; 0.45-μm pore size; Biodyne, Pensacola, FL). The membrane was processed and developed with a chemiluminescent nucleic acid detection module (Pierce) in accordance with the manufacturer's instructions. Reactions on the membrane were then visualized and imaged with the LabWorks 4.5 imaging system (UVP Products, Upland, CA).
HA inhibition test.
HA inhibition (HI) was performed to prove that the selected aptamers recognized the active site of the HA protein of swH3 IAV and could inhibit the viral infectivity. Turkey red blood cells (RBC) were diluted to 0.5% in PBS. The HA test was performed in a microtiter 96-well plate with 50-μl samples containing virus and 50 μl of 0.5% RBC. The samples were incubated at room temperature for 30 to 45 min, and the agglutination of RBC was inspected. HI was performed with 500 pmol of each aptamer added to the virus before the addition of the RBC, followed by incubation at room temperature for 30 min.
DNase I footprinting assay.
A DNase I footprinting assay was performed to identify the binding site(s) of the selected aptamer (31, 32). The recombinant swH3 protein (4 to 6 μg) was mixed with 1 pmol of HA68 labeled with 6-carboxyfluorescein (FAM) at the 5′ end in 1× binding buffer (LightShift; Pierce), nuclease-free water was added to a final volume 50 μl, followed by incubation at room temperature for 1 h. After incubation, the samples were treated with 0.2 U of DNase I (amplification-grade; Invitrogen), followed by incubation at 37°C for 5 min. To inactivate the DNase I, 2 mM EDTA was added to each sample, followed by incubation at 70°C for 10 min. Recombinant protein from an H1N1 human influenza virus (A/Solomon Islands/3/06 [human H1]) was used as a negative control and treated under identical conditions. A negative control (without protein) was applied using nuclease-free water in place of protein. After the reaction, the samples were purified by using a MiniElute PCR purification kit (Qiagen) and eluted with 14 μl of nuclease-free water. Approximately 12 μl of purified samples were submitted to the BMGC for fragment analysis on a 3130XL genetic analyzer. The binding region of aptamer was analyzed by Peak Scanner software (v.1.0; Applied Biosystems, Foster City, CA) with Liz500 as an internal size standard.
Specificity of each aptamer against recombinant HA proteins of different subtypes of IAVs.
The aptamer dot blot assay was modified and performed to test the specificity of each aptamer by using recombinant HA proteins in different subtypes of IAVs from the BEI resource: H1, H2, H3, H5, H6, H7, and H9 (Table 2). Each protein was diluted with nuclease free-water to a concentration of 50 ng/μl, and 2 μl each was blotted onto nitrocellulose membrane (Protran; Whatman/Pierce). The membrane was blocked with 5% fish gelatin (Sigma, St. Louis, MO) in 1× PBST at 4°C. After 12 h of incubation, the membrane was incubated with 5′ digoxigenin (DIG)-labeled aptamer at the final concentration 100 nM in 1% fish gelatin at room temperature with gentle shaking for 1 h and then washed five times with 1× PBST in 5-min increments. The membrane was incubated with peroxidase-conjugated IgG fraction monoclonal mouse anti-DIG (Jackson Immunoresearch Laboratories, Inc., West Grove, PA) in 1:12,500 dilutions in 1% fish gelatin in 1× PBST at room temperature for 30 min. The membrane was then washed once with 1× PBS and developed with luminal enhancer and stable peroxide solution in a 1:1 dilution (LightShift; Thermo) at room temperature for 5 min. The membrane was visualized and imaged by LabWorks 4.5 software (UVP Products).
Binding affinity of selected aptamers.
