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Phenotypic effects of mutations observed in the neuraminidase of human origin H5N1 influenza A viruses [1]

['David Scheibner', 'Institute Of Molecular Virology', 'Cell Biology', 'Friedrich-Loeffler-Institut', 'Federal Research Institute For Animal Health', 'Greifswald-Insel Riems', 'Ahmed H. Salaheldin', 'Department Of Poultry Diseases', 'Faculty Of Veterinary Medicine', 'Alexandria University']

Date: 2023-02

Global spread and regional endemicity of H5Nx Goose/Guangdong avian influenza viruses (AIV) pose a continuous threat for poultry production and zoonotic, potentially pre-pandemic, transmission to humans. Little is known about the role of mutations in the viral neuraminidase (NA) that accompanied bird-to-human transmission to support AIV infection of mammals. Here, after detailed analysis of the NA sequence of human H5N1 viruses, we studied the role of A46D, L204M, S319F and S430G mutations in virus fitness in vitro and in vivo. Although H5N1 AIV carrying avian- or human-like NAs had similar replication efficiency in avian cells, human-like NA enhanced virus replication in human airway epithelia. The L204M substitution consistently reduced NA activity of H5N1 and nine other influenza viruses carrying NA of groups 1 and 2, indicating a universal effect. Compared to the avian ancestor, human-like H5N1 virus has less NA incorporated in the virion, reduced levels of viral NA RNA replication and NA expression. We also demonstrate increased accumulation of NA at the plasma membrane, reduced virus release and enhanced cell-to-cell spread. Furthermore, NA mutations increased virus binding to human-type receptors. While not affecting high virulence of H5N1 in chickens, the studied NA mutations modulated virulence and replication of H5N1 AIV in mice and to a lesser extent in ferrets. Together, mutations in the NA of human H5N1 viruses play different roles in infection of mammals without affecting virulence or transmission in chickens. These results are important to understand the genetic determinants for replication of AIV in mammals and should assist in the prediction of AIV with zoonotic potential.

Avian influenza viruses (AIV) including viruses of the H5Nx Goose/Guangdong-lineage caused hundreds of human infections and fatalities worldwide and pose a continuous zoonotic and pandemic threat. It is important to understand the role of mutations that support AIV-infection in mammals after bird-to-human transmission. Here, we describe the prevalence and the role of mutations in the viral neuraminidase (NA), which is mainly responsible for virus release from infected cells, preferentially selected in human H5N1 viruses. Compared to their avian ancestors, human-like H5N1 had less NA in virus particles and exhibited lower NA activity conferred mainly by the L204M substitution. Furthermore, NA mutations increased the binding of H5N1 to human-type receptors, acted synergistically to confer high virus replication in avian and human cells and/or modulated virulence and replication in mice, but not in chickens. This study is important to understand the genetic and biological properties of human AIV and will help to predict zoonotic potential AIV.

Funding: This project was partially funded by grants from the Deutsche Forschungsgemeinschaft (VE780/1-1, AB 567) to EMA and the DELTA-FLU Project (ID: 727922) funded by the European Union to EMA and TCM as well as the German Centre for Infection Research (DZIF), partner site Giessen, Germany (TTU 01.806 Broad-spectrum Antivirals and FF 01.901 Nucleoside-booster to Co-PI SP), DFG funded SFB 1021 (RNA viruses: RNA metabolism, host response and pathogenesis, TP C01 to Co-PI SP and TP BO2 to Co-PI MM), and National Research Centre (TT110801 and 12010126 to AM). LMZ and SF were supported by the Federal Excellence Initiative of Mecklenburg Western Pomerania & the European Social Fund (ESF) Grant KoInfekt (ESF/14-BM-A55-0002/16). The glycan microarray experiments are supported by the Netherlands Organization for Scientific Research (NWO TOPPUNT 718.015.003) to G.-J.B., R.P.dV is a recipient of ERC starting grant 802780 and a Beijerinck Premium of the Royal Dutch Academy of Sciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2023 Scheibner et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Here, we analyzed all NA gene sequences of H5N1 viruses of human- and poultry-origin from Eurasia and Africa. We generated several recombinant viruses with mutations in the NA and studied their effect in vitro and in vivo in chickens, mice and ferrets. We found that NA mutations in human H5N1 play diverse roles in virus replication in mammals without affecting virus fitness in birds.

