Potential genomic reassortments with canonical influenza virus cannot be ruled out and should be assessed.

Shorebirds and waterfowls are believed to be the reservoir hosts for influenza viruses, whereas swine putatively act as mixing vessels. The recent identification of two influenza-like virus genomes (designated H17N10 and H18N11) from bats has challenged this notion. A crucial question concerns the role bats might play in influenza virus ecology. Structural and functional studies of the two major surface envelope proteins, hemagglutinin (HA) and neuraminidase (NA), demonstrate that neither has canonical HA or NA functions found in influenza viruses. However, putative functional modules and domains in other encoded proteins are conserved, and the N-terminal domain of the H17N10 polymerase subunit PA has a classical structure and function. Therefore, potential genomic reassortments of such influenza-like viruses with canonical influenza viruses cannot be excluded at this point and should be assessed.

In this review we summarize recent work on the functions and structures of both HA and NA derived from H17N10 and H18N11. In addition, all influenza proteins are examined using bioinformatics, and proteins from the six internal genes of the H17N10/H18N11 genomes are shown to conserve known structural and functional modules or domains. We also further discuss our recent work on the N-terminal domain of the polymerase subunit PA. We believe that the functions and structures of all the encoded proteins should be examined in detail to understand these influenza-like viruses better. Moreover, their reassortment potential should be assessed by reverse genetics experiments.

In 2012, an astonishing story published in Proc. Natl. Acad. Sci. USA [] caused some concerns because a new influenza virus genome (H17N10) was isolated from bats by next-generation sequencing (NGS). This raised a serious scientific and public health question as to the origin and evolution of influenza virus. If this genome can produce a real influenza virus that can cause human or animal infections, then the situation for influenza virus-caused diseases would be more complicated because bats are known to harbor many viruses and are regarded as a reservoir host for many human- and animal-infecting viruses, including the SARS (severe acute respiratory syndrome) coronavirus []. If this is the case, then our understanding of the influenza virus ecology will need to be rewritten. Therefore research on this NGS-identified novel genome is urgently needed for the sake of public health. Studies on the two surface envelope proteins, HA and NA (NA-like) [], demonstrate that neither protein has the corresponding canonical influenza virus functions or structures []. Therefore the new genome does not represent a ‘true’ influenza virus, and it should be renamed influenza-like virus, at most. Whether or not the genomic segments will reassort with canonical influenza A virus genomes should be vigorously tested in the near future. Recently a similar virus genome, H18N11, was again identified by NGS, and neither HA nor NA have canonical structures or functions [].

Bat-derived influenza hemagglutinin H17 does not bind canonical avian or human receptors and most likely uses a unique entry mechanism.

Influenza A virus is an enveloped negative-stranded RNA virus, with a segmented genome of 8 pieces, which encode a total of 14 proteins []. There are three major envelope proteins embedded in the virus surface: hemagglutinin (HA), neuraminidase (NA), and matrix protein 2 (M2) []. HA is responsible for virus binding to susceptible cells, which harbor sialylated proteins (as virus receptors), eventually resulting in fusion to and entry into the cells. NA is a sialidase that enables mature viruses to be released from infected cells []. To date 16 HA subtypes and nine NA subtypes have been identified ( Figure 1 ), and the different reassorted combinations are used to name the viruses, for example, the common seasonal flu viruses H1N1, H2N2, H3N2, and the sporadic human infections with H5N1 and the recent H7N9 [].

Phylogenetic trees of the hemagglutinin (HA) and neuraminidase (NA) genes of all known influenza A virus subtypes including H17/18 and NA-like N10/11. (A) The categorized HA molecules can be grouped into two groups, group 1 and group 2. The bat-derived H17 and H18 (each marked with a star) belong to group 1, displaying a typical sequence feature of group 1 HA molecules. (B) All the known NAs could also be grouped into two groups: group 1 and group 2. The bat-derived N10 and N11 (each marked with a star) do not belong to either group. They are NA homologs or could be called NA-like molecules. Here we propose that they be categorized as influenza A-like group 3.