The recombinant swH3 protein was prepared at a concentration 25 ng/μl by dilution with 50 mM Tris–250 mM NaCl (pH 8.2) (protein buffer). Nitrocellulose membranes (Protran) were blotted with 2 μl of the protein (50 ng) in triplicate and dried at room temperature for 1 h. The membranes were blocked with 5% fish gelatin in 1× PBST at 4°C. After 12 h of incubation, the membranes were incubated with different concentrations of 5′ Bio-aptamer in 2-fold dilutions (1 to 1,024 nM) in 1% fish gelatin in 1× PBST at room temperature for 1 h with gentle shaking. The membranes were washed with 1× PBST for 5 min with gentle shaking. After five washes, the membranes were incubated with NeutrAvidin protein and horseradish peroxidase (Thermo) conjugated in 1:12,500 dilutions in 1% fish gelatin in PBST at room temperature for 30 min. The membranes were washed once with 1× PBS and developed with luminol enhancer and stable peroxide solution in 1:1 dilution (LightShift) at room temperature for 5 min. The membranes were visualized and imaged by LabWorks 4.5 software. The chemiluminescence intensity was calculated by using ImageJ 1.45s software. The dissociation constant (Kd) was calculated based on a nonlinear regression equation.
Influenza live virus detection by aptamer dot blot assay.
All processes involving work with live viruses were performed in a class II biosafety cabinet. IAV isolates from North American swine belonging to phylogenetic lineages of H3 were obtained from the University of Minnesota Veterinary Diagnostic Laboratory (UMVDL) as blind samples in minimal essential medium (MEM). AIV H3N2 in allantoic fluid was used as the representative for H3 cluster I (A/mallard/South Dakota/SE128/2007) (33). Detailed descriptions of the reference viruses used in the present study are given in Fig. 1 and Table 3. Samples containing live viruses were centrifuged at full speed (13,000 rpm) for 5 min prior use. Then, 2 μl of each culture (supernatant) was blotted onto the nitrocellulose membranes in triplicate (Protran). The membranes were dried at room temperature for 1 h and blocked with 5% fish gelatin (Sigma) in 1× PBST at 4°C. After 12 h of incubation, denatured sheared salmon sperm DNA (Ambion, Austin, TX) was added to the membranes with blocking buffer at a concentration of 10 ng/μl, followed by incubation at room temperature for 20 min. Membrane was incubated with DIG-aptamers (100 nM) in 1% fish gelatin at room temperature for 1 h and then washed five times with 1× PBST. The washing and developing steps were performed as described above.
Fig 1
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 (more ...)
Table 3
Table 3
Detail descriptions of reference influenza live viruses used in this study and results of each aptamer detected by aptamer dot blot assay
Selection of DNA aptamers against recombinant swH3 protein.
Sequencing of 80 transformants containing aptamer inserts (round 8) revealed a tendency toward sequence saturation, and thus seven additional iterations of SELEX were performed. After 15 rounds of SELEX, enriched aptamer pool showed high affinity against recombinant swH3 protein by gel shift assay (data not shown). Sequencing of 95 transformants containing aptamer inserts (round 15) were performed. DNA sequences and the percent redundancy of each sequence from the aptamer pool of round 15 of SELEX are shown in Table 4. The sequence similarities were compared, and five aptamer candidates from the selected pool were chosen for further characterization based on the differences in nucleotides (Fig. 2). DNA sequences of the enriched aptamer pool of rounds 8 and 15 were also compared to show the evolutionary change in the sequences (see Fig. S1 in the supplemental material). Secondary structure predictions of five selected DNA sequences are shown in Fig. S2 in the supplemental material. By aptamer dot blot assay, four aptamer candidates were shown to be specific to recombinant swH3 protein, and one (HA1c) did not bind to any proteins.
Table 4
Table 4
DNA sequences and number of clones from the aptamer pool of the 15th round of SELEX
Fig 2
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 (more ...)
Specificity of four candidate aptamers.
An aptamer dot blot assay was performed to test the specificity of the selected four aptamers. The results show that all aptamers are highly specific to H3 subtype compared to 10 other recombinant HA proteins representing seven different HA subtypes (Fig. 3).
Fig 3
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 (more ...)
Aptamer binding to recombinant swH3 protein.
Aptamers HA68 and HA7 were chosen to test using EMSA to show that aptamer specificity was for recombinant swH3 protein and not for an extraneous His6-tagged recombinant protein (rNP). The results show that the aptamers specifically recognized recombinant swH3 protein in a dose-dependent fashion (Fig. 4).
