Rapid and broad detection of H5 hemagglutinin by an immunochromatographic kit using novel monoclonal antibody against highly pathogenic avian influenza virus belonging to the genetic clade 2.3.4.4

<div><p>Highly pathogenic avian influenza viruses (HPAIVs) of H5 subtype have persistently caused outbreaks in domestic poultry and wild birds worldwide and sporadically infected humans. Rapid and accurate diagnosis is one of the key strategies for the control of H5 HPAIVs. However, the sensitivity of the diagnosis of H5 HPAIVs has gradually reduced due to extensive antigenic variation during their evolution. Particularly, the previously developed immunochromatographic diagnosis kit for H5 viruses, Linjudge Flu A/H5, exhibits reduced detection of H5 HPAIVs isolated in recent years. In the present study, we established a new advanced H5 rapid immunochromatographic detection kit (New Linjudge Flu A/H5) by a combination of two anti-H5 hemagglutinin monoclonal antibodies, A64/1 previously applied in the Linjudge Flu A/H5 and A32/2, a novel monoclonal antibody generated from a clade 2.3.4.4 H5 HPAIV. The new kit broadly detected all classical and recent H5 influenza viruses and showed a higher specificity and sensitivity than the original Linjudge Flu A/H5 with recently circulating H5 HPAIVs. Furthermore, the applicability of the New Linjudge Flu A/H5 was demonstrated by detecting antigens from the swabs and tissue homogenates of naturally infected birds and experimentally infected chickens with H5N6 HPAIVs belonging to the genetic clade 2.3.4.4. Our study, therefore, can provide an effective point-of-care rapid antigen detection kit for the surveillance of H5 avian influenza viruses and as a prompt countermeasure against the current widespread of the clade 2.3.4.4 H5 HPAIVs in domestic and wild birds.</p></div

Antigen detection from the swabs and tissue homogenates of naturally infected birds and experimentally infected chickens with H5N6 HPAIVs.

<p>Antigen detection from the swabs and tissue homogenates of naturally infected birds and experimentally infected chickens with H5N6 HPAIVs.</p

Specificity of the New Linjudge Flu A/H5, Linjudge Flu A/H5 and ImunoAce Flu (NP) with IAVs and influenza B viruses.

<p>Specificity of the New Linjudge Flu A/H5, Linjudge Flu A/H5 and ImunoAce Flu (NP) with IAVs and influenza B viruses.</p

Detection limit of the New Linjudge Flu A/H5, Linjudge Flu A/H5 and ImunoAce Flu (NP) to detect H5 HA antigens.

<p>Detection limit of the New Linjudge Flu A/H5, Linjudge Flu A/H5 and ImunoAce Flu (NP) to detect H5 HA antigens.</p

Comparison of primary structures of mlEFL35p and ebolavirus VP35s.

<p>The same amino acid residues that are found in VP35s and mlEFL35p are highlighted in orange. Residues highlighted in green represent amino acids that are grouped together in the same classes, based on their physical/chemical properties. The blue rectangles show sequence gaps found between mlEFL35 and VP35s. The NP binding domain consisting of the residues 20–48 (EBOV numbering), termed NPBP, is highlighted in yellow. Amino acid residues indicated by red dots have been identified to be important for VP35 homo-oligomerization as well as viral replication and transcription [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186450#pone.0186450.ref035" target="_blank">35</a>]. Amino acid residues indicated by blue dots and green dots have been shown to be critical for the dsRNA binding and polymerase cofactor activities, respectively [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186450#pone.0186450.ref031" target="_blank">31</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186450#pone.0186450.ref036" target="_blank">36</a>]. The VP35 homo-oligomerization domain and IFN inhibitory domain are indicated by arrows and arrowheads, respectively. Asterisks indicate cysteine residues in VP35s. Black and red stars indicate the amino acids that form the FBP and the CBP regions on 3-dimensional structure of the VP35 IID, respectively.</p

Expression of the mlEFL35p and VP35s in HEK 293T cells.

<p><b>(A)</b> Expression of each protein was confirmed by western blotting. HA-tagged mlEFL35p (HA-mlEFL35p), HA-tagged EBOV VP35 (HA-ZVP35) and HA-tagged RESTV VP35 (HA-RVP35) were detected as 30, 37, and 40 kDa proteins, respectively. <b>(B)</b> Distribution of each protein is visualized by an immunofluorescence assay with anti-HA antibodies. Cells were counterstained with DAPI.</p

Immunoprecipitation assay of mlEFL35p, VP35s and NP.