Since 1997, highly pathogenic (HP) AIV Goose/Guangdong (GsGd) H5Nx viruses continue to cause high losses in poultry and pose a serious pandemic risk [ 11 ]. The NA of GsGd H5N1 viruses usually possesses a NA-stalk deletion, a major determinant for adaptation of AIV in poultry [ 12 ], and the HA has evolved into 10 phylogenetic clades (clade 0 to 9) and tens of subclades. Clade 2 viruses, including the recent panzootic clade 2.3.4.4, are still endemic in Asia and Egypt [ 13 ]. Since 2006, Egypt has reported the highest number of GsGd-H5N1-infections in humans worldwide with ~42% (359/861) of infections and ~26% (120/455) of global fatal cases [ 14 ]. Compared to avian-origin H5N1 viruses, the Egyptian human-like (HL) H5N1 clade 2.2.1.2 viruses possessed HA mutations which enhanced virus affinity to the human-type 2,6-SA receptors but retained its interaction with avian-type 2,3-SA receptors [ 15 ], and mutations in the viral polymerase complex (PB2, PB1, PA, NP) which increased viral replication in human cells [ 16 ]. In 2014–2015, a novel group in clade 2.2.1.2 spread in poultry and transmitted to at least 165 humans [ 17 , 18 ]. Compared to the parental clade 2.2.1 in 2006 (designated avian-like, AL), the NA gene of HL-H5N1 in 2008 (HL-08; referring to human-origin H5N1 viruses in 2008) and 2015–2016 (HL-16; referring to human-origin H5N1 viruses in 2015/2016) viruses possessed 4 and 16 NA missense mutations, respectively, with unknown biological relevance [ 17 ].

Influenza A viruses (IAV) possess a negative-sense RNA genome composed of eight segments, which encode at least 11 viral proteins. IAV are classified according to the variation of the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA), which occur in 18 (for HA) and 11 (for NA) subtypes. IAV infect a wide range of species including birds and humans. Human influenza viruses belong to H1-H3 subtypes, while aquatic birds are the reservoir for H1-H16 avian influenza viruses (AIV) [ 1 ]. Although AIV generally do not replicate efficiently in humans, some strains are able to cross the species barrier and infect humans inducing infections which range from self-limiting flu-like illness to death [ 1 ]. Importantly, most of the pandemic human influenza viruses carried gene segments encoding HA, NA and/or polymerase basic-1 (PB1) from AIV [ 2 , 3 ]. Animal-to-human transmission of AIV has been accompanied by genetic alterations, which enabled efficient virus replication in human cells. While mutations linked to human-adaptation of AIV in PB2, PB1 and HA are well characterized [ 4 , 5 ], surprisingly little is known about the role of NA in human infections with AIV. NA is a tetrameric mushroom-like glycoprotein complex with each monomer composed of N-terminal, transmembrane, stalk and head domains [ 6 ]. The head domain contains the sialidase activity, which cleaves sialic acid (SA) to release progeny virions or digests mucin in the respiratory tract to enable systemic virus spread. The NA enzymatic pocket is stabilized by highly conserved catalytic and framework residues [ 6 ]. Therefore, neuraminidase inhibitors (NAI) are used successfully to control influenza viruses in humans [ 7 ]. Mutations in NA are thought to evolve to maintain a meticulous functional balance with the receptor-binding activity of the HA [ 8 ] or are selected for by external pressure (e.g. antivirals or vaccination) [ 9 , 10 ].

Results

Unique mutations co-evolved in the NA of human H5N1 viruses Sequence analysis of Egyptian H5N1 viruses (n = 509) isolated from humans (n = 126) and poultry (n = 383) from 2006 to 2016 revealed a particularly high prevalence of four missense mutations, i.e. A46D, L204M, S319F and S430G (N2 numbering equivalents: 48, 224, 339 and 451), in human-origin viruses compared to viruses from poultry (p < 0.005) (Fig 1A). Interestingly, we rarely observed these mutations individually, except for S319F in 2006–2007. Viruses with all four NA- mutations dominated after 2008 (HL-08) and those isolated in 2015–2016 (HL-16) possessed twelve novel NA mutations. Analysis of NA-N1 genes from poultry (n = 3311) and humans (n = 324) in Asia from 1997 to 2016 demonstrated low prevalence of these markers (Fig 1A), indicating enrichment of these mutations in human-H5N1 viruses in Egypt, where the highest number of H5N1-human infections are reported. Analysis of non-H5N1 sequences revealed 100% conservation of L204, except for influenza viruses of H8 subtype, which carry M204. A46D resides in the stalk domain, while the other mutations are in the head domain (Fig 1B). S319F is located in an antigenic site [19], while L204M resides between the sialidase catalytic and framework sites [20]. S430G is not part of any particular site hitherto identified. Molecular docking predicted that L204M alone or in combination with the other three mutations might decrease the binding of NA to the SA ligand (S1 Fig). PPT PowerPoint slide