Fig 4
Fig 4
Two selected aptamer candidates, HA68 and HA7, were screened by using EMSA.
HI results.
Since the aptamers were enriched and selected by using recombinant protein, we wanted to show that these aptamers can bind to live virus. An HI assay was performed to show the neutralization activity and specificity of the four selected aptamers to the HA protein of A/swine/Minnesota/SG-00235/2007 (H3N2) compared to no inhibition of the unselected library (WAP40). At a concentration of 2.5 μM, the four aptamers HA68, HA7, HA2a, and HA2b were able to completely inhibit the agglutination of RBC at 16, 8, 16, and 4 HA U/50 μl, respectively (Fig. 5). In addition, we show that none of selected aptamers bound to a heterologous virus (A/swine/Minnesota/SG-00239/2007 [H1N2]). The result indicates that the aptamers bind specifically to HA protein likely at or around the receptor-binding site (34, 35) of H3N2 IAV of swine and do not bind to the H1N2 IAV of swine.
Fig 5
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).
Determination of the dissociation constants (Kd) of each aptamer.
To describe the strength of the binding affinity between aptamer and recombinant swH3 protein, dissociation constants (Kd) of each aptamer were calculated from the dot blot chemiluminescence intensities based on a nonlinear regression analysis. A concentration of each aptamer that saturates 50% of binding sites on the target protein was determined as the Kd. The results of the binding curve were fit to a modified equation (36) as follows: AB = (ABmax [A])/(Kd + [A]). In this modified equation, AB represents the fraction bound as measured by the chemiluminescence intensity, [A] is the concentration of the aptamer, and Kd is the dissociation constant. The binding affinities of HA2b, HA68, HA2a, and HA7 were 3.7, 7.1, 16.0, and 22.3 nM, respectively (Fig. 6).
Fig 6
Fig 6
The dissociation constant (Kd) of each DNA aptamer was used to describe the strength of the binding affinity between aptamer and recombinant swH3 protein. The Kd was calculated from the dot blot chemiluminescence intensity based on a nonlinear regression (more ...)
Binding region of aptamer HA68 to recombinant swH3 protein.
A DNase I footprinting assay was performed to identify the binding region of the aptamer to the recombinant swH3 protein. In the present study, HA68 was chosen for this purpose. The binding region of HA68 was identified to be between aptamer residues 8 and 40 (Fig. 7). An identical result was obtained using recombinant human H3 protein derived from A/Uruguay/716/2007 (H3N2). In addition, HA68 did not bind to recombinant human H1 protein (A/Solomon Islands/2/2006 [H1N1]) (see Fig. S3 in the supplemental material).
Fig 7
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 (more ...)
Use of aptamers to detect influenza live viruses.
A dot blot assay was performed to show the aptamer could be used to detect live viruses. Archived H3N2 IAVs from North American swine were provided by the UMVDL and used as representatives of each H3 cluster. The results showed that HA68 was able to bind to all lineages of the H3 subtype unequivocally, whereas the other three aptamers specifically identified H3 live viruses belonging to the phylogenetic clusters I, II/III, and IV, especially the virus that is closely related to the recent H3N2v (sample 4 in Fig. 1 and and8).8). It is likely that this application was affected by the virus titer in the cultured samples (see Fig. S4 in the supplemental material). A sample of H3N2v obtained from the CDC also tested positive in a dot blot assay, suggesting the possible application of selected aptamers in detecting zoonotic transmission.
Fig 8
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 (more ...)
Neutralizing DNA aptamers with high affinity against HA protein of H3N2 IAVs of swine origin were developed, and the methods for selection and characterization were also described. In the present study, four aptamer candidates were identified and characterized by using the SELEX method. The results of the aptamer dot blot assay showed that selected DNA aptamers can be used to detect and differentiate the subtypes of IAVs. Furthermore, all four aptamers showed an inhibitory effect of in vitro viral infectivity by HI test, suggesting that the aptamers were capable of binding to HA protein likely at or around the receptor-binding site required for penetration into the host cells of H3N2 IAVs (34, 35). This ability may apply to further study for therapeutic potential for the selected aptamers.