Figure 1 Phylogenetic trees of the hemagglutinin (HA) and neuraminidase (NA) genes of all known influenza A virus subtypes including H17/18 and NA-like N10/11. (A) The categorized HA molecules can be grouped into two groups, group 1 and group 2. The bat-derived H17 and H18 (each marked with a star) belong to group 1, displaying a typical sequence feature of group 1 HA molecules. (B) All the known NAs could also be grouped into two groups: group 1 and group 2. The bat-derived N10 and N11 (each marked with a star) do not belong to either group. They are NA homologs or could be called NA-like molecules. Here we propose that they be categorized as influenza A-like group 3.

Influenza virus is a member of the Orthomyxoviridae family. There are three types of influenza virus based on its internal proteins of nucleoprotein and matrix protein, namely A, B, and C []. Among these, influenza A virus is the most prevalent pathogen for both humans and animals, causing the so-called seasonal flu. Influenza A virus was also the causative agent of four major pandemics, in other words the 1918 Spanish flu, 1957 Asian flu, 1968 Hong Kong flu, and the 2009 swine-origin pandemic flu (2009 pH1N1), as well as of the relatively milder pandemic, the 1977 Russian flu []. For the origin, genesis and ecology of influenza viruses, migratory birds (shorebirds and waterfowls) are regarded as reservoir hosts, providing a large pool of virus gene segments that can contribute to novel reassortant viruses []. Meanwhile, swine are believed to be ‘mixing vessels’ or at least intermediate hosts that influenza A viruses can utilize to ‘jump’ from poultry to humans [].

HAs H17 and H18 do not bind to the canonical sialic acid receptors

12 Tong S.

et al. A distinct lineage of influenza A virus from bats. 19 Tong S.

et al. New world bats harbor diverse influenza A viruses. 20 Air G.M. Sequence relationships among the hemagglutinin genes of 12 subtypes of influenza A virus. 21 Nobusawa E.

et al. Comparison of complete amino acid sequences and receptor-binding properties among 13 serotypes of hemagglutinins of influenza A viruses. 22 Gamblin S.J.

Skehel J.J. Influenza hemagglutinin and neuraminidase membrane glycoproteins. 12 Tong S.

et al. A distinct lineage of influenza A virus from bats. 16 Sun X.

et al. Bat-derived influenza hemagglutinin H17 does not bind canonical avian or human receptors and most likely uses a unique entry mechanism. 17 Zhu X.

et al. Hemagglutinin homologue from H17N10 bat influenza virus exhibits divergent receptor-binding and pH-dependent fusion activities. 19 Tong S.

et al. New world bats harbor diverse influenza A viruses. HA is the receptor binding protein of the influenza A virus, and is responsible for virus entry into host cells. Before the discovery of the H17 and H18 genes there were 16 subtypes of HA described, H1–H16. Based on their primary sequences, these HA molecules can be categorized into two groups ( Figure 1 ): group 1 (H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, and H18) and group 2 (H3, H4, H7, H10, H14, and H15) []. Bat-derived H17 and H18 should be placed into group 1 based on their primary sequences ( Figure 1 ) [].

22 Gamblin S.J.

Skehel J.J. Influenza hemagglutinin and neuraminidase membrane glycoproteins. 16 Sun X.

et al. Bat-derived influenza hemagglutinin H17 does not bind canonical avian or human receptors and most likely uses a unique entry mechanism. 23 Lu X.

et al. Structure and receptor binding specificity of hemagglutinin H13 from avian influenza A virus H13N6. 16 Sun X.

et al. Bat-derived influenza hemagglutinin H17 does not bind canonical avian or human receptors and most likely uses a unique entry mechanism. 23 Lu X.

et al. Structure and receptor binding specificity of hemagglutinin H13 from avian influenza A virus H13N6. As expected from their primary sequences, the H17 and H18 structures resemble the structures of group 1 HAs rather than those of group 2. Previously solved HA structures demonstrate that there are group-specific features at sites where extensive conformational changes occur for HA activation, including the conformation of the interhelix loop and the rigid body orientation of the globular domain []. Taking H17 as an example, it displays a similar interhelix loop conformation to the HAs from group 1, and this is consistent with the phylogenetic analysis. Superimposition with other solved HA structures by means of the long central α-helices of HA2 revealed that the globular domains fall into three subgroups []: subgroup 1, including H1, H2, H5, and H9; subgroup 2, including H3, H7, and H14; and subgroup 3, consisting of H13, H16, H17 and H18. These differences may result from subtle variation in the interhelix loops among different HA subtypes and could signify different mechanisms during HA activation [].