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TIFF original image Download: Fig 1. Prevalence of NA mutations in H5N1 of human and poultry-origin, structural modelling and replication in primary avian and human cell culture. Prevalence of N1-NA mutations in human (blue) and poultry (black) origin sequences of H5N1 in Egypt and Asia in GenBank and GISAID at 31.12.2016 were retrieved and analyzed using Geneious (A). Predicted location of NA mutations (depicted in red) on the NA tetramer generated by SWISS Model using the amino acid sequence of A/chicken/Egypt/06207/2006 (accession number ACR56180); one of the earliest viruses introduced into Egypt in February 2006. Functional sialidase residues R98, D131, R132, R205, E257, R273, R348 and Y382 (N1 numbering) are in cyan and framework sites E99, R136, W159, S160, D179, I203, E208, H255, E258 and E405 are in yellow. A46D is located in the stalk domain and is not visible in this model (B). Virus replication assays in primary chicken embryo kidney cells (CEK) (C) and primary normal human bronchial epithelial cells (NHBE) (D) infected at an MOI of 0.001 for indicated time points were done in three independent replicates. Results are shown as mean and standard deviation. Asterisks refer to statistical significance compared to rg-AL at p < 0.05 (see main text for details). Dashed lines in panels C and D indicate the predicted detection limit of plaque and foci assays in this study. https://doi.org/10.1371/journal.ppat.1011135.g001 Based on these analyses, we generated seven recombinant viruses using Egyptian HL-H5N1 virus isolated in 2016 (designated rg-HL-16) as backbone. HL-16 is an avian isolate with 12 mutations that are found in isolates that have infected humans. Therefore, this isolate is used to represent human-isolates. In addition to rg-HL-16, six recombinant viruses carrying seven segments from rg-HL-16 and different NA segments were generated. These viruses include an H5N1 carrying NA resembling parent “avian-like” clade 2.2.1 (2006 virus, designated “rg-AL”), four viruses carrying avian-like-NA with single mutations designated as rg-AL-46D, rg-AL-204M, rg-AL-319F or rg-AL-430G, and an H5N1 carrying all four mutations resembling “human-like” 2.2.1.2 viruses from 2008 (rg-HL-08). None of the H5 viruses in this study was directly isolated from human cases.

Single NA mutations reduced virus replication in avian, mammalian and human cell culture Viruses carrying all four mutations (rg-HL-16 and rg-HL-08) or lacking all of them (rg-AL) replicated similarly in primary chicken cells (CEK) (p > 0.1) (Fig 1C) and canine MDCK-II (p > 0.1) or human A549 cell lines (p > 0.06) at 8 to 48 hpi (S2 Fig). Conversely, viruses carrying single mutations, particularly L204M, replicated less efficiently than rg-HL-16 and rg-AL (p < 0.05) in these cells, particularly at 8 hpi. In differentiated normal human bronchial epithelial (NHBE) cells, only three of the NA variants were tested: rg-AL, rg-HL-16 and rg-AL-204M. As controls, human (H1N1 and H3N2) and avian (H7N1) viruses were used (Fig 1D). Interestingly, after multiple cycle replication, rg-HL-16 replicated to significantly higher titers than human H1N1/H3N2 viruses and other recombinant H5N1 viruses tested in this experiment (p < 0.04). While the replication of rg-AL-204M was significantly lower than human H1N1/H3N2 viruses at 12 and 24 hours post-infection (hpi) (p < 0.05), they reached comparable titers 48 hpi. Together, the single NA mutations reduced H5N1 virus replication in different cells, with the lowest replication efficiency for rg-AL-204M. High replication efficiency in cell lines was restored by the combination of the mutations in human-like viruses (rg-HL-08 and rg-HL-16) or by avian-like viruses lacking these mutations (rg-AL), further indicating the interdependence and synergistic effect. In contrast, human-like H5N1 virus (rg-HL-16) replicated to higher levels than all other tested viruses in primary human airway epithelia.

