Abstract
Highly pathogenic avian influenza (HPAI) H5N1 viruses, which have emerged in poultry and other wildlife worldwide, contain a characteristic multi-basic cleavage site (CS) in the hemagglutinin protein (HA). Because this arginine-rich CS is unique among influenza virus subtypes, antibodies against this site have the potential to specifically diagnose pathogenic H5N1. By immunizing mice with the CS peptide and screening a phage display library, we isolated four antibody Fab fragment clones that specifically bind the antigen peptide and several HPAI H5N1 HA proteins in different clades. The soluble Fab fragments expressed in Escherichia coli bound the CS peptide and the H5N1 HA protein with nanomolar affinity. In an immunofluorescence assay, these Fab fragments stained cells infected with HPAI H5N1 but not those infected with a less virulent strain. Lastly, all the Fab clones could detect the CS peptide and H5N1 HA protein by open sandwich ELISA. Thus, these recombinant Fab fragments will be useful novel reagents for the rapid and specific detection of HPAI H5N1 virus.
Highlights
Influenza is a highly contagious disease caused by viruses that belong to the family Orthomyxoviridae [1,2]
We report several unique recombinant Fab fragments obtained from an immunized phage display library that target the cleavage site (CS) peptide of HA derived from highly pathogenic avian influenza (HPAI) H5N1 virus (HA331), and we discuss their potential applications in diagnostics
Mice were immunized with the HA331-bovine serum albumin (BSA) conjugate
Summary
Influenza is a highly contagious disease caused by viruses that belong to the family Orthomyxoviridae [1,2]. HA is a glycoprotein responsible for virus binding to sialic acid on the host cell surface, and mediates the fusion of the viral and cellular membranes after endocytosis [7,8]. Because the cleavage of HA0 into HA1 and HA2 is required for the fusion of viral and cellular membranes, the expression patterns of host proteases determine the viral organ tropism [10,11]. The HA0 cleavage site (CS) of the highly pathogenic avian influenza (HPAI) viruses contains a multi-basic sequence, which is cleaved by ubiquitous intercellular endoproteases including PC6 and furin [10,11,12]. HPAI viruses are able to enter multiple cell types and organs, and cause systemic infections
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