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.
Highlights
Avian influenza virus (AIV) H9N2 subtype infecting turkeys was first reported in the United States in 1966 [1] and has widely spread to Europe and Asia since the 1990s [2,3]
Indirect Enzyme-linked immunosorbent assay (ELISA) with 7 hybridoma cell lines producing monoclonal antibodies (McAbs) was conducted for six avian influenza A virus subtypes at 1000 HAU/mL
Four hybridoma cell lines (A27-9, A37-C9, A39-G10, and A45D5) reacted with the H9N2 subtype with high signal and had no cross-reactivity with other subtype viruses
Summary
Avian influenza virus (AIV) H9N2 subtype infecting turkeys was first reported in the United States in 1966 [1] and has widely spread to Europe and Asia since the 1990s [2,3]. In China, the H9N2 subtype virus was isolated from diseased chickens in Guangdong province in 1994 and later infected domestic poultry in other provinces in China [2,4,5,6]. The first field outbreak of the H9N2 virus in Korea was reported in March 1996 caused by A/chicken/Korea/96006/96 (H9N2) with a 20% mortality rate and a severe (98.1%) drop in egg production. H9N2 is a low pathogenic avian influenza (LPAI) virus, which circulates primarily among wild birds and domestic poultry, resulting in great economic losses to the poultry industry because of declined egg production or moderate to high mortality [9,10,11,12]. Given the great economic losses, as well as the fear that H9N2 viruses may become a pandemic through repeated interspecies transmission from poultry to humans, efficient surveillance of H9N2 is essential
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