24 Sauter N.K.

et al. Hemagglutinins from two influenza virus variants bind to sialic acid derivatives with millimolar dissociation constants: a 500 MHz proton nuclear magnetic resonance study. 25 Takemoto D.K.

et al. A surface plasmon resonance assay for the binding of influenza virus hemagglutinin to its sialic acid receptor. 26 Gambaryan A.S.

et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl(N-acetyllactosamine). 24 Sauter N.K.

et al. Hemagglutinins from two influenza virus variants bind to sialic acid derivatives with millimolar dissociation constants: a 500 MHz proton nuclear magnetic resonance study. 25 Takemoto D.K.

et al. A surface plasmon resonance assay for the binding of influenza virus hemagglutinin to its sialic acid receptor. 26 Gambaryan A.S.

et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl(N-acetyllactosamine). 22 Gamblin S.J.

Skehel J.J. Influenza hemagglutinin and neuraminidase membrane glycoproteins. 27 Bullough P.A.

et al. Structure of influenza haemagglutinin at the pH of membrane fusion. 28 Harrison S.C. Viral membrane fusion. 29 Skehel J.J.

Wiley D.C. Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. 30 Wiley D.C.

Skehel J.J. The structure and function of the hemagglutinin membrane glycoprotein of influenza virus. 22 Gamblin S.J.

Skehel J.J. Influenza hemagglutinin and neuraminidase membrane glycoproteins. 27 Bullough P.A.

et al. Structure of influenza haemagglutinin at the pH of membrane fusion. 28 Harrison S.C. Viral membrane fusion. 29 Skehel J.J.

Wiley D.C. Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. 30 Wiley D.C.

Skehel J.J. The structure and function of the hemagglutinin membrane glycoprotein of influenza virus. 31 Wilson I.A.

et al. Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 Å resolution. Figure 2 Configurations of avian and human receptor analogs. (A) The avian receptor analog. The Sia-1 (sialic acid, SA) is linked to Gal-2 (galactose) via an α2,3-glycosidic bond. The glycans extend forward and the hydrophilic glycosidic oxygen atom is exposed to the receptor binding site. (B) The human receptor analog. The Sia-1 is linked to Gal-2 via an α2,6-glycosidic bond. The glycans fold back and the hydrophobic C6 atom is exposed to the receptor binding site. In both (A) and (B) N-acetylglucosamine is abbreviated as GlcNAc-3; black arrows indicate the orientations of the glycosidic linkage. Influenza A virus enters susceptible cells through endocytosis after binding to cell surface receptors []. The receptors for influenza A viruses are sialic acids (SA) linked to cell surface glycolipids or glycoproteins []. After entering the cells, the virus fuses with the endosomal membrane under low pH and subsequently the genetic material is released into the cell []. Avian- and human-adapted influenza A viruses harbor distinct HA molecules that have different capacities to bind specifically linked SA-moieties. In Figure 2 , the configurations of the α-2,3-linked SA and α-2,6-linked SA are shown. The molecular basis of the interaction of these specific receptors with the virus-derived HA has been extensively studied []. In the trimeric structure of the HA, resolved as early as in 1981 in an outstanding collaborative work by Wilson, Wiley, and Skehel [], the HA head domain of each monomer was shown to be responsible for SA binding. We now know that there are three secondary elements and one base element involved directly in SA binding. The three secondary elements (the 130-loop, the 190-helix, and the 220-loop, in H3 numbering) form the edge portion, and four conserved residues (Y98, W153, H183, and Y195) form the base portion. These two portions usually form a shallow cavity to accommodate sialylated glycans. Typically, the SA moiety of the sialylated glycan forms several conserved hydrogen bonds with the 130-loop and the base residue Y98, and the remaining glycan moieties interact with the 220-loop or 190-helix.