<p><b>(A)</b> To examine whether mlEFL35p interacted with mlEFL35p itself, EBOV and RESTV VP35s, FLAG-tagged mlEFL35p, EBOV and RESTV VP35s (FLAG-ZVP35 and FLAG-RVP35, respectively) expressed in HEK 293T cells were immunoprecipitated (IP) with HA-tagged mlEFL35p and VP35s of EBOV and RESTV (HA-ZVP35 and HA-RVP35, respectively). Precipitated proteins were detected by western blotting with anti-FLAG tag antibodies. <b>(B)</b> EBOV NP (ZNP) and RESTV NP (RNP) were expressed in HEK 293T cells and immunoprecipitated with HA-tagged VP35 of EBOV and RESTV (HA-ZVP35 and HA-RVP35) or HA-tagged mlEFL35p. HA-tagged proteins were detected by western blotting with an anti HA-tag antibody. ZNP and RNP were similarly detected with rabbit antisera specific to NPs.</p

Inhibition of the RIG-I-mediated signaling pathway by mlEFL35 and VP35s.

<p><b>(A)</b> The RIG-I-mediated signaling pathway is shown. The human IFN-β promoter is activated through RIG-I, IPS-1 and TBK1. The IFN-β promotor activity was measured by luciferase reporter assays. <b>(B)</b> HEK 293 cells were transfected with each plasmid expressing HA-tagged influenza A virus NS1 (IAVs-NS1-HA), EBOV VP35 (HA-ZVP35), RESTV VP35 (HA-RVP35) or mlEFL35p (HA-mlEFL35p) and the plasmids for the reporter gene expression along with the RIG-I CARD domain vector, IPS-1 or TBK1 expression vector. NS1 and VP35 are known as IFN antagonists. Western blotting was performed to examine the expression of NS1, VP35s, and mlEFL35p. Each HA-tagged protein (IAVs-NS1-HA, HA-ZVP35, HA-RVP35, and HA-mlEFL35p) was detected with an anti-HA-tag antibody. (<b>C</b>) Transfected cells were solubilized and luciferase assays were performed. Relative luciferase activities were calculated by setting the values given by the cells transfected with a control empty plasmid expressing the HA tag alone. Significantly lower values compared to control cells (Empty) are indicated by asterisks (*p < 0.05, **p < 0.01). (<b>D</b>) Concentrations of IFN-β in the supernatants of cells transfected with the indicated plasmids (1000 ng) were measured by ELISA. Means and standard deviations of five independent experiments are shown. Significantly lower values compared to control cells (Empty) are indicated by asterisks (<b>*</b><i>p</i> < 0.05, <b>**</b><i>p</i> < 0.01).</p

BLAST search data of mlEFL35.

<p>BLAST search data of mlEFL35.</p

Luciferase expression from the Ebola virus minigenome with mlEFL35p.

<p><b>(A)</b> HEK 293T cells were transfected with the indicated amounts of plasmids for the expression of the HA tag alone, HA-tagged mlEFL35p (HA-mlEFL35p), or EBOV VP35 (HA-ZVP35) along with plasmids for the expression of NP, VP30, L, the T7 polymerase and p3E5E-luc. Relative luciferase activities were determined by setting the values of control cells transfected with the HA-ZVP35-expressing plasmid to 100%. Means and standard deviations of three independent experiments are shown. Significant differences from control cells (HA ZVP35) are indicated by asterisks (<b>*</b><i>p</i> < 0.05). Between the empty control and mlEFL35p, there was no significant difference. <b>(B)</b> HEK 293T cells were transfected with the indicated amounts of plasmids for the expression of the HA tag alone, HA-tagged mlEFL35p (HA-mlEFL35p), or EBOV VP24 (ZVP24) along with plasmids for the expression of NP, VP35, VP30, L, the T7 polymerase and p3E5E-luc. ZVP24 was used as a positive control. Means and standard deviations of three independent experiments are shown. Significantly lower values compared to control cells (Empty) are indicated by asterisks (<b>**</b><i>p</i> < 0.01). <b>(C)</b> Expression of each protein was detected by western blotting. HA-tagged proteins (HA-ZVP35 and HA-mlEFL35p) were detected with an anti-HA tag antibody. ZVP24 were detected with a VP24-specific mouse antiserum produced with the synthetic peptide corresponding to amino acid positions 3–15 (KATGRYNLISPKK) of EBOV VP24. β actin were detected with an anti-β actin antibody.</p