Since 1998, H3N2, initially isolated from North American swine, has become endemic in pig populations. Although pigs can be infected with many subtypes of IAVs, H1N1 and H3N2 are the most important and the most frequently isolated from pigs worldwide (12). Currently, most diagnostic tools rely on HI and virus isolation, both of which are time-consuming and require extensive laboratory resources, including RBCs or embryos from specific-pathogen-free chicken and cell-culturing facilities (21).
On the other hand, aptamers show promise as ideal candidates for molecular targeting applications. Aptamers can be chemically synthesized, and all processes were performed by in vitro techniques. Aptamers can be applied on a wide range of matrices for a primary clinical specimen. This addresses the primary advantage of an aptamer over an antibody, since the former can reduce the physiological variations from animals and replace animal systems (37). Moreover, aptamers can be easily and inexpensively synthesized without batch-to-batch variation (37, 38). They are also stable during long-term storage and easy to transport at an ambient temperature. Similar to antibodies, aptamers can also be labeled with reporter molecules such as fluorescein, biotin, and digoxigenin, which increases their applicability for further applications (3945). DNA and RNA aptamers have shown similar function and performance in terms of affinity and specificity (46). RNA molecules are susceptible to enzymatic degradation but can be stabilized by base modifications. DNA aptamers are easier to prepare, are stable, and can be amplified in one step by PCR and manipulated for the selection process. Current applications are primarily DNA based (47).
IAVs attach to host cells via sialic acid receptor. This receptor is also found on the RBC of several species of animals. Turkeys, guinea pigs, type O-human blood, and chicken RBC are traditionally used for HI tests (4850). Due to the presentation of sialic acid receptor on RBC, IAVs can agglutinate RBC (34). Since the aptamers in the present study weren developed based on recombinant swH3 protein, HI tests were performed to show that the aptamers bound specifically to viral HA protein.
IAVs have been identified in mixed infections due to the genomic heterogeneity identified by next-generation sequencing and exhibit significant genetic diversity (51, 52). HA protein is the part of the virus that is recognized by the host immune system. Thus, incremental variations largely result in antigenic drift (51, 53, 54), and ligands with high precision would be necessary to accurately detect infecting viruses.
Several studies of aptamers for IAVs have been reported, e.g., RNA aptamers for H5N2 (55) and H9N2 (56), DNA aptamers for human H1N1, H2N2, and H3N2 (28), and an RNA aptamer to distinguish between pathogenic human H3N2 and other pathogenic viruses from low-pathogenicity viruses (27). However, these reports have not demonstrated that these aptamers could be used to determine homologous and heterologous viral subtypes. In the present study, high-affinity DNA aptamers against SIV H3N2 were selected and characterized. The results of aptamer-dot blot assays showed that these aptamers can be used to detect and differentiate IAV subtypes. Aptamer HA68 has shown high sensitivity and binding affinity to all H3 clusters in the study. Three other aptamers (HA7, HA2a, and HA2b) have shown high sensitivity and specificity to clusters I, II/III, and IV, especially the virus that is closely related to the recent H3N2v (sample 4 in Fig. 1 and and8).8). Moreover, our results show that selected aptamers, especially HA68, bind not only to H3 IAV of swine but also human H3 and avian H3 (Fig. 3 and and8).8). For future applications, HA68 can be used to detect and subtype H3N2 IAVs, and other aptamers may be used to determine the phylogenetic cluster.
Supplementary Material
Supplemental material
ACKNOWLEDGMENTS
This study was funded by College of Veterinary Medicine, University of Minnesota, funds awarded to S.S. We acknowledge a Chulalongkorn University Dutsadi Phiphat scholarship providing financial support to M.W.
Footnotes
Published ahead of print 17 October 2012
Supplemental material for this article may be found at http://dx.doi.org/10.1128/JCM.02118-12.
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