Generation and characterization of nucleic acid aptamers targeting the capsid P domain of a human norovirus GII.4 strain

AbstractHuman noroviruses (NoV) are the leading cause of acute viral gastroenteritis worldwide. Significant antigenic diversity of NoV strains has limited the availability of broadly reactive ligands for design of detection assays. The purpose of this work was to produce and characterize single stranded (ss)DNA aptamers with binding specificity to human NoV using an easily produced NoV target—the P domain protein. Aptamer selection was done using SELEX (Systematic Evolution of Ligands by EXponential enrichment) directed against an Escherichia coli-expressed and purified epidemic NoV GII.4 strain P domain. Two of six unique aptamers (designated M1 and M6-2) were chosen for characterization. Inclusivity testing using an enzyme-linked aptamer sorbent assay (ELASA) against a panel of 14 virus-like particles (VLPs) showed these aptamers had broad reactivity and exhibited strong binding to GI.7, GII.2, two GII.4 strains, and GII.7 VLPs. Aptamer M6-2 exhibited at least low to moderate binding to all VLPs tested. Aptamers significantly (p<0.05) bound virus in partially purified GII.4 New Orleans outbreak stool specimens as demonstrated by ELASA and aptamer magnetic capture (AMC) followed by RT-qPCR. This is the first demonstration of human NoV P domain protein as a functional target for the selection of nucleic acid aptamers that specifically bind and broadly recognize diverse human NoV strains

Selection, Characterization and Application of Nucleic Acid Aptamers for the Capture and Detection of Human Norovirus Strains

<div><p>Human noroviruses (HuNoV) are the leading cause of acute viral gastroenteritis and an important cause of foodborne disease. Despite their public health significance, routine detection of HuNoV in community settings, or food and environmental samples, is limited, and there is a need to develop alternative HuNoV diagnostic reagents to complement existing ones. The purpose of this study was to select and characterize single-stranded (ss)DNA aptamers with binding affinity to HuNoV. The utility of these aptamers was demonstrated in their use for capture and detection of HuNoV in outbreak-derived fecal samples and a representative food matrix. SELEX (Systematic Evolution of Ligands by EXponential enrichment) was used to isolate ssDNA aptamer sequences with broad reactivity to the prototype GII.2 HuNoV strain, Snow Mountain Virus (SMV). Four aptamer candidates (designated 19, 21, 25 and 26) were identified and screened for binding affinity to 14 different virus-like particles (VLPs) corresponding to various GI and GII HuNoV strains using an Enzyme-Linked Aptamer Sorbant Assay (ELASA). Collectively, aptamers 21 and 25 showed affinity to 13 of the 14 VLPs tested, with strongest binding to GII.2 (SMV) and GII.4 VLPs. Aptamer 25 was chosen for further study. Its binding affinity to SMV-VLPs was equivalent to that of a commercial antibody within a range of 1 to 5 µg/ml. Aptamer 25 also showed binding to representative HuNoV strains present in stool specimens obtained from naturally infected individuals. Lastly, an aptamer magnetic capture (AMC) method using aptamer 25 coupled with RT-qPCR was developed for recovery and detection of HuNoV in artificially contaminated lettuce. The capture efficiency of the AMC was 2.5–36% with an assay detection limit of 10 RNA copies per lettuce sample. These ssDNA aptamer candidates show promise as broadly reactive reagents for use in HuNoV capture and detection assays in various sample types.</p></div

Aptamer sequences obtained from 4^th, 7^th and 9^th round of SELEX for SMV.

<p>Bolded sequences were used in characterization studies and applications.</p><p>Aptamer sequences obtained from 4^th, 7^th and 9^th round of SELEX for SMV.</p

Equilibrium dissociation curves for select aptamers.

<p>GII.2. SMV VLPs (3 µg/ml) were screened with different concentrations (10 nM, 100 nM, 500 nM, 1 µM, 2 µM) of each aptamer using ELASA. To estimate K_d, plots of the T/N ratios (absorbance at 450 nm) as a function of the aptamer concentration were fitted to a non-interacting binding sites model with the equation <i>Y</i> = Bmax <i>X</i>/Kd+<i>X</i>. The negative controls consisted of PBS. Results correspond to aptamers SMV 19 (A), SMV 21 (B), SMV 25 (C) and SMV 26 (D).</p

Predicted structural folding of select ssDNA aptamers for SMV.

<p>Common motifs identified in boxes.</p

Performance of the AMC-RT-qPCR method (using aptamer SMV 25) as applied to artificially contaminated lettuce samples.

<p>Lettuce samples were inoculated with varying concentrations of a 20% suspension of HuNoV GII.4 fecal stock. They were pre-treated for virus concentration and purification using a combined elution-PEG precipitation method prior to AMC-qPCR. The negative controls consisted of the AMC using blocked beads in the absence of the aptamer. Experiments were done in triplicate. Statistically significant differences in recovery efficiencies are designated with an asterisk (<i>p</i><0.05).</p

Binding of selected aptamers to hepatitis A virus (HAV) and poliovirus (PV).

<p>Cell culture lysates of poliovirus and HAV were tested using ELASA with candidate aptamers. Positive controls consisted of SMV-VLPs; negative controls consisted of PBS alone. Results are expressed as ratios between absorbance readings for test sample versus negative control (T/N). Experiments were done in triplicate. Statistically significant differences between the ratios obtained from the positive control and the samples are designated with an asterisk (p<0.05).</p

Comparison of ELASA and ELISA for detection of SMV-VPLs using aptamer 25.

<p>SMV-VLPs (1 to 5 µg) were suspended in partially purified 20% HuNoV-negative stool suspension, serially diluted, and detected using aptamer 25 (1 µM) (ELASA) or anti-GII.2 antibody (1∶5000) (ELISA). Negative controls consisted of PBS. Results are expressed as ratios between absorbance readings for test sample versus negative control (T/N). Experiments were done in triplicate. Statistically significant differences between the ratios obtained are designated with an asterisk (p<0.05).</p

Binding affinity of selected aptamers against a broad panel of HuNoV VLPs.

1<p>Values indicate the ratio of absorbance readings for the test sample (T) versus negative control (N) using ELASA. Per convention <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106805#pone.0106805-Ebel1" target="_blank">[17]</a>, results less than 2.0 are considered negative (−). Low (+/−), medium (+) or strong (++) binding were interpreted for ratios between 2 and 5; >5 and 10; and >10, respectively. All experiments were done in triplicate.</p><p>Binding affinity of selected aptamers against a broad panel of HuNoV VLPs.</p