Development of a Rapid Fluorescent Diagnostic System to Detect Subtype H9 Influenza A Virus in Chicken Feces

Abstract

The circulation of the H9N2 virus results in significant economic losses in the poultry industry, and its zoonotic transmission highlights the need for a highly sensitive and rapid diagnostic and detection system for this virus. In this study, the performance of lateral flow test strips for a fluorescent immunochromatographic test (FICT) was optimized for the diagnosis of H9N2 virus-infected animal samples. The novel monoclonal antibodies (McAbs) against influenza A H9 viruses were developed, and two categories of McAbs with linear and conformational epitopes were compared for the performance of rapid diagnostic performance in the presence of feces sample at different time points (2, 4, and 6 days) post-infection (dpi). The limit of detection (LOD) of FICT and Kd values were comparable between linear and conformational epitope McAbs. However, superior performance of linear epitope McAbs pairs were confirmed by two animal studies, showing the better diagnostic performance showing 100% relative sensitivity in fecal samples at 6 dpi although it showed less than 80% sensitivity in early infection. Our results imply that the comparable performance of the linear epitope McAbs can potentially improve the diagnostic performance of FICT for H9N2 detection in feces samples. This highly sensitive rapid diagnostic method can be utilized in field studies of broiler poultry and wild birds.

Development of a Rapid Fluorescent Diagnostic System for Early Detection of the Highly Pathogenic Avian Influenza H5 Clade 2.3.4.4 Viruses in Chicken Stool

“…However, a positive signal was not detected in the three cloacal samples collected from the same H5N6-infected group. This result corroborated the lower viral load of cloacal samples compared to fecal samples reported for AIVs [ 53 , 54 ], as this trend was observed for the H5N6 and H9N2 viruses included in the present study. We also observed more mucus in cloacal than in fecal samples, and thus our system should be further optimized to detect viruses in cloacal samples.…”

Cysteamine-Gold Coated Carboxylated Fluorescent Nanoparticle Mediated Point-of-Care Dual-Modality Detection of the H5N1 Pathogenic Virus

“…Similarly, earlier reports suggest that the EuNPs have surface-functionalized carboxylated groups with strong stability that easily attach to amines and thiol ligands through covalent bond interactions [ 48 ]. The EuNPs obtained from Bangs Laboratories are well-established fluorescence chelates, and our research group has previously developed EuNP-mediated FICT kits for the detection of AIV [ 22 , 26 , 49 , 50 ]. Nonetheless, both of the types of nanoparticle have thus far been used only in single mode detection approaches.…”

“…Both of the assays were prepared, as per the optimized procedure that was previously developed by our group with minor modifications [ 22 ]. For the FICT, 4 μL of conjugate solution was diluted 40-fold, and the EuNPs-Ab conjugation and the Cys Au-EuNPs-Ab complex dropped onto a conjugate pad.…”

“…The metals such as Au-Ag, Au-Pt, Pt-Pd, and QDs have been used in RDT kits; however, so far improvements to the LOD have only been obtained using these methods with single-mode approaches [ 15 , 16 , 17 , 18 , 19 , 20 , 21 ]. Although our group has previously developed RDT devices for single-mode detection [ 22 ], several commercial EuNPs, such as florescent dyes [ 23 ], aliphatic amine latex beads [ 24 ], CdSe/CdS/ZnS QDs, and latex coated CdSe/CdS/ZnS QDs [ 17 ], show good sensitivity (93%) and specificity (100%). Our group has previously focused on enhancing the LOD with single-mode detection.…”