L204M promoted plasma membrane accumulation of NA of human-like viruses and increased viral cell-to-cell spread We further investigated the correlation between NA activity and virus distribution after infection of MDCK-II cells. Using confocal microscopy, we visualized the distribution of the NA in infected cells, 8 and 24 hpi with anti-N1 antibodies without permeabilization. rg-AL-204M and rg-HL-08 showed more abundant labelling on the plasma membrane than rg-HL-16, rg-AL and PR8 suggesting virus accumulation (Fig 6). These results suggested that the L204M has a role in the accumulation of human-like H5N1 NA on the cell membrane. Nonetheless, the effect of L204M could be compensated by other NA mutations as seen in rg-HL-16. PPT PowerPoint slide

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TIFF original image Download: Fig 6. Cellular localization of NA in infected cells. Cellular localization of NA in infected MDCK-II cells without permeabilization. MDCK-II cells were infected with indicated viruses with an MOI of 1 for 8 (A) and 24 h (B). Cells were fixed and analyzed via confocal microscopy. Cell nuclei were counterstained with Hoechst33342. NA was detected by rabbit anti-N1 polyclonal antibodies. Confocal images were acquired with a Leica DMI6000 TCS SP5 confocal laser-scanning microscope (Leica Microsystems, Germany). Representative images of three independent experiments were processed using arivis vision4D (v3.4.0). Scale bar: 25 μm. https://doi.org/10.1371/journal.ppat.1011135.g006 To confirm these results, we determined the relation between membrane bound NA and the amount of virus released into the supernatant of infected cells by flow cytometry and plaque test, respectively, at 8 and 24 hpi. As expected, accumulation of NA of rg-HL-16, rg-HL-08 and rg-AL-204M with low NA-activity on the plasma membrane was generally increased over rg-AL and PR8 with high NA-activity (p < 0.02) (Fig 7A). At 24hpi the amount of rg-AL-204M NA on the cell membrane was highest followed by rg-HL-08 and rg-HL-16 (p < 0.005). Notably, virus release as measured by plaque test inversely correlated with surface NA accumulation. Compared to PR8 and rg-AL, the titers of rg-AL-204M, rg-HL-08 and rg-HL-16 in the supernatant were significantly lower (p < 0.001) (Fig 7B). These data further suggest that L204M plays a role in virus release into the supernatant of infected cells possibly due to an accumulation on the plasma membrane. PPT PowerPoint slide

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TIFF original image Download: Fig 7. NA accumulation, virus release, cell-to-cell spread and affinity to modified turkey erythrocytes. MDCK-II cells were infected with the indicated viruses with an MOI of 1 for 8 and 24 h and the accumulation of NA on the plasma membrane was measured by flow cytometry and polyclonal anti-N1 antibodies. The results are the mean and standard deviation of three independent replicates (A). Virus release into the supernatant was determined by plaque test. The results are the mean and standard deviation of three independent replicates (B). Cell-to-cell spread of indicated viruses was assessed by measuring the size of 100 plaques induced in MDCK-II at 72 hpi using NIS Nikon Software (C). Viruses were adjusted to 32 HA units using chicken erythrocytes. Shown is the HA titer of indicated viruses against resialiated turkey erythrocytes carrying avian 2,3- or human 2,6-sialic acid (SA). The HA test was conducted in three independent experiments (D). Asterisks indicate significant differences compared with rg-AL at p < 0.05. https://doi.org/10.1371/journal.ppat.1011135.g007 To study whether NA accumulation on the plasma membrane can promote viral cell-to-cell viral spread, plaque diameters induced by the different viruses in MDCK-II cells were measured (Fig 7C). rg-HL-16 and rg-AL-204M produced significantly larger plaques than rg-AL (p < 0.0001). Likewise, also the other viruses, except rg-AL-430G, had significantly larger plaques than rg-AL (p < 0.0001). Together, human-like mutations in the NA (except S430G) alone or combined increased H5N1 cell-to-cell spread.

