Accurate and rapid diagnosis of highly pathogenic avian influenza A H5N1 is of critical importance for the effective clinical management of patients. Here, we developed a rapid and simultaneous detection toolkit for influenza A H5 subtype viruses in human samples based on a bioconjugate of quantum dots (QDs) assembly and a smartphone-based rapid dual fluorescent diagnostic system (SRDFDS).Methods: Two types of QDs were assembled on a latex bead to enhance the detection sensitivity and specificity of influenza A infection (QD580) and H5 subtype (QD650). The dual signals of influenza A and H5 subtype of H5N1-infected patients were detected simultaneously and quantified separately by SRDFDS equipped with two emission filters.Results: Our results showed a high sensitivity of 92.86% (13/14) and 78.57% (11/14), and a specificity of 100% (38/38, P < 0.0001) and 97.37% (37/38) for influenza A and H5 subtype detection, respectively.Conclusion: Therefore, our multiplex QD bioconjugates and SRDFDS-based influenza virus detection toolkit potentially provide accurate and meaningful diagnosis information with improved detection accuracies and sensitivities for H5N1 patients.

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<b><i>Background:</i></b> When infected with the chikungunya virus (CHIKV), 3% to 28% of CHIKV-infected individuals remain asymptomatic, necessitating the development of improved high-throughput screening methods to overcome the limitations of molecular diagnostics or enzyme-linked immunosorbent assays (ELISAs). <b><i>Objective:</i></b> In this study, two novel monoclonal antibodies (mAbs) targeting envelope 1 (E1) of CHIKV were developed and applied in a fluorescence-linked immunosorbent assay (FLISA) using coumarin-derived dendrimer as the fluorophore. <b><i>Methods:</i></b> The performance of the FLISA was compared with that of ELISA. <b><i>Results:</i></b> Using the two novel mAbs (2B5 and 2C8), FLISA could detect 1 × 10⁵ PFU/mL of CHIKV, exhibiting a 2-fold lower limit of detection (LOD) compared to ELISA. The LOD of FICT corresponded to a comparative threshold value of 23.95 and 4  ×  10⁶ of RNA copy number/µL. In the presence of human sera and blood, virus detection by FLISA was 3-fold better than ELISA, with an LOD of 2 × 10⁵ PFU/mL. Sera and blood interfered with the ELISA, resulting in 6 × 10⁵ PFU/mL as the LOD. <b><i>Conclusions:</i></b> FLISA using two novel mAbs and coumarin-derived dendrimer is a superior diagnostic assay for detecting CHIKV in human sera and blood, compared to conventional ELISA.

Low-pathogenicity avian influenza viruses (LPAIV) introduced by migratory birds circulate in wild birds and can be transmitted to poultry. These viruses can mutate to become highly pathogenic avian influenza viruses causing severe disease and death in poultry. In March 2019, an H7N3 avian influenza virus—A/Spot-billed duck/South Korea/WKU2019-1/2019 (H7N3)—was isolated from spot-billed ducks in South Korea. This study aimed to evaluate the phylogenetic and mutational analysis of this isolate. Molecular analysis revealed that the genes for HA (hemagglutinin) and NA (neuraminidase) of this strain belonged to the Central Asian lineage, whereas genes for other internal proteins such as polymerase basic protein 1 (PB1), PB2, nucleoprotein, polymerase acidic protein, matrix protein, and non-structural protein belonged to that of the Korean lineage. In addition, a monobasic amino acid (PQIEPR/GLF) at the HA cleavage site, and the non-deletion of the stalk region in the NA gene indicated that this isolate was a typical LPAIV. Nucleotide sequence similarity analysis of HA revealed that the highest homology (99.51%) of this isolate is to that of A/common teal/Shanghai/CM1216/2017 (H7N7), and amino acid sequence of NA (99.48%) was closely related to that of A/teal/Egypt/MB-D-487OP/2016 (H7N3). An in vitro propagation of the A/Spot-billed duck/South Korea/WKU2019-1/2019(H7N3) virus showed highest (7.38 Log10 TCID50/mL) virus titer at 60 h post-infection, and in experimental mouse lungs, the virus was detected at six days’ post-infection. Our study characterizes genetic mutations, as well as pathogenesis in both in vitro and in vivo model of a new Korea H7N3 viruses in 2019, carrying multiple potential mutations to become highly pathogenic and develop an ability to infect humans; thus, emphasizing the need for routine surveillance of avian influenza viruses in wild birds.

Influenza A virus subtype H1N1 has caused global pandemics like the “Spanish flu” in 1918 and the 2009 H1N1 pandemic several times. H1N1 remains in circulation and survives in multiple animal sources, including wild birds. Surveillance during the winter of 2018–2019 in Korea revealed two H1N1 isolates in samples collected from wild bird feces: KNU18-64 (A/Greater white-fronted goose/South Korea/KNU18-64/2018(H1N1)) and WKU19-4 (A/wild bird/South Korea/WKU19-4/2019(H1N1)). Phylogenetic analysis indicated that M gene of KNU18-64(H1N1) isolate resembles that of the Alaskan avian influenza virus, whereas WKU19-4(H1N1) appears to be closer to the Mongolian virus. Molecular characterization revealed that they harbor the amino acid sequence PSIQRS↓GLF and are low-pathogenicity influenza viruses. In particular, the two isolates harbored three different mutation sites, indicating that they have different virulence characteristics. The mutations in the PB1-F2 and PA protein of WKU19-4(H1N1) indicate increasing polymerase activity. These results corroborate the kinetic growth data for WKU19-4 in MDCK cells: a dramatic increase in the viral titer after 12 h post-inoculation compared with that in the control group H1N1 (CA/04/09(pdm09)). The KNU18-64(H1N1) isolate carries mutations indicating an increase in mammal adaptation; this characterization was confirmed by the animal study in mice. The KNU18-64(H1N1) group showed the presence of viruses in the lungs at days 3 and 6 post-infection, with titers of 2.71 ± 0.16 and 3.71 ± 0.25 log10(TCID50/mL), respectively, whereas the virus was only detected in the WKU19-4(H1N1) group at day 6 post-infection, with a lower titer of 2.75 ± 0.51 log10(TCID50/mL). The present study supports the theory that there is a relationship between Korea and America with regard to reassortment to produce novel viral strains. Therefore, there is a need for increased surveillance of influenza virus circulation in free-flying and wild land-based birds in Korea, particularly with regard to Alaskan and Asian strains.