Characterization of the glycoproteins of novel fish influenza B-like viruses

Abstract

Novel influenza-like virus sequences previously identified in fish and amphibians were found to cluster as a sister clade of influenza B viruses, but have thus far remained uncharacterized. We demonstrate that salamander influenza-like virus (SILV) HA is functionally divergent from influenza B virus HA and does not bind to α 2,3- and α2,6-linked sialic acids. However, the HAs of Siamese algae-eater influenza-like virus (SAEILV) and chum salmon influenza-like virus (CSILV) bind to α2,3 linked sialic acid. Furthermore, SAEILV HA binds to sialyated Lewis X, is activated by human airway enzymes and is fusogenic at a wide range of pH conditions. SAEILV NA has a highly conserved active site and a similar structure to other known NAs. We also determined the cryo-electron microscopy structure of the HA of a previously described virus from the same sister clade, the Wuhan spiny eel influenza virus (WSEIV). Importantly, no cross-reactive antibodies against these HAs or NAs were found in the human serum, suggesting that humans are immunologically naïve to these viruses.

Fig. 1. Phylogenetic and comparative sequence analysis of SILV, SAEILV and CSILV glycoproteins.

Phylogenetic analysis of the salamander influenza-like virus (SILV), Siamese algae-eater influenza like virus (SAEILV) and chum salmon influenza-like virus (CSILV) HA and NA amino acid sequences and representative sequences from influenza A and B HAs (A) and NAs (B) were performed by the maximum likelihood method. The scale bar shows estimated amino acid substitutions per site. Conserved amino acid residues of the SILV HA and NA (C, F), SAEILV HA and NA (D, G), CSILV HA and NA (E, H) respectively relative to influenza B virus HA and NA are highlighted in blue on top of the publicly available structure of influenza B/Brisbane/60/2008 HA (PDB# 4FQM) and NA (PDB# 4CPL) by ChimeraX. Amino acid sequence alignment of the SILV HA and NA, SAEILV HA, NA and, CSILV HA and NA and WSEIV HA and NA are displayed against influenza B/Malaysia/2506/2004 HA and NA, (I) and (J). Key features are shown in different colors. Asterisks indicate identical amino acids. SDS-PAGE analysis of recombinant expressed SILV, SAEILV and CSILV HAs (K) and SAEILV NAs (L) and influenza B/Malaysia/2506/2004 HA and NA in deglycosylated and non-deglycosylated conditions.

Fig.2. Functional profile of SILV, SAEILV and CSILV HAs.

(A) Hemagglutination assays with recombinant HA proteins from salamander influenza-like virus (SILV), Siamese algae-eater influenza like virus (SAEILV) and chum salmon influenza-like virus (CSILV) were performed with turkey and chicken red blood cells (RBCs). Samples were added at an initial concentration of 10 μg/ml and serially diluted two-fold. (B-E). Sialic acid binding of recombinant HA proteins of SILV, CSILV and SAEILV was tested on a glycan microarray containing synthetic glycans with α2,3- and α2,6-linked sialic acid (Neu5Ac) presented as linear structures, bi-and tri-antennary glycans including sialyl lewis X (SLe^x) structures. B/Netherlands/2914/2015 served as positive controls (B-F). Glycans A-M and O-W represent linear and bi-antennary structures respectively with indicated backbones. Glycans 1–22 represents tri-antennary N-glycans, either the MGAT4 or MGAT5 arm was elongated to two LacNAc repeating units. Glycans were terminating without a NeuAc (yellow), or with α2,6-linked NeuAc (pink), α2,3-linked NeuAc (white), Le^x (blue), or SLe^x (black). Bars with two colors indicate glycans terminating in different epitopes on different arms. Mean relative fluorescence unit (RFU) ± SD are shown. (G) Experiment setup: HEK 293T cells were co-transfected with two plasmids, one encoding the full length of corresponding HA and one encoding human airway proteases. The HA cleavage state was determined after a 48-hour transfection. Representative western blots show the bands of uncleaved HA0 and cleaved HA1. Cells transfected with pCAGGS HA, either untreated or exposed to TPCK-treated trypsin, served as assay controls. (H) Experiment setup: HeLa cells were transfected with pCAGGS plasmid expressing full length of corresponding HA and were treated trypsin, and exposed to an acidic buffer, and allowed to recover to determine for cell-to-cell fusion. (I) Representative photographs showing polykaryon formation in cells transfected with pCAGGS SILV, SAEILV, CSILV, and B/Malaysia/2506/2004 HA.