NA mutations increased H5N1-affinity to human-type receptors Besides the HA, NA can also bind directly to cellular receptors affecting virus entry [23,24]. Therefore, we firstly studied the receptor binding affinity to turkey erythrocytes (TRBCs) expressing either avian 2,3-SA or human 2,6-SA in standard HA test [5]. As expected, human H1N1 bound efficiently to 2,6-SA but not 2,3-SA (p < 0.001), while avian H4N2 did not show affinity to 2,6-SA (Fig 7D). All H5N1 viruses showed comparable affinity to 2,3-SA (p = 0.9). However, human-like viruses (rg-HL-16 and rg-HL-08) exhibited ≥ four-fold higher binding affinity to 2,6-SA than rg-AL and rg-AL-204M (p < 0.04). Insertion of A46D, S319F or S430G into rg-AL significantly increased binding-affinity to 2,6-SA (p < 0.01). Interestingly, only rg-AL-319F and rg-AL-430G had similar binding-affinity to 2,3-SA and 2,6-SA. To confirm these results, we tested the receptor specificity of all H5N1 viruses using a glycan microarray presenting preferred receptors for human viruses [25]. The viruses were screened on the array with and without oseltamivir (OS) to disentangle HA specificity and the influence of NA on receptor binding. All viruses bound to avian-like 2,3-linked Neu5Ac glycans (Fig 8) overall signal was decreased without OS indicated contributions of NA. rg-AL fails to convincingly bind human-type receptors, which was even more clear in the presence of OS, however, indicating that this NA might has a sialic acid binding function. For rg-HL-16 bound to some human-like 2,6-linked Neu5Ac was observed in the absence of OS (Fig 8). Interestingly, the shift in binding specificity of rg-HL-16 was probably due to NA S430G (Fig 8). NA S430G was especially interesting as it binds human-type receptors in both the absence and presence of OS, while the HA matches the other viruses. Thus, the enzymatic site is not responsible for the binding to the human-like 2,6-linked Neu5Ac. In summary, human-like viruses (rg-HL-16 and rg-HL-08) had dual binding affinity to avian- and human-type receptors, and NA S430G mutation plays a role in the affinity to human-type receptors. PPT PowerPoint slide

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TIFF original image Download: Fig 8. The impact of NA on binding specificity to avian- and human-like glycan moieties. Shown in numbers are N-glycan compounds terminating in gal (#1–3), avian type receptors (#4–6, black bars) and human type receptors (#7–15, white bars) (A). Glycolipids presentation of avian-type receptors are shown as #A-M magenta bars [25,80] (B). Viruses were applied to the array surface in the absence (C, E, G) or presence of oseltamivir (OS) (D, F, H) at concentration of 200 nM (n = 4). Representative results for three independent experiments are shown for rg-AL (C and D), rg-HL-16 (E and F) and rg-AL-430G (G and H). https://doi.org/10.1371/journal.ppat.1011135.g008

NA retained specificity and cleavage of avian-type sialic acid receptors during virus entry The release of human-like virus from infected cells can be also affected by the density of sialic acid on the cell surface. To explore the efficiency and specificity of NA to remove SA-receptors during virus entry, an underappreciated role of the NA, we infected MDCK-II cells which contain both 2,6-SA and 2,3-SA, and genetically modified MDCK-SIAT1 cells, which express elevated amount of 2,6-SA and reduced amount of 2,3-SA [26]. Cells were infected with rg-HL-16 and rg-AL for two hours only and the amount of 2,3-SA and 2,6-SA was measured using flow cytometry (S5 Fig). Compared to non-infected cells, we found that both viruses were efficient in removal of 2,3-SA (p < 0.0001). Interestingly, in MDCK-II, but not MDCK-SIAT1, cells rg-AL was more efficient than rg-HL-16 to cleave 2,3-SA and 2,6-SA (p < 0.03). These results indicate that NA retained specificity to cleave avian-type receptors during entry, which is probably essential in reducing the density of SA bound to the HA at late stage of release. The presence of high amount of 2,6-SA on cell surface may further explain the inability of rg-HL-16, which has a higher affinity/specificity to this receptor, to be efficiently released compared to rg-AL.

Altered NA activity did not affect the tropism of H5N1 viruses to non-ciliated airway epithelia Previous studies showed that some human influenza viruses preferred ciliated epithelial cells (ECs) with both 2,3- and 2,6-SA, while an AIV H5N1 preferentially infected non-ciliated mucin-producing goblet cells rich in 2,3-SA [27, 28]. To determine whether Egyptian human-like H5N1 viruses attach either to ciliated or non-ciliated epithelial cells and whether NA mutations alter viral airway epithelia tropism, differentiated primary ferret airway ECs (FAECs) were infected with rg-AL, rg-AL-204M, rg-HL-16 or human H3N2 (S6 Fig). All H5N1 viruses infected the FAECs and were mainly detectable in non-ciliated ECs. Conversely, human H3N2 infected efficiently both ciliated and non-ciliated cells. These results indicate that Egyptian H5N1 viruses can infect cells resembling the respiratory tracts of mammals, but less efficiently than human H3N2 virus. Altered NA activity apparently does not affect tropism to the airway epithelia.

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