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

Abstract

Rapid diagnosis is essential for the control and prevention of H5 highly pathogenic avian influenza viruses (HPAIVs). However, highly sensitive and rapid diagnostic systems have shown limited performance due to specific antibody scarcity. In this study, two novel specific monoclonal antibodies (mAbs) for clade 2.3.4.4 H5Nx viruses were developed by using an immunogen from a reversed genetic influenza virus (RGV). These mAbs were combined with fluorescence europium nanoparticles and an optimized lysis buffer, which were further used for developing a fluorescent immunochromatographic rapid strip test (FICT) for early detection of H5Nx influenza viruses on chicken stool samples. The result indicates that the limit of detection (LoD) of the developed FICT was 40 HAU/mL for detection of HPAIV H5 clade 2.3.4.4b in spiked chicken stool samples, which corresponded to 4.78 × 104 RNA copies as obtained from real-time polymerase chain reaction (RT-PCR). An experimental challenge of chicken with H5N6 HPAIV is lethal for chicken three days post-infection (DPI). Interestingly, our FICT could detect H5N6 in stool samples at 2 DPI earlier, with 100% relative sensitivity in comparison with RT-PCR, and it showed 50% higher sensitivity than the traditional colloidal gold-based rapid diagnostic test using the same mAbs pair. In conclusion, our rapid diagnostic method can be utilized for the early detection of H5Nx 2.3.4.4 HPAIVs in avian fecal samples from poultry farms or for influenza surveillance in wild migratory birds.

Identification of specific neutralizing antibodies for highly pathogenic avian influenza H5 2.3.4.4b clades to facilitate vaccine design and therapeutics

“…293 T cells were generously provided by Dr. Chris MOK from The University of Hong Kong. Recombinant H5 viruses containing HA from A/Anas/KR/2017/2.3.4.4b, RG/A/Swan/MG/2020/2.3.4.4 h, and RG/A/VN/2014/2.3.2.1c, referred to as wild-type viruses (Table S1), and internal gene segments from A/Puerto Rico/8/34 (H1N1) (PR8) were generated as previously described [ 20 , 21 ]. The recommended H5 mutants of A/Anas/KR/2017/2.3.4.4b and RG/A/Swan/MG/2020/2.3.4.4 h were generated via site-directed mutagenesis of the plasmids using the primers described in Table S2.…”

“…An indirect ELISA was performed as previously described [ 20 , 23 ]. Briefly, wild-type and mutated viruses were diluted to 1280 HAU/mL in coating buffer (bicarbonate/carbonate 100 mM, pH 9.6).…”

“…In our previous study, two novel (#11.4 and #23.3) specific mAbs targeting the HA of the H5N6 2.3.4.4b subclade viruses were developed and applied to diagnostic systems [ 20 ]. Here, we report the neutralization abilities of both mAbs against the H5 2.3.4.4b clade viral challenge in mouse models and Madin-Darby Canine Kidney cell line (MDCKs).…”

Identification of novel biomarkers for anti-Toxoplasma gondii IgM detection and the potential application in rapid diagnostic fluorescent tests

“…Following centrifugation at 27,237× g for 10 min, the precipitated bioconjugates were collected, washed once, and resuspended in 400 μL of 2 mM borax pH 9.0 containing 0.1% BSA. Finally, the bioconjugates were stored in the dark at 4°C for subsequent use ( Duong et al, 2022 ).…”