Fig. 3. WSEIV HA cryoEM structure resembles influenza B HA.

(A). CryoEM map and model of WSEIV HA showing the HA1/HA2 subunits, fusion peptide, RBS pocket, and N-linked glycans. (B). Models of WSEIV HA and B/Brisbane/60/2008 HA (PDB 4FQM) are aligned by the central helix of HA2. (C). The surface of WSEIV HA and B/Brisbane/60/2008 HA are colored by electrostatic potential using ChimeraX. (D). Key residues within the RBS of WSEIV HA and B/Brisbane/60/2008 HA. Ab-initio reconstruction maps and selected 2D classes of SAEILV HA (E) and SILV HA (F) from negative stain EM

Fig. 4. NA specific activities and cleavage specificities of the SAEILV NA.

(A) Neuraminidase enzymatic activity of recombinant SAEILV and B/Malaysia/2506/2004 NA proteins was examined via enzyme linked lectin assay (ELLA) using fetuin, at four different temperatures: 4, 20, 33 and 37°C. The curves indicate absorbance measured at 490 nm with error bars indicating standard deviation. (B) Specific enzymatic activity (inverse of half-maximum lectin binding) is shown for each individual NA at each temperature. (C) Inhibitory susceptibility to three NA inhibitors (oseltamivir, peramivir and zanamivir) was evaluated in an ELLA-based neuraminidase inhibition assay. (D) Specific activity of SAEILV and B/Malaysia/2506/2004 NA as determined by ELLA using different glycoproteins-lectin combinations normalized to the fetuin-ECA.

Fig. 5. SAEILV NA cryoEM structure resembles influenza B NA

(A). CryoEM map of SAEILV NA showing the tetrameric NA head, active site, and N-linked glycan. (B). Aligned models of SAILV NA, WSEIV NA (PDB 7XVU), and B/Brisbane/60/2008 NA (PDB 4CPL) showing conserved secondary structures, key active site residues, and underside glycans among 3 NAs. (C). The surface and the active site pockets of SAEILV NA, WSEIV NA, and B/Brisbane/60/2008 NA are colored by electrostatic potential using ChimeraX. (D). CryoEM map and model showing the conserved calcium binding pocket in SAEILV NA. (E). Aligned models of SAEILV NA and 1G01 + A/California/04/2009 (H1N1) showing 1G01 HCDR3 clashes with SAEILV NA Y239.

Fig. 6. Binding profiles of the anti-influenza B virus HA and NA monoclonal antibodies (mAbs).

(A and C) Broadly cross-reactive anti-influenza B virus HA and NA human and mouse monoclonal antibody binding to recombinant protein (SILV, SAEILV, CSILV and B/Malaysia/2506/2004 HA) and (SAEILV and B/Malaysia/2506/2004 NA) in enzyme linked immunosorbent assay. (B) Anti-influenza B virus human and mouse monoclonal antibody binding to cell surface expressed NAs of SILV, SAEILV, CSILV and B/Malaysia/2506/2004 by flow cytometry. (D) Cross reactive antibody profile of sera from post-seasonal influenza vaccination recipients. Antibodies against SILV, SAEILV and CSILV HA and SAEILV NA were determined via ELISA. Mopeia virus glycoprotein was used as negative control for baseline establishment.