30 Wiley D.C.

Skehel J.J. The structure and function of the hemagglutinin membrane glycoprotein of influenza virus. 32 Liu J.

et al. Structures of receptor complexes formed by hemagglutinins from the Asian Influenza pandemic of 1957. 33 Zhang W.

et al. Molecular basis of the receptor binding specificity switch of the hemagglutinins from both the 1918 and 2009 pandemic influenza A viruses by a D225G substitution. 34 Gamblin S.J.

et al. The structure and receptor binding properties of the 1918 influenza hemagglutinin. 35 Zhang W.

et al. An airborne transmissible avian influenza H5 hemagglutinin seen at the atomic level. 36 Xiong X.

et al. Receptor binding by a ferret-transmissible H5 avian influenza virus. 37 Shi Y.

et al. Structures and receptor binding of hemagglutinins from human-infecting H7N9 influenza viruses. 38 Xiong X.

et al. Receptor binding by an H7N9 influenza virus from humans. Substitution of the residues in the three secondary elements is important for the HA protein to obtain avian or human SA receptor preference. Different HA subtypes use different substitutions to achieve this goal. For H2 and H3 HAs, Q226L and G228S substitutions in the 220-loop are responsible for the switch between avian and human receptor binding specificities [] whereas, for H1 HA, different combinations of substitutions at residues 190 and 225 are important for the SA binding preference []. For H5 HA, a single Q226L substitution is enough to change the receptor binding preference []. By contrast, for H7 HAs, Q226L substitution is not solely responsible for the acquisition of human receptor binding, and other amino acid substitutions also contribute to the receptor binding switch, especially G186 V substitution [].

16 Sun X.

et al. Bat-derived influenza hemagglutinin H17 does not bind canonical avian or human receptors and most likely uses a unique entry mechanism. 17 Zhu X.

et al. Hemagglutinin homologue from H17N10 bat influenza virus exhibits divergent receptor-binding and pH-dependent fusion activities. 19 Tong S.

et al. New world bats harbor diverse influenza A viruses. 16 Sun X.

et al. Bat-derived influenza hemagglutinin H17 does not bind canonical avian or human receptors and most likely uses a unique entry mechanism. 19 Tong S.

et al. New world bats harbor diverse influenza A viruses. 16 Sun X.

et al. Bat-derived influenza hemagglutinin H17 does not bind canonical avian or human receptors and most likely uses a unique entry mechanism. 19 Tong S.

et al. New world bats harbor diverse influenza A viruses. Figure 3 Receptor-binding ‘head’ domains of all the hemagglutinins (HAs) with known structures (including influenza A and B) showing the SA binding sites. This figure clearly shows that H17 and H18 have a smaller SA-binding cavity – with two acidic amino acids (labeled in red) in comparison to the non-charged amino acids in all the other HA molecules (labeled in yellow). Note, the base of the receptor binding groove is labeled in green; both H17 and H18 show a ‘closed’ conformation, and that of H18 is even smaller. Multiple lines of evidence, including surface plasmon resonance (SPR) experiments, MDCK cell binding assays, and glycan microarray analysis, revealed that the bat-derived H17 and H18 do not bind to canonical human or avian receptors []. This lack of canonical receptor binding is likely due to specific structural features in the putative receptor binding sites of H17 and H18 HA. In the H17 and H18 structures ( Figure 3 ) there is no obvious cavity to accommodate the sialylated glycans, due to strong interactions among three secondary elements (130-loop, 190-helix, and 220-loop) through a hydrogen bond and salt bridge network formed by residues D136, Q190, H226, and D228 []. Furthermore, the negatively charged D136 in the 130-loop (all canonical influenza HAs have an uncharged threonine or serine at this position) could result in a charge conflict with the negatively charged carboxyl group of SA, which is unfavorable for SA receptor binding []. Moreover, residue 98 (usually a conserved tyrosine) in the base of the receptor binding site is a phenylalanine in H17 and H18, and this could also affect SA receptor binding capacity. Thus, these five key residues likely contribute to the lack of SA receptor binding by H17 and H18 and make the putative binding cavity a much smaller, pseudo-binding site (a ‘closed’ site) ( Figure